Novel Globular Polymeric Supports for Membrane-Enhanced Peptide

Article pubs.acs.org/Macromolecules

Novel Globular Polymeric Supports for Membrane-Enhanced Peptide Synthesis Vida Castro,† Christian Noti,‡ Wenqian Chen,‡ Michele Cristau,‡ Andrew Livignston,§ Hortensia Rodríguez,*,†,∥ and Fernando Albericio*,†,⊥,#,% †

Institute for Research in Biomedicine (IRB Barcelona), 08028 Barcelona, Spain Lonza Ltd., CH-3930 Visp, Switzerland § Imperial College London, London, U.K. ∥ School of Chemistry, Yachay Tech, Yachay City of Knowledge, Urcuqui, Ecuador ⊥ Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain # Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 08028 Barcelona, Spain % School of Chemistry, University of KwaZulu Natal, Durban 4000, South Africa ‡

S Supporting Information *

ABSTRACT: Membrane-enhanced peptide synthesis (MEPS), a technique that combines liquid-phase peptide synthesis (LPPS) with organic solvent nanofiltration (OSN), has emerged as a new methodology to tackle current challenges in solid-phase peptide synthesis (SPPS), the current strategy of choice for the preparation of peptides. This new technology platform is scalable beyond kilogram scale, automatable, and compatible with established Fmoc chemistry, as it combines chemistry in solution with expedient membrane purification. Here we screened novel highly rejected soluble polymeric supports and studied their application for the preparation of a model peptide. Our findings make a significant contribution to the development of MEPS.

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INTRODUCTION Peptides are relevant molecular entities. The pharmaceutical market’s demands for these molecules have grown rapidly in recent years due to an increasing number of therapeutic targets and the development of improved delivery methods.1−5 Like most organic molecules, peptides were synthesized for many years in solution. However, solid-phase peptide synthesis (SPPS) has gained widespread acceptance and is now the most common method used to produce peptides.6−8 The main drawback of SPSS is the use of excess amino acid and reagents, resulting in a high cost of raw materials. In this regard, parallel to the development of the SPPS strategies, the so-called liquid phase peptide synthesis (LPPS) was studied. LPPS is based on a growing peptide chain attached to a polymer, such as poly(ethylene glycol) (5000−20000 MW)9−20 and polystyrene,21−24 which are soluble in the reaction solvents but precipitate in ether and alcohols. Thus, the reaction takes place in solution, and the resulting peptide−polymer is isolated by either precipitation or centrifugation.25−27 However, the LPPS strategy is hampered by the lack of a continuous method for the isolation of the peptide−polymer to facilitate the automatization of the process. In this regard, organic solvent nanofiltration (OSN)28 is a pressure-driven method that achieves efficient molecular © XXXX American Chemical Society

separations. Applications of OSN include the following: (a) concentration, (b) solvent exchange, (c) purification, (d) peptide solution reconcentration,29 and (e) amino acid recovery.30,31 At laboratory scale, OSN separations are typically performed in two modes, namely (i) dead-end filtration, where the entire solvent volume passes through the membrane perpendicularly under applied hydrostatic or gaseous pressure, and (ii) cross-flow filtration, where the solvent passes parallel to the surface of the membrane and only part of the solvent volume passes through the membrane in response to the pressure applied.32 OSN has become important for LPPS because of the development of new polymeric and ceramic membranes that are resistant to organic solvents and have a molecular weight cutoff (MWCO) that is adjustable to polymers. Compared to traditional methods for purifying compounds, membrane filtration has the advantage that it operates at ordinary temperatures, avoids phase transition, and has low energy consumption.33,34 The combination of LPPS and OSN has led to membraneenhanced peptide synthesis (MEPS), which could be a viable Received: October 29, 2016 Revised: February 7, 2017

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DOI: 10.1021/acs.macromol.6b02258 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. Structures of novel branched polymers with a molecular weight of 8.2 kDa for DPEG, 7.6 kDa for DNPEG, and 6.2 kDa for PyPEG.

Scheme 1. DPEG Total Synthesisa

a

Reagents and conditions: (i) I (0.16 equiv), II (0.002 equiv), CuBr (0.8 equiv), diethylamine (8 equiv), sodium ascorbate (0.8 equiv), DCM-ACN, rt, 16 h.

typically around 1 nm,66−69 and the pores are not cylindrical. Nevertheless, these insights from ultratfiltration and nanpores are broadly borne out by data for linear and branched PEG chains passing through PBI nanofiltration membranes, where it was found that rejection of a PEG star with an aromatic ring at the core was higher than the same molecular weight linear PEG, in the range 2000−8000 g mol.58−70 Therefore, the anchor molecules for MEPS were designed to be four- or five-arm branched (star-shape) molecule. To overcome the aforementioned problems associated with the use of linear PEG, globular polymers should be used. Here we designed and prepared three globular PEG-based polymers and tested their compatibility with OSN, one of them in front MEPS.

alternative to SPPS. MEPS applies the concept of membraneassisted purification to liquid-phase synthesis, offering advantages over SPPS by combining polymer-supported chemistry with the purification concept of SPPS. OSN-assisted LPPS has been reported only by Livingston et al.28,35,36 for the synthesis of 1−5-mer model peptides at laboratory scale, using PEG linear polymers as support and ceramic membranes to purify the intermediate products after each step. However, linear polymers, which have also been used in several examples of LPPS12,13,15−17,24,37−56 have a limited application in MEPS.28 In this regard, the morphological characteristics of linear polymers (i.e., flexible chains in random coil) result in a significant loss of polymer through membranes during OSN, regardless of polymer size.57 Previous experiments have shown that branched polymer (star-shape) molecules are able to pass through the pores of ultrafiltration membranes with cylindrical pores, even when the pores are smaller than the hydrodynamic radius of the polymer.58 This is because the molecules can deform under a flow field. Interestingly, for cylindrical pores it has also been shown that as flow is reduced, branched (star-shape) molecules become less able to pass nanopores than linear molecules of the same molecular weight.59 The experimental and modeling results of linear and branched (star-shape) molecules through cylindrical pores in ultrafiltration membranes provide useful insights for the design of anchor for MEPS.58−65 It was shown that the arm number of branched (star-shape) molecule plays a more important role than the arm length in the rejection of the molecule.65 In the MEPS process the nanofiltration membranes have considerably smaller pores than in these previous studies,

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RESULTS AND DISCUSSION

The design of the three soluble polymeric supports [DPEG, DNPEG, and PyPEG (Figure 1)] was based on chains of diamine-functionalized propyl-poly(ethylene glycol) (1)71 with a low molecular weight (1.5 kDa) attached to a core molecule. DPEG and DNPEG were constructed on the pentetic acid core [diethylenetriaminepentaacetic acid (DTPA)], while the pyromellitic acid core was used for PyPEG (Figure 1). The PEG arms were attached via click chemistry using the Cu(I)catalyzed azide/alkyne cycloaddition (CuAAC) for DPEG and via amide bond for DNPEG and PyPEG. The way in which PEG arms were attached to the select core was modified from the DPEG (through CuAAC) to DNPEG and PyPEG (through amide bond formation) because of the synthesis using azide B


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Macromolecules Scheme 2. Syntheses of I and IIa,b

a Reagents and conditions: (i) 1 (0.004 equiv), TfN3 (3.2 equiv) DCM−MeOH (98:2), rt, 2 h. bReagents and conditions: (ii) 2 (0.8 equiv), 4 (4.6 equiv), PyPOB (9.6 equiv), DIEA, DCM−DMF, pH = 8, rt, 1 h.

Figure 2. (a) HPLC chromatogram of DPEG. Gradient: from 5% to 100% ACN over 8 min. (b) DPEG structure.

Scheme 3. DNPEG Synthesisa

a

Reagents and conditions: (i) 1 (5.3 equiv), 2 (1.0 equiv), PyBOP (3.3 equiv), DIEA, DCM Anh, pH = 8−9, rt, 1 h.

chemistry has to be avoid in the industry with consideration for security issues. DPEG Polymer. The DPEG, with a theoretical molecular weight of 8.2 kDa, presents five aminopropyl-PEG branches attached to the DTPA core. It was prepared through click chemistry between azido propyl-PEG-aminopropyl derivative (I) and DTPA-propargyl (II) using CuBr, diethylamine, sodium ascorbate, and DCM-ACN as solvent (Scheme 1). The synthesis of azidopropyl-PEG-aminopropyl derivative (I) was carried out by selective diazotransfer reaction of O,O′bis(3-aminopropyl)poly(ethylene glycol) (1, 1.5 kDa) using triflyl azide (TfN3), CuSO4, and K2CO3 in DCM−H2O as solvent. I was obtained as the major product and O,O′-bis(3azidopropyl)poly(ethylene glycol) (III) as byproduct (Scheme 2, reaction 1). The mixture of azidopropyl derivatives I and III was used without further purification. The DTPA-propargyl (II) was obtained through the reaction of DTPA (2) and propargylamine (4) in the presence of benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (PyBOP), DIEA as base, and DCM-DMF as solvent (Scheme 2, reaction 2). The reaction was followed by HPLC and HPLCMS. After purification in reverse phase (RediSep Normal-

reverse Silica Flash Columns-Isco C18 of 43g), II was obtained with high purity (95%). The preparation of DPEG (Scheme 1) was followed by IR spectroscopy, and the reaction was stopped when the corresponding band to the azide group present in I disappeared from the IR spectra (see Supporting Information). After 24 h of reaction, the solvents were removed, and the crude product was dissolved in water. EDTA was added, and this mixture was dialyzed against water for 2 days using a membrane (MWCO of 2 kDa). Finally, the solution was lyophilized, obtaining DPEG in 50% yield. The DPEG HPLC profile showed a broad peak, as to be expected for a polydisperse sample (Figure 2). The IR spectrum of DPEG showed a signal at 1632 cm−1 corresponding to carbonyl amide groups and also the characteristic aminopropyl-PEG bands at 3438, 2882, and 1111 cm−1 (see Supporting Information). 1H NMR analyses allowed corroboration of the DPEG structure by detecting the signal corresponding to amine protons (HA) and protons of triazole moieties (HB) (Figure 2b). The five amide protons (HA) appeared as a broad signal at 8.5 ppm. The protons corresponding to the triazole moieties (HB) appeared as a C


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thereby corroborating this assignation (see Supporting Information). PyPEG Polymer. PyPEG, which has theoretical molecular weight of 6.2 kDa, was built on the hydrophobic pyromellitic acid core, which holds four aminopropyl-PEG branches. PyPEG was synthesized in a one-step reaction between pyromellitic acid (3) and O,O′-bis(3-aminopropyl)poly(ethylene glycol) (1.5 kDa, 8 equiv) under toluene reflux for 8 h (Scheme 4). The reaction was followed by HPLC in conjunction with 1H NMR techniques. The starting pyromellitic acid (3) had a retention time in HPLC of 3.9 min (Figure 4a), and the chromatogram corresponding to final product showed the total consumption of the pyromellitic acid and a broad peak at 10.1 min corresponding to PyPEG (Figure 4b).

singlet at 7.9 ppm. The rest of the signals corresponded to the nature of the aliphatic protons present in this globular polymer. MALDI-TOF mass spectra also supported the polymer characterization, which displayed the mass peak corresponding to DPEG molecular weight (Mw) at 8272 Da, very closed to the calculate average Mw (8200 Da). Mass higher than those corresponding to DPEG molecular weight were not detected, corroborating the absence of cross-linking byproducts (see Supporting Information). DNPEG Polymer. DNPEG polymer, with a theoretical molecular weight of 7.6 kDa, presented the same DTPA core as DPEG, and also five aminopropyl-PEG branches, but these were attached through amide bonds instead through triazole moieties (Scheme 3). The polymer synthesis was carried out by reaction between O,O′-bis(3-aminopropyl)poly(ethylene glycol) (1.5 kDa) and DTPA anhydride (2) with PyBOP and DCM as solvent (Scheme 3). The reaction pH was adjusted to 8−9 by addition of DIEA. After 1 h, the solvent was removed, and DNPEG was purified by OSN using an Inopor 750 (MWCO 750 Da) ceramic membrane. The reaction was followed by HPLC, and DNPEG was also analyzed by HPLC. As expected, a profile with a broad peak at 4.5 min retention time was obtained (Figure 3).

Figure 4. HPLC chromatograms: (a) commercial product pyromellitic acid (3) and (b) PyPEG synthesis. Gradient: from 5% to 95% ACN over 25 min. Figure 3. HPLC chromatogram of DNPEG. Gradient: from 5% to 100% ACN over 8 min.

Two different reaction conditions were tested in order to optimize PyPEG formation (Figure 5). The 1H NMR spectrum obtained when the reaction was carried out at 55 °C for 8 h (Figure 5, RI: 55 °C, 8 h, spectrum 2) showed more than one signal in the aromatic zone, leading to the conclusion that some PEG branches were not incorporated. A mixture of polymers with one, two, three, and four branches was obtained under these conditions. When the reaction temperature was increased to 125 °C, the 1H NMR spectrum (Figure 5, RII: 125 °C, 8 h, spectrum 3) presented a unique signal at 7.73 ppm, which confirmed total incorporation of all PEG branches (Figure 5).

