KR101945899B1 - Preparation method of hyperbranched polyaminoglycerol based on amino glycidyl ether - Google Patents

Abstract

The polyglycidyl ether-based monomer having a protected secondary amine group is added to the polyol initiator to perform a single polymerization reaction followed by deprotection. The polyglycerol skeleton of the higher-resolution type has high biocompatibility and has an amine functional group , It is possible to bond with various biological molecules. In particular, the amine functional group is concealed inside the high-diffusive polyglycerol skeleton, so that high biocompatibility can be maintained.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for preparing a polyglycidyl ether of a high-diffusive polyamino glycerol based on aminoglycidyl ether,

The present invention relates to a hyperbranched polyaminoglycerol based homopolymer having an amine functional group so that it can be used as a carrier in a biological / biomedical field and a method for producing the homopolymer. The present invention also relates to a monomer compound used in the production of a highly branched polyamino glycerol, and a process for producing the same.

Polyamines are widely used in industrial fields such as biotechnology and have attracted much attention. Polymers containing amine groups such as polyethyleneimine and polypropyleneimine are attracting interest as CO ₂ adsorbents having a high amine content. Polyamines are useful as chelating agents for dissolving metal ions in organic solvents and are also used as curing agents for epoxy resins.

Polyamines are important compounds that are related to the structure, arrangement and function of many major biological molecules, including nucleic acids and proteins. Spontaneous polyamines such as spermine and spermidine are involved in cell growth, maintenance of membrane stability, regulation of cellular uptake, and free radical scavenging. In addition, polyamines have high potential as mediators of gene therapy because of their cationic nature in their physiological conditions. In general, polyethyleneimine has been used as a gene transfer medium with high transfection efficiency due to its proton-adsorbing ability, but its high charge density has sometimes caused fatal toxicity to cells. Accