The invention relates to branched soluble polymeric supports for membrane enhanced peptides synthesis (MEPS), and their use in MEPS.

BACKGROUND OF THE INVENTION

In the following text, the following meanings are used, if not otherwise stated:

AA amino acid

AAA amino acid analysis

ACN acetonitrile

benzotriazole- 1 -yl-sulfonyl azide COSY correlated spectroscopy

CuAAC copper-catalyzed azide-alkyne cycloaddition

2-CTC 2-chloro trityl chloride

DCM dichloromethane

DIEA N,N-ethyldiisopropylamine

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

DIPCDI N,N'-diisopropylcarbodiimide

DTPA diethylenetriaminepentaacetic acid

EDC-HC1 N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDTA ethylenediaminetetraacetic acid

eq equivalent

ESMS electrospray mass spectrometry

2-ethylimidazole- 1 -sulfonyl azide FDA Food and Drug Administration

FT-IR fourier transform infrared spectrometry

HBTU 0-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-pho sphate, CAS

94790-37-1

HOBt 1-hydroxybenzotriazole

HOSu N-hydroxysuccinimide

HPLC high-performance liquid chromatography

*salt

imidazole- 1 -sulfonyl azide

MsOH and HBF ₄ kDa kilo Dalton;

LPPS liquid phase peptide synthesis

MEPS membrane enhanced peptide synthesis

MWCO molecular weight cut-off

MW molecular weight

m/z mass-to-charge ratio

NMR 1H, 13C nuclear magnetic resonance proton and carbon

NMP N-methylpyrrolidone

OSN organic solvent nanofiltration

Oxyme Pure ethyl cyano(hydroxyimino)acetate

PEG polyethylene glycol

PEEK pol(ether ether ketone)

PEI polyethlyene imide

PMDA pyromellitic dinahydride

PYBOP benzotriazol- 1 -yloxytripyrrolidinophosphoniumhexafluorophosphate

SPPS solid phase peptide synthesis

THF tetrahydrofuran

TFA trifluoroacetic acid

triflylazide, trifluoromethanesulfonyl azide, triisopropylsilan

TMSN ₃ trimethylsilylazide. tR retention time

UV ultraviolet

delta chemical shift

J coupling constant

v frequency

Scale-up of peptide manufacturing is one of the bottle neck steps to bring peptide-based drugs to market. In general, chemical peptide synthesis methods are divided into solid phase and liquid phase peptide synthesis (LPPS). Although, the first commercial peptides were manufactured using the LPPS, solid phase peptide synthesis (SPPS) has been rapidly gaining acceptance over the years. The main drawback associated to SPPS is that reactions take place in a heterogeneous medium. In this sense, liquid phase peptide synthesis methodologies, which use a soluble support, could be a good alternative. The most serious challenges faced by SPPS can be overcome with the use of LPPS and more recently, with combination of LPPS and organic solvent nanofiltration. This new technology platform is known as

Membrane Enhanced Peptide Synthesis (MEPS), and applies the concept of membrane assisted purification coupled to liquid phase peptide synthesis, offering advantages over SPPS by combining LPPS with the purification concept of the solid phase method.

The use of soluble supports in LPPS avoids the problems generated by the heterogeneous media in SPPS. However, purification methods to remove the remaining reagents in LPPS have been the major drawback in the overall LPPS process.

MEPS is a technology platform that combines organic solvent nanofiltration (OSN) with LPPS. OSN is a membrane process capable of producing efficient molecular scale separations. OSN separations are typically performed in one of two modes: (a) dead-end filtration, where the entire solvent volume passes through the membrane under applied hydrostatic or gaseous pressure; or (b) 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 due to the applied pressure.

Membranes used in OSN are classified as polymeric or ceramic membranes. In general, they must have excellent long term stability when used with reaction solvents and high selectivity between soluble supports and their derivatives, byproducts, and excess reagents, such as unreacted amino acids, activators and deprotection reagents.

The selectivity of OSN membranes is commonly characterized and determined by molecular weight cut-off (MWCO), which is the molecular weight (MW) for which 90% rejection of the solute is achieved by the membrane. In other words, the MWCO describes the retention capabilities of a membrane and refers to the molecular mass of a solute where the membranes have a rejection greater than 90%. MWCO values are often supplied by the manufacturer and provide an initial indication of the membrane operating range. However, MWCO is highly dependent on the solvent-solute system used for membrane characterization. Because different methods are being employed by different manufacturers, caution must be applied before relying on these values.

In filtration processes, the permeate is the fraction of the feed stock that passes through the filter, whereas the fraction which is rejected by the filter is known as the retentate. The rejection measure and MWCO are closely related.

In order to determinate the efficiency in any OSN procedure, the rejection R(%) can be used. The rejection R(%) can be calculated with the following equation: C(permeate) \

R(%) = i - _r C ₍ (re _t ten ₁ ta ₁ te J) /

The concentrations C(permeate) and C(retentate) can be determined e.g. with HPLC. The ideal soluble support should be stable during all stages of synthesis, and should have chemically stable functional groups, allowing peptidic chain growth. The support should be stable in a broad range of solvents and coupling reagents, and should show higher rejection on polymeric or ceramic membranes. US 2011/245460 Al discloses the use of linear soluble polymeric support based on

MeO-PEG-NH ₂ (methylated amino poly(ethylene glycol). However, its application in MEPS would be limited due to its behavior under pressure. When high pressure is applied to filtrate this kind of polymer, higher quantities of polymer are lost due to its morphological characteristics. This behavior has been previously reported for similar systems by Patterson et al, Desalination, 2008, 218, 248-256.

There was a need for soluble polymeric supports for use in MEPS, that show high rejection.

SUMMARY OF THE INVENTION

Subject of the invention is a branched soluble polymeric support SOLSUPP;

SOLSUPP comprises a residue of formula (RES-APOLETHGLYC)

(RES-APOLETHGLYC) wherein none or only one of the bonds denoted with (1) and (2) is covalently connected to H;

SOLSUPP is selected from the group consisting of compound of formula (SOLPEG) and compound of formula (PYPEG); (SOLPEG)

(PYPEG)

Rl is a residue of formula (RES-TRIAZPEG) or a residue of formula (RES-PEG);

R2 is residue of formula (RES-PEG);

EG)

(*) denotes the connecting bond that covalently connects the NH residue of residue of formula (RES-TRIAZPEG) or a residue of formula (RES-PEG) respectively with the C=0 residue of compound of formula (SOLPEG) or of compound of formula (PYPEG) respectively; nl is an integer from 22 to 38;

compound of formula (SOLPEG), wherein Rl is the residue of formula (RES-TRIAZPEG), is called compound of formula (DPEG); compound of formula (SOLPEG), wherein Rl is the residue of formula (RES-PEG), is called compound of formula (DNPEG).

DETAILED DESCRIPTION OF THE INVENTION

Preferably, SOLSUPP is compound of formula (DNPEG) or compound of formula (PYPEG); more preferably, SOLSUPP is compound of formula (PYPEG);

Further subject of the invention is a method for preparation of compound of formula (DPEG), by a reaction REACl, in REACl a compound of formula (DTPA-PROPARGYL) is reacted with a compound of formula (Rl-AZIDO) in the presence of a Cu(I) salt.

(DTPA-PROPARGYL)

(Rl-AZIDO)

Θ

Reacl is also called CuAAC.

Preferably, the molar amount of compound of formula (Rl-AZIDO) is from 5 to 20 times, more preferably from 5 to 15 times, based on the molar amount of compound of formula (DTPA-PROPARGYL). Preferably, the Cu(I) salt is selected from the group consisting of CuBr, Cul, CuCl, and mixtures thereof, or the Cu(I) salt is prepared in situ by reduction of a Cu(II) salt, preferably the Cu(II) salt being of CuS0 ₄ ;

preferably, the reduction in situ of the Cu(II) salt is done by sodium ascorbate;

more preferably the Cu(I) salt is of CuBr.

Preferably, the molar amount of the Cu(I) salt is from 4.5 to 7.5 times based on the molar amount of compound of formula (DTP A-PROP ARG YL) .

Preferably, REACl is done in the presence of diethylamine.

Preferably, the molar amount of diethylamine is from 4.5 to 7.5 times based on the molar amount of compound of formula (DTP A-PROP ARG YL).

Preferably, REAC l is done in the presence of sodium ascorbate.

Preferably, the molar amount of sodium ascorbate is from 4.5 to 7.5 times based on the molar amount of compound of formula (DTP A-PROP ARG YL).

Preferably, REAC l is done in a solvent SOLVl .

Preferably, SOLVl is selected from the group consisting of DMF, ACN, THF, DCM, toluene H20, NMP, DMSO, Ci_ ₄ alcohol, and mixtures thereof; more preferably of DMF ACN, THF, DCM, toluene, and mixtures thereof; even more preferably of DCM, ACN and mixtures thereof; especially a mixture of DCM and ACN.

The Ci_ ₄ alcohol is preferably MeOH, EtOH or tertBuOH

Preferably, the reaction time of REACl is from 1 h to 48 h, more preferably from 12 h to 36 h.

Preferably, the reaction temperature of REACl is from 0 to 50°C, more preferably from 10 to 35 °C.

After REACl , any solvent can be removed, preferably by distillation, resulting in a crude.

Then preferably said crude can be dissolved in water, followed by addition of EDTA, followed by a dialysis against water or against SOLVl . The dialysis can be done with a membrane with a MWCO of 2 kDa. Dialysis can take any time needed, preferably it take 1 to 5 days. The product can be finally isolated by evaporation of any water or solvent, or lyophilization. Preferably, compound of formula (DTPA-PROPARGYL) is prepared by a reaction REAC2- DTPA, in REAC2-DTPA a compound of formula (DTPA-ANHYD) is reacted with compound of formula (PROP AM).

(DTPA-ANHYD)

^* NH ₂ (PROPAM)

Preferably, the molar amount of compound of formula (PROPAM) is from 5 to 10 times, more preferably from 5 to 7.5 times, based on the molar amount of compound of formula (DTPA-ANHYD).

Preferably, REAC2-DTPA is done in the presence of PYBOP.

Preferably, the molar amount of PYBOP is from 5 to 14 times based on the molar amount of compound of formula (DTPA-ANHYD).

Preferably, REAC2-DTPA is done in the presence of DIEA.

Preferably, REAC2-DTPA is done at a pH of from 7.5 to 9; more preferably, the pH of from 7.5 to 9 in REAC2-DTPA is adjusted by the addition of DIEA.

Preferably, the molar amount of DIEA is from 0.001 to 0.1 times based on the molar amount of compound of formula (DTPA-ANHYD).

Preferably, REAC2-DTPA is done in a solvent SOLV2-DTPA.

Preferably, SOLV2-DTPA is preferably selected from the group consisting of DCM, DMF, and mixtures thereof; more preferably SOLV2-DTPA is a mixture of DCM and DMF; even more preferably SOLV2-DTPA is a mixture of DCM and DMF with a ratio from 8 : 3 to 6 : 3. Preferably, the reaction time of REAC2-DTPA is from 10 min to 5 h, more preferably from 30 min to 2 h.

Preferably, the reaction temperature of REAC2-DTPA is from 10 to 50°C, more preferably from 15 to 30°C.

After REAC2-DTPA the obtained compound of formula (DTPA-PROPARGYL) can be

purified by chromatography.

Preferably, compound of formula (Rl-AZIDO) is prepared by a reaction REAC2-AZIDO, REAC2-AZIDO a compound of formula (Rl-APOLETHGLYC) is reacted with a compound DIAZOCOMP;

DIAZOCOMP is selected from the group consisting of sodium azide, TMSN3, TfN ₃ ,

benzotriazole-l-yl-sulfonyl azide, 2-ethylimidazole-l-sulfonyl azide, and imidazole- 1- sulfonyl azide.

Preferably, DIAZOCOMP is TfN ₃ .

Preferably, the molar amount of DIAZOCOMP is from 0.5 to 1 times, more preferably from 0.5 to 0.85 times, based on the molar amount of compound of formula (Rl- APOLETHGLYC).

Preferably, TfN ₃ is prepared in situ from trifluoromethanesolfonic anhydride and sodium

azide.

Preferably, the molar amount of sodium azide is from 2 to 10 times, more preferably from 3 to 7.5 times, based on the molar amount of trifluoromethanesolfonic anhydride. Preferably, REAC2-AZIDO is done in the presence of a compound METSALT, METSALT is selected from the group consisting of Cu(II) salt, Ni(II) salt, and Zn(II) salt;

more preferably, METSALT is CuS0 ₄ .

Preferably, the molar amount of METSALT is from 1 to 10 mol%, more preferably from 1.5 to 7.5 mol%, the mol% based on the molar amount of DIAZOCOMP.

Preferably, REAC-AZIDO is done in the presence of K ₂ C0 ₃ .

Preferably, the molar amount of K ₂ C0 ₃ is from 0.1 to 2 times based on the molar amount of DIAZOCOMP. Preferably, REAC2-AZIDO is done in a solvent SOLV2-AZIDO.

Preferably, SOLV2-AZIDO is preferably selected from the group consisting of DCM, water and mixtures thereof; more preferably SOLV2-AZIDO is a mixture of DCM with water. Preferably, the reaction time of REAC2-AZIDO is from 1 h to 24 h, more preferably from 5 h to 18 h.

Preferably, the reaction temperature of REAC2-AZIDO is from 0 to 50°C, more preferably from 0 to 30°C.

The product from REAC2-AZIDO can be used without further purification as substrate for REACl .

A by-product of REAC2-AZIDO is compound of formula (Rl-BISAZIDO).

The mixture of compound of formula (Rl-AZIDO) and compound of formula (Rl- BISAZIDO), that can result from REAC2-AZIDO, can also be used in REACl, also without further purification or separation, instead of the compound of formula (Rl- AZIDO).

Further subject of the invention is a method for preparation of compound of formula

(DNPEG), by a reaction REACIN, in RE AC IN compound of formula (DTPA- ANHYD is reacted with compound of (Rl-APOLETHGLYC).

(Rl-APOLETHGLYC)

Preferably, the molar amount of compound of (Rl-APOLETHGLYC) is from 5 to 10 times, more preferably from 5 to 7.5 times, based on the molar amount of compound of formula (DTP A- ANH YD) . Preferably, REACIN is done in the presence of PYBOP.

Preferably, the molar amount of PYBOP is from 2 to 6 times, more preferably from 3 to 5 times, based on the molar amount of compound of formula (DTPA-ANHYD).

Preferably, REACIN is done in the presence of DIEA.

Preferably, REACIN is done at a pH of from 8 to 9; more preferably, the pH of from 8 to 9 in REACIN is adjusted by the addition of DIEA.

Preferably, REACIN is done in a solvent SOLV1N; SOLV1N is DCM.

Preferably, the reaction time of REACIN is from 30 min to 2 h.

Preferably, the reaction temperature of REACIN is from 0 to 50°C, more preferably from 10 to 30°C.

After REACIN, any solvent can be removed, preferably by distillation, resulting in a crude.

Then preferably said crude can be dissolved in water or in a solvent such as SOLV1N, preferably followed by addition of EDTA, followed by a dialysis against water of against a solvent, such as SOLV1N. The dialysis can be done with a membrane with a MWCO of 450 or 750 Da. Dialysis can take any time needed, preferably it take 1 h to 5 days. The product can be finally isolated by evaporation of any water or solvent, or by lyophilization.

Further subject of the invention is a method for preparation of compound of formula

(PYPEG), by a reaction REAC1PY, in REACIPY compound of formula (PMDA) is reacted with compound of formula (Rl-APOLETHGLYC).

(PMDA) Preferably, the molar amount of compound of formula (Rl-APOLETHGLYC) is from 4 to 12 times, more preferably from 6 to 9 times, based on the molar amount of compound of formula (PMDA).

Preferably, REACIPY is done in a solvent SOLV1PY; SOLV1PY is toluene.

Preferably, the reaction time of REACIPY is from 30 min to 24 h, more preferably from 2 h to 18 h, even more preferably from 4 h to 12 h.

Preferably, the reaction temperature of REACIPY is from 50 to 150 °C, more preferably from

80 to 140°C, even more preferably from 100 to 140°C.

After REACIPY, any solvent can be removed, preferably by distillation, resulting in a crude.

Then preferably said crude can be dissolved in water or a solvent, such as SOLV1PY, preferably followed by addition of EDTA or diethylamine, followed by a dialysis against water of against a solvent, such as SOLV1PY. The dialysis can be done with a membrane with a MWCO of 450 or 750 Da. Dialysis can take any time needed, preferably it take 1 h to 5 days. The product can be finally isolated by evaporation of any water or solvent, or by lyophilization.

Further subject of the invention is the use of SOLSUPP as support in membrane enhanced peptide synthesis.

Further subject of the invention is a method for preparation of a peptide by membrane

enhanced peptide synthesis employing liquid phase peptide synthesis and organic solvent nanofiltration;

wherein SOLSUPP is used as the support for the growing peptide chain.

The method comprises

coupling in liquid phase a first amino acid, a first dipeptide or first tripeptide, onto SOLSUPP; continuing the preparation of the peptide by coupling of the remaining amino acid residues of the peptide in form of amino acids, dipeptides or tripeptides, onto the growing peptide chain;

and using organic solvent nanofiltration employing a membrane for removing impurities

during the liquid phase peptide synthesis. The organic solvent nanofiltration can be applied at any time of the liquid phase peptide synthesis, it can be applied between or after any of the reactions and between or after any of the steps of the liquid phase peptide synthesis. The organic solvent nanofiltration is done with a membrane, preferably with a polymeric or ceramic membrane, more preferably with a ceramic membrane.

Preferably, the membrane has a molecular weight cut-off that provides 75 to 100% rejection, more preferably 80 to 100% rejection, even more preferably 90 to 100% rejection, especially 95 to 100% rejection, more especially 97.5 to 100% rejection, even more especially 99 to 100%, of SOLSUPP.

Preferably, the membrane has a molecular weight cut-off of from 0.1 to 3 kDa, more

preferably of from 0.2 to 2 kDa, even more preferably of from 0.3 to 1 kDa, especially of from 0.3 to 0.9 kDa, more especially from 0.3 to 800 kDa.

EXAMPLES

Methods:

IR spectra were determined in an FT-IR Nexus (Termo Nicolet 760).

All analytical HPLCs were performed in three systems:

1) HPLC PDA 2695 Alliance using two different Sunfire columns: RP-C18 column (10 mm, 4.6 nm x 100 nm reverse phase column), solvent A (H ₂ 0) - solvent B (ACN), gradient 5% to 100% of ACN of the ACN (0.036% TFA) into H20 (0.045% TFA) were run at

0.3mL/min flow rate over 8 min;

2) RP-C18 column (lOmn, 4,6mm x 150nm reverse phase column), Isopropanol (solvent A).

0.1 vol% TFA/Acetonitrile (solvent B); 0.1 vol% TFA/H ₂ 0 (solvent C); the eluent flow was kept constant at 1 ml for the 25 min required for complete elution. The composition of solvents was changed first 5% solvent B and 95% solvent C until 95% solvent B and 5% solvent C for the elution, and

3) HPLC PDA Acquity UPLC Binary Sol MGR Waters using a BioBasic-18 RP-C18 column (5 micrometer, 2.1 x 150 nm reverse phase column), solvent A(H ₂ 0)-solvent B(ACN), gradient from 5% to 95% of ACN of the ACN (0.036% TFA) into H ₂ 0 (0.045% TFA) were run at 0.3mL/min flow rate over 8 min.

Absorbance was detected at 220 nm.

RP-HPLC-ESMS were performed using two systems: Waters Micromass ZQ spectrometer, gradient from 100% of ACN of the ACN (0.07% formic acid) into H ₂ 0 (0.1% formic acid) were run at 0.3 mL/min flow rate over 8 min, mass spectra were recorded using an

Electron Spray Ionization (ESI) technique on a Micromass ZQ, Waters SN: MAA 076; and Acquity UPLC Binary Sol MGR (Waters Corporation), linear gradients of ACN (0.1% formic acid) into H ₂ 0 (0.1% formic acid) were run at 100 microL/min flow rate over 9 min, mass spectra were recorded using a LCT-Premier (Waters) (TOF analyzer). NMR spectra were recorded using two systems: Bruker DPX 400 (400 MHz-lH, 75.4-13C), and Bruker DPX 500 (500 MHz-lH, 75.4-13C). Chemical shifts are given as delta values against tetramethylsilane as the internal standard and J values are given in Hz.

Materials The OSN membranes used in this work were: polymeric membrane; DuraMem® 500

(MWCO of 500 g-mol-1), DuraMem® 700 (MWCO of 700 g-mol-1) and PEEK (MWCO not known) flatsheet membranes provided by Imperial Collegue (UK) and MET company (UK). Ceramic membranes: Inopor® 450 (MWCO of 450 g-mol-1) and Inopor® 750 (MWCO of 700 g-mol-1) provided by Inopor company HITK (Germany).

All Fmoc-L-AA-OH and Fmoc-Rink- Amide, HOBt and HBTU were purchased from Iris Biotech. K ₂ C0 ₃ was purchased from Acros. CuBr, sodium ascorbate, DIEA, DIPCDI, DTPA-ANHYD, propargylamine, Tf0 ₂ , NaN ₃ , CuS0 ₄ , methoxypolyethylene glycol amine (5 kDa), 0,0'-Bis(3-aminopropyl)polyethylene glycol (1.5 kDa), diethylamine, piperidine, EDTA, PMDA and TIS were purchased from Sigma- Aldrich and all were used without purification unless otherwise noted. All other reagents were purchased from Carlo Erba, Romil and Lonza and used without further purification. Membrane Properties

Two types of membranes were used: polymeric membranes and ceramic membranes. Physical and chemical properties of the membranes are shown in Table 1.

Table 1 : Membranes supplied by the inopor GmbH, 98669 Veilsdorf, Germany, and Evonik MET Ltd., UK. Abbreviations used in this table: Molecular weight cut off (MWCO), No Data (N.D.), Titanium dioxide (Ti0 ₂ )

Polymeric Membrane: Filtration experiments were conducted using membranes in a stainless steel, SEPA ST (Osmonics, USA), dead end nanofiltration cell. Membrane coupons with an effective membrane area of 14 cm2 were cut and placed on a sintered metal plate. An O-ring was used to seal the feed solution from the permeate side. N ₂ at 10 bar was used as the driving force for filtration. A stirrer was used in all experiments to minimize the effects of concentration polarization on the membrane's surface.

The membranes were pre-conditioned with pure solvent until steady state fluxes were achieved. Thereafter, 100 ml of test solution (polymer) was charged to the cell. For each filtration, 50 ml were allowed to permeate through the membrane. DuraMemb® 450 and 750 are a trademark of MET. One milliliter from each of the feed and retentate samples was taken for analysis. A 1 ml sample of the permeate sample was taken at the end of the concentration phase. In between filtrations with different test solutions, the cell was washed thoroughly with the solvent to be tested and pre-conditioned prior to the addition of the test mixture.

Ceramic membrane: Filtration experiments were conducted using ceramic membranes Inopor 450 and 750 in a stainless steel, Natan GmbH, dead end nanofiltration cell. N ₂ at 10 bar was used as the driving force for filtration. The membranes were pre-conditioned with pure solvent until steady state fluxes were achieved. Next, 500 ml of test solution (polymer) was charged to the cell. For each filtration, 500 ml were allowed to permeate through the membrane. DuraMemb® 450 and 750 are a trademark of MET.

Between change of differents crudes with test solutions, the cell was washed thoroughly with the solvent to be tested and pre-conditioned prior to the addition of the test mixture.

One milliliter from each of the feed and retentate samples . and 1 ml sample of the permeate sample were taken at the end of the concentration phase, for HPLC analysis (Method 2).

Example 1 - Compound of formula (DPEG)

Compound of formula (DTPA-ANHYD) (300 mg, 0.83 mmol) was dissolved in DCM-DMF 7:3 (300 mL) and PYBOP (2.4 g, 9.6 mmol) was added. DIEA (1.5 mL, 0.009 mmol) was added to the reaction until a pH of 8 was obtained. Next, compound of formula (PROP AM) (317 microL, 4.6 mmol) was added. The reaction was stirred for one hour and subsequently, the solvent was removed under reduced pressure. Purification of the crude was achieved by reverse phase chromatography (RediSep® Normal-reverse Silica Flash Columns-Isco CI 8 of 43 g) Solvent A(H ₂ 0)-solvent B(ACN), gradient from 0 to 100% of ACN of the ACN (0.036% TFA) into H20 (0.045% TFA) were run at 40 mL/min flow rate over 60 min, that is ISCO Combiflash using a reverse phase column (CI 8)) to obtain compound of formula (DTP A-PROP ARG YL) . Purity was 95%. Triflylazide preparation (0.4 mmol): A solution of sodium azide (lg, 15.9 mmol) was dissolved in distilled H ₂ 0 (2.6 mL) with CuS0 ₄ (2 mol%), K ₂ C03 (1 eq) in DCM (4.4 mL) and cooled in an ice bath. Triflyl anhydride (539 microL, 3.2 mmol) was added slowly over 5 min followed by stirring for 2 h. The mixture was placed in a separatory funnel and the CH ₂ C1 ₂ phase removed. The aqueous portion was extracted with DCM (two times with 1.5 mL each). The organic fractions, containing the triflylazide, were pooled and washed once with saturated Na ₂ C0 ₃ and used without further purification.

0,0'-Bis(3-aminopropyl)polyethylene glycol (1.5 kDa, representing compound of formula (Rl-APOLETHGLYC) with nl being 22-38, 6 g, 4 mmol) was dissolved in DCM (8 mL). Then, CuS0 ₄ (3.9 mg, 0.016 mmol) and K ₂ C0 ₃ (239 mg, 1.72 mmol) were dissolved in distilled H20 (5 mL), and the two combined solutions were added. The previously obtained TfN3 was added and the mixture was stirred at room temperature overnight. Subsequently, the aqueous phase was removed and the organic solvent was removed under reduced pressure to obtain the respective azidopropyl-PEGaminopropyl derivative (6 g, representing compound of formula (Rl-AZIDO)) crude without further purification.

To a solution of said azidopropyl-PEGaminopropyl derivative (3 g, ca. 2 mmol) in DCM* (60 mL), diethylamine (831 microL, 0.8 mmol), sodium ascorbate (159 mg, 0.8 mmol) as a 1% solution in ACN* and compound of formula (DTP A-PROP ARG YL) . (93 mg, 0.16 mmol) dissolved in ACN* were added. Then, the mixture was bubbled with Argon for one minute, and CuBr (114 mg, 0.8 mmol) dissolved in purged acetonitrile (4 mL) was added.

Subsequently, the reaction vessel was sealed and the reaction stirred at room temperature for 24 h. After this time, the corresponding band to the azide group present in said azidopropyl- PEGaminopropyl derivative at ca. 2098 cm ^_1 had disappeared from the IR spectra. Then the solvent was removed under reduced pressure. The crude product was dissolved in distilled H ₂ 0, EDTA (2 eq for each eq of CuBr) was then added and the mixture dialyzed against water (two changes over four days, the membrane had a MWCO of 2 kDa). After that, the sample was lyophilized to obtain compound of formula (DPEG). Yield 50%.

The compound of formula (DPEG) was analyzed by HPLC (Method 1) gradient: from 5% to 100%) ACN over 8 min), and its structure was confirmed by spectroscopic techniques such as IR and 1H-NMR. The HPLC profile showed a broad peak, as is to be expected for a poly disperse sample, at 4.8 min of retention time. The IR spectrum of compound of formula (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 .

1H-NMR analysis allowed corroboration of the structure. The five amide protons appeared as a broad signal at 8.5ppm. This assignation was confirmed when a few drops of D20 were added and its resonance signal in the NMR spectrum disappeared due to the rapid exchange of amide protons with heavy water. The protons corresponding to the triazole moieties appeared as a singlet at 7.9 ppm. *DCM and acetonitrile were purged of molecular oxygen by bubbling with Argon for 40 min.

IR(KBr): 3438, 2882, 1632, 1469, 1344, 1283, 1111, 963, 841 cm ^"1

1H-NMR (400 MHz, DMSO-d6): delta 8.52 (s, 5H, HB), 7.88 (s, 5H, HA) ppm

Molecular weight ca. 8.2 kDa.

Example 2 - Compound of formula (DNPEG)

To a suspension of compound of formula (DTPA-ANHYD) (222 mg, 0.62 mmol, 1 eq) in dry DCM (17 mL) was added PYBOP (1.1 g, 2.04 mmol, 3.3 eq) and a solution of 0,0'-Bis(3- aminopropyl)poly ethylene glycol (1.5 kDa, representing compound of formula

(Rl-APOLETHGLYC) with nl being 22-38, theoretical loading: 0.66 mmol NH ₂ /g, 5 g, 3.3 mmol, 5.3 eq) in DCM (23 mL).

The pH was adjusted to 8 to 9 by the addition of DIEA, and the resulting mixture was stirred at room temperature for 1 h. Subsequently, the solvent was removed under reduced pressure to obtain the crude of compound of formula (DNPEG). Theoretical loading: 0.61 mmol NH ₂ /g. Purification of the crude was achieved by OSN using ceramic membranes Inopor 750 (MWCO: 750 Da) in a stainless steel, dead end nanofiltration cell. N ₂ at 10 bar was used as the driving force for filtration. The membranes were pre-conditioned with DCM as pure solvent until steady state fluxes were achieved. Next, 500 ml of test solution the crude of compound of formula (DNPEG) (5 g in 500 mL of DCM) was charged to the cell. For each filtration, 500 ml was allowed to permeate through the membrane, 60 diafiltrations were carried out to obtain a purified compound of formula (DNPEG).