The IR and 1H NMR spectra confirmed DNPEG formation. When IR spectra of starting O,O′-bis(3-aminopropyl)poly(ethylene glycol) (1.5 kDa) and DNPEG were compared (see Supporting Information), a new band corresponding to νCO of new amide bonds generated in DNPEG was detected at 1646 cm−1. The 1H NMR analysis evidenced the presence of amide bonds on DNPEG by the typical broad signal of amide protons at 8.1 ppm. This signal disappeared with the addition of D2O, Scheme 4. PyPEG Synthesisa

a

Reagents and conditions: (i) 1 (8.0 equiv), 3 (1.0 equiv), toluene, Δ, 8 h. D


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Figure 5. PyPEG synthesis and 1H NMR spectra (in MeOD) comparison of the aromatic zone. Spectrum 1, red line: O,O′-bis(3aminopropyl)poly(ethylene glycol) (1.5K Da); spectrum 2, green line: conditions RI (55 °C, 8 h); spectrum 3, blue line: conditions RII (125 °C, 8 h).

Figure 6. Zoomed area of 1H NMR spectrum of PyPEG in DMSO-d6.

assignation (spectra not shown). To upfield, signals at 2.8 and 1.7 ppm were assigned to methylenes (HD and HE protons), respectively. These assignations were corroborated by COSY experiments (see Supporting Information). In order to corroborate the number of branches present in the PyPEG structure, the ratio between the integration of the amide proton (HC) signal in the 1H NMR spectrum and that of benzene protons (HA and HB) was determined as 2:1. This result allowed us to confirm the coupling of four branches per benzene ring (Figure 6). These signals were selected due to the fact that HA and HB belonged to a core, whereas HC was a part of a new bond between each branch and the core. MALDI-TOF mass spectra also supported the polymer characterization, which displayed the mass peak corresponding to PyPEG molecular weight (Mw) at 6422 Da, very closed to the calculate average Mw (6200 Da). Masses higher than those corresponding to PyPEG molecular weight were not detected, corroborating the absence of cross-linking byproducts (see Supporting Information). Rejection of Polymers. In order to demonstrate the potential of the three novel polymers as support for MEPS, the rejections of all polymers (%) were measured, using two ceramic membranes with the cross-flow configuration. Additionally, DPEG rejection was also determined by applying three polymeric membranes (Table 1).

After PyPEG synthesis under optimum conditions (RII), the polymer was purified by OSN using an Inopor membrane (MWCO = 750 Da). The optimal number of diafiltrations was determined by 1H NMR, by comparing the integration of the signal corresponding to the protons of the benzene core (Figure 5, HA and HB; δ,7.7 ppm) with that of the methylene closest to the primary amine (Figure 5, HE; δ, 2.9 ppm). When the proportion was close to 1:4, the polymeric reaction was complete (Figure 5). After 60 diafiltrations with DCM, PyPEG was obtained with 96.8% purity. PyPEG was characterized by IR, 1H NMR, 13C NMR, and mass spectroscopy. The IR spectrum of PyPEG, in comparison with compound 1, showed a new vibrational mode at 1649 cm−1, corresponding to the carbonyl group (νCO) of the amide bonds present (see Supporting Information). The 1H NMR study, including mono- and bidimensional experiments, was carried out to corroborate the structure of PyPEG. First of all, the 1H NMR PyPEG spectrum, with and without previous presaturation of methylene signals corresponding to the PEG fragments, was carried out (see Supporting Information). This technique was used to obtain better resolution in other signals and to facilitate structural analysis. To downfield, a broad signal corresponding to four amide protons (HC) and a singlet corresponding to the equivalents benzene protons (HA and HB) were observed at 8.3 and 7.5 ppm, respectively. The signal intensity reduction at 8.3 ppm when D2O drops were added, thus corroborating this E


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Macromolecules Table 1. Rejection (%) of Three Novel Polymers Supports (DPEG, DNPEG, and PyPEG) by Ceramic and Polymeric Membranes % rejectionc membranes

typea

MWCOb (Da)

DPEG

DNPEG

PyPEG

Inopor 450 Inopor 750 Vicote PEEK DuraMem 500 DuraMem 900

C C P P P

450 750

99 96 78 78 78

100 100

100 100

500 900

a

Type of membranes: C = ceramic and P = polymeric. bMWCO: molecular weight cutoff. cRejection = (1 − permeate concentration/ retentate concentration) × 100%.28

As an example, HPLC chromatograms of retentate and permeate samples obtained in the Inopor 750 Da membrane (Figure 7) showed that PyPEG did not cross this membrane.

Figure 8. HPLC chromatograms: (a) PyPEG polymer, tr = 10.1 min; (b) Fmoc-Rink-PyPEG, tr = 19.6 min; (c) H-Rink-PyPEG, tr = 10.5 min. Gradient: from 5% to 95% ACN over 25 min.

Rink-PyPEG was carried out by OSN with a ceramic membrane (Inopor 750) and DCM as solvent to obtain Fmoc-RinkPyPEG with adequate purity (Figure 8b). Fmoc deprotection was achieved using piperidine 20% in DMF for 30 min, after which the product was purified by OSN under the same conditions as before. After 40 diavolumes (450 mL per diavolume), H-Rink-PyPEG was obtained in high purity and then used to start LPPS of the model peptide Fmoc-RADANH2 (Figure 8c). OSN-assisted LPPS of RADA-NH2 was accomplished, starting from H-Rink-PyPEG and using sequential steps of Fmoc/t-Bu strategy similar to those used in SPPS. The Fmoc protecting group was removed by treatment of polymer derivatives with piperidine 20% in DMF for 30 min. Fmocamino acid coupling reactions were carried out with HBTU and DIEA as coupling reagents and DCM-DMF (9:1) as solvent at room temperature for 2 h (Scheme 5). Coupling/deprotection

Figure 7. HPLC chromatograms: (a) retentate sample and (b) permeate sample. Gradient: from 5% to 95% ACN over 25 min.

The rejection data showed that the ceramic membranes (i.e., Inopor 450 and Inopor 750) had significantly higher rejection than polymeric membranes (Vicote PEEK, DuraMem 500 and DuraMem 900), even though the MWCO were similar. All three globular molecules had high rejection values, thereby demonstrating their potential as supports for MEPS. Based on reaction conditions, safety, and costs, PyPEG was further tested for MEPS. MEPS of RADA. To corroborate the potential of PyPEG as a support for MEPS, we synthesized the tetramer RADA. PyPEG was funtionalized with Fmoc-Rink amide-linker via amide bond formation by reacting of PyPEG with Fmoc-Rink amide-linker in the presence of N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU) and DIEA in DCM as solvent. This first coupling reaction was monitored by HPLC and confirmed by the Kaiser test. The PyPEG derivative sample was previously precipitated with diethyl ether and gave a clear negative result after 2 h of reaction (see Supporting Information). HPLC profiles showed the total disappearance of the peak corresponding to the starting product (Figure 8a) and a new peak at a higher retention time due to the formation of Fmoc-Rink-PyPEG (Figure 8b). The purification of Fmoc-

Scheme 5. MEPS of RADA on H-Rink-PyPEGa

a

Reagents and conditions: (i) H-Rink-PyPEG (1.0 equiv), Fmoc-AAOH (1.5 equiv), HBTU (1.5 equiv), DIEA (3.0 equiv), DCM−DMF (9:1), rt, 2 h.

reactions were monitored using the Kaiser test. After each deprotection or coupling step, the resulting functionalized polymer was purified by OSN with the same ceramic membrane used for purifying Fmoc-Rink-PyPEG and H-RinkPyPEG. The product obtained in each step was followed by HPLC. The HPLC chromatograms showed that 15 diavolumes were sufficient to obtain the polymer derivatives with adequate F


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postreaction mixture. The liquid-phase reactions were less constrained by mass-transfer limitations and hence required a smaller excess of reagents than SPPS. The optimization of the OSN step and solvent recovery will result in savings in solvents and improved process economics. Thus, MEPS provides a useful alternative for the synthesis of peptides and soluble supports on an industrial scale. Here we have demonstrated that globular molecules such as PyPEG can play a pivotal role in MEPS and also in the synthesis of other organic molecules using a similar approach.

purity. After the last coupling, HPLC of totally protected peptide−polymer showed a broad peak at 4 min of retention time (Figure 9).