Molecular weight ca. 7.6 kDa. When IR spectra of starting 0,0'-Bis(3-aminopropyl)polyethylene glycol and compound of formula (DNPEG) were compared, a new band corresponding to v(C=0) of the newly formed amide bonds was detected at 1646 cm ^"1 .

IR(KBr): 3427, 2874, 1646, 1457, 1351, 1299, 1250, 1104, 925, 843 cm ^"1

The 1H-NMR analysis evidenced the presence of amide bonds on compound of formula (DNPEG) by the typical broad signal of amide protons at 8.1ppm. This signal disappeared with the addition of D ₂ 0, corroborating this assignation. The rejection of compound of formula (DNPEG) was determined for two ceramic

membranes: Inopor® 450 and Inopor® 750. Compound of formula (DNPEG) showed quantitative rejection (100%) in both membranes.

Example 3 - Compound of formula (PYPEG)

0,0'-Bis(3-aminopropyl)polyethylene glycol (1.5 kDa, representing compound of formula (Rl-APOLETHGLYC) with nl being 22-38, theoretical loading; 0.66 mmol NH ₂ /g, 30 g, 20 mol, 8 eq) was dissolved in toluene (300 mL), and compound of formula (PMDA) (0.54 g, 2.5 mol, 1 eq) was added. The mixture was placed in 250 mL vessel equipped with a Dean Stark and a reflux column. The reaction was stirred and heated at 125°C for 8 hours. Subsequently, the solvent was removed under reduced pressure to obtain crude compound of formula (PYPEG) with a theoretical loading of 0.65 mmol NH ₂ /g. Purification of the crude was achieved by OSN using ceramic membranes Inopor® 750 in a stainless steel, dead end nano filtration cell. N ₂ at 10 bar was used as the driving force for filtration. The membranes were pre-conditioned with DCM as pure solvent until steady state fluxes were achieved. For OSN diethylamine (2 g, 27 mmol, 2 eq) in DCM was added to the solution of crude of compound of formula (PYPEG) (34g in 500ml of DCM). Next, 500 ml of said solution of crude of compound of formula (PYPEG) was charged to the cell. For each filtration, 500 ml were allowed to permeate through the membrane, 60 diafiltrations were realized to obtain purified compound of formula (PYPEG) (96.8% purity). Subsequently, the solvent was removed under reduced pressure, and the solid was again dissolved in DCM (100 mL) and evaporated (10 repeats).

Molecular weight ca. 6.2 kDa When IR spectra of starting 0,0'-Bis(3-aminopropyl)polyethylene glycol and compound of formula (PYPEG) were compared, a new band corresponding to v(C=0) of the newly formed amide bonds was detected at 1646 cm ^"1 .

IR(KBr): 3427, 2871, 1653, 1456, 1351, 1299, 1250, 1106, 925, 846cm ^"1

1H-NMR (500 MHz, D6-DMSO): delta = 8.30 (s, 4H), 7.7 (s, 2H), 3.65 to 3.25 (m), 2.9 (m, 2H), 1.7 (m, 2H)

The ratio between the integration of amide protons with delta 8.30 signal in the 1H-NMR

spectrum and that of benzene protons signals with delta 7.7 was determined as 2: 1, showing the presence of four branches per each benzene ring.

13C-NMR (500 MHz, D6-DMSO): delta = 28.6, 29.2, 36.5, 37.5, 60.2, 67.6, 68.1, 69.8, 72.3, 127.0, 136.8, 166.9 ppm. The rejection of compound of formula (PYPEG) was determined for two ceramic membranes: Inopor® 450 and Inopor® 750. Compound of formula (PYPEG) showed quantitative rejection (100%) in both membranes.

Example 4 (MEPS with compound of formula DPEG)

Fmoc-Gly-OH and Fmoc-Asp(tBu)-Rink-Gly-OH were coupled onto compound of formula (DPEG), prepared according to example 1, using standard conditions (1 eq of compound of fomrula (DPEG), 1.2 eq of Fmoc-AA-OH, 1.2 eq of HBTU, 1.2 eq of DIEA in DCM for 16 h at room temperature), and the reactions were followed by HPLC (Method 1). After purification by dialysis against water, the final products Fmoc-Gly-DPEG and Fmoc-Asp- Rink-Gly-DPEG were analyzed by HPLC (Method 1) and in each case, HPLC peaks showed a characteristic UV profile corresponding to the Fmoc group, corroborating the coupling.

The rejection of DPEG, Fmoc-Gly-DPEG, and Fmoc-Asp(tBu)-Rink-Gly-DPEG was measured using an OSN system, with three polymeric membranes. Vicote® PEEK,

DuraMem® 500 and DuraMem® 900 were tested (Table 3, Entries 1-9). DPEG showed a rejection of 78%. In general, the rejection increased for Fmoc-Gly-DPEG and Fmoc- Asp(tBu)-Rink-Gly-DPEG, and as expected, the best results were obtained when membranes with MWCO of 500 Da were used instead of those with a MWCO of 900 Da. DPEG rejection in two ceramic membranes was also measured (Table 3, Entries 10 and 11) showing quantitative values of 96% and 99% that were higher than those obtained for polymeric membranes, independently of their MWCO ^' s.

Example 5 (MEPS on compound of formula PYPEG)

Compound of formula (PYPEG), prepared according to example 3, was reacted with an Fmoc-Pvink Amide Linker in the presence of HBTU and DIEA in DCM as solvent:

Fmoc-Pvink Amide Linker (18 g, 33 mol, 3 eq) was dissolved in DCM (300 mL) and DIEA (8.5 g, 66 mol, 6 eq) was added. HBTU (12.5 g, 33 mol, 3 eq) was dissolved in DMF (120 mL). Compound of formula (PYPEG) (17 g, 2.7 mmol. 1 eq, theoretical loading; 0.65 mmol NH ₂ /g) was dissolved in DCM (80 mL), then the solution of Fmoc-Rink Amide Linker and DIEA in DCM was added and then the HBTU solution in DMF was added. The reaction was stirred for 2 hours at room temperature. The coupling reaction was followed by HPLC (Method 2). HPLC profiles showed the total disappearance of the peak corresponding to the starting material and a new peak at a higher retention time due to the formation of Fmoc- Rink-PYPEG.

The purification of Fmoc-Rink-PYPEG was carried out by OSN with Inopor® 750 and DCM as solvent: The obtained solution (ca. 500 ml) of Fmoc-Rink-PYPEG was charged to the cell and was purified by OSN using Inopor® 750 in a stainless steel, dead end nanofiltration cell. N ₂ at 10 bar was used as the driving force for filtration. For each filtration, 500 ml of DCM were allowed to permeate through the membrane, 40 diafiltrations were carried out to obtain pure Fmoc-Rink-PYPEG. Purification was analyzed by HPLC (Method 2).

The Fmoc deprotection was carried out using piperidine 20% in DMF for 30 min. Next, the polymer was purified by OSN under the same conditions as the Fmoc-protected derivative. After 40 diafiltrations, H ₂ N-Rink-PYPEG was obtained.

OSN-assisted LPPS of RADA-NH ₂ was accomplished, starting from H ₂ N-Rink-PYPEG and using sequential steps of Fmoc/tBu strategy. OSN was always carried out using using the Inopor® 750.

In general, the protecting Fmoc group was always removed by treatment with piperidine 20% in DMF for 30 min. Amino acid coupling reactions were carried out in the presence of HBTU and DIE A as coupling reagents, and DCM-DMF (9: 1) as solvent at room temperature for two hours (Scheme 1).

Scheme 1

HBTU, DIEA

DCM-DMF (9:1)

2h

After each deprotection or coupling steps, the resulting functionalized polymer was purified by OSN. The purification of each sequential step was followed by HPLC (Method 2). The HPLC chromatograms allowed concluding that 15 diafiltrations were enough to obtain the polymer derivatives with adequate purity. After the last coupling, HPLC of Fmoc-RADA- Rink-PYPEG showed one broad peak at.

The details of the MEPSwere as follows:

Fmoc-AA-OH (1.5 eq) was dissolved in DCM (400 mL) and DIEA (3.3 g, 25 mmol, 3 eq) was added. HBTU (5 g, 13 mmol, 1.5 eq) was dissolved in DMF (50 mL). H ₂ N-Rink-PYPEG (13 g, 2 mmol. 1 eq, theoretical loading; 0.65 mmol NH ₂ /g) was dissolved in DCM (50 mL), then the solution of Fmoc-AA-OH and DIEA in DCM and the HBTU solution in DMF were both added. The reaction mixture was stirred for 2 hours at room temperature. The reaction was followed by HPLC (Method 2 and 3). 500 ml of the Fmoc-AA-Rink-PYPEG solutions were purified by OSN using Inopor® 750 in a stainless steel, dead end nanofiltration cell. N ₂ at 10 bar was used as the driving force for filtration. The membranes were pre-conditioned with pure solvent until steady state fluxes were achieved. For each filtration, 500 ml were allowed to permeate through the membrane, 10 diafiltrations were carried out to obtain purified Fmoc-AA-Rink-PyPEG.

The Fmoc-RADA-Rink-PYPEG was treated with a standard cocktail of TFA-H ₂ 0-TIS (95:2.5:2.5) for one hour, and precipitated with (C ₂ H ₅ ) ₂ 0. After the cleavage procedure, the peptide was purified by reverse phase chromatography (RediSep® Normal-reverse Silica Flash Columns-Isco CI 8 of 43 g,) Solvent A(H ₂ 0)-solvent B(ACN), gradient from 0 to 100% of ACN of the ACN (0.036% TFA) into H20 (0.045% TFA) were run at 40 mL/min flow rate over 40 min that is ISCO Combiflash using a reverse phase column (CI 8). The peak corresponding to Fmoc-RADA-NH ₂ was collected and lyophilized. HPLC and HPLC-MS analysis and an aminoacid analysis (AAA) were done. The results allowed concluding that 0.2 mmol/g of RAD A peptide on compound of formula (PYPEG) was synthesized. The AAA unequivocally confirmed Fmoc-RADA-NH ₂ formation.

Calculation for C ₃ iH4oN ₈ 0 ₈ :652.71, ESI-MS (M +H): 653.31