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ASSOCIATED CONTENT

S Supporting Information *

Figure 9. HPLC chromatogram of Fmoc-Arg(Pbf)-Ala-Asp(tBu)-AlaRink-PyPEG. Gradient: from 5% to 95% ACN over 9 min.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b02258. Experimental details (PDF)

The protected peptide was cleaved from PyPEG using an acid treatment with a standard cocktail of TFA-H2O-TIS (95:2.5:2.5) for 1 h. After the cleavage procedure, the peptide was purified by semipreparative HPLC, and the peak corresponding to Fmoc-RADA-NH 2 was collected and lyophilized. The peptide was characterized by HPLC, HPLCMS, and amino acid analysis (AAA). The AAA showed that 0.2 mmol/g of peptide was synthesized on the PyPEG. MEPS vs SPPS. The SPPS of the same sequence (RADA) was carried out on Rink amide-ChemMatrix resin, by using DIPCDI/Oxyma-Pure as coupling reagent. Ten equivalents of each Fmoc protected amino acids and coupling reagents (DIPCDI and Oxyma-Pure) at 45 °C for 2 h were used, which are the optimized coupling conditions stablished in previous work for the same sequence.72 As we explain before, the MEPS allowed using much lower quantities of coupling reagents (1.5 equiv of Fmoc-AA-OH, 3 equiv of DIEA, and 1.5 equiv of HBTU). At the end, the peptidyl resin was treated with TFA/ TIS/H2O (95:2.5:2.5) for 2 h. HPLC/ES-MS confirmed the desired (Fmoc-Arg-Ala-Asp-Ala-NH2) product. The crude Fmoc protected 4-mer (RADA) was obtained with a purity of 69% under SPPS, in comparison with 84% when MEPS protocols was used. The loading for the three polymers is 0.61 mmol/g for DPEG, 0.66 mmol/g for DNPEG, and 0.64 mmol/ g for PyPEG. These values represent that approximately there are 3.7 × 1020 reactive sites per gram of polymer. This value is of the same order than the one found in the solid supports used for conventional SPPS. Regarding spent solvent volumes, greater quantities are used under actual MEPS protocols; however, it is important highlight that this new technology platform has been conceived as an important alternative for peptide production at industrial scale.26 On the other hand, further optimization of the separation step and wash solvent volume would be possible allowing solvent savings and improved process more efficient.

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AUTHOR INFORMATION

Corresponding Authors

*(H.R.) E-mail [email protected]. *(F.A.) E-mail [email protected]. ORCID

Wenqian Chen: 0000-0001-8867-3012 Fernando Albericio: 0000-0002-8946-0462 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was carried out in the authors’ laboratories. The study was partially funded by the CICYT (CTQ2012-30930), the Generalitat de Catalunya (2014 SGR 137), and the Institute for Research in Biomedicine Barcelona (IRB Barcelona).

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ABBREVIATIONS MEPS, membrane-enhanced peptide synthesis; OSN, organic solvent nanofiltration; SPPS, solid-phase peptide synthesis; LPPS, liquid-phase peptide synthesis; MWCO, molecular weight cutoff; PEG, poly(ethylene glycol); DMF, dimethylformamide; ACN, acetonitrile; DCM, dichloromethane; DMF, N,N-dimethylformamide; ACN, acetonitrile; MeOH, methanol; DIEA, N,N-diisopropylethylamine; TEA, triethylamine; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate; TFA, trifluoroacetic acid; CuAAC, Cu(I)-catalyzed azide−alkyne cycloaddition; DTPA, diethylenetriaminepentaacetic acid; pyromellitic acid, 1,2,4,5-benzenetetracarboxylic acid; HPLC, high-performance liquid chromatography; HPLC-MS, liquid chromatography− mass spectrometry; NMR, nuclear magnetic resonance spectroscopy; COSY, correlation spectroscopy; IR, infrared spectroscopy; AAA, amino acid analysis.

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CONCLUSIONS Three novel globular soluble supports, DPEG, DNPEG, and PyPEG, were designed and synthesized. They exhibited high rejection (96%−100%) by ceramic membranes and are therefore suitable for MEPS. On the basis of reaction conditions, safety, and costs, we selected PyPEG for the synthesis of a model peptide (Fmoc-RADA-NH2). The synthesis was performed successfully, thereby demonstrating the potential of this kind of polymer for MEPS. The MEPS of Fmoc-RADA-NH2 combines the advantages of liquid peptide synthesis with membrane purification of the

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REFERENCES

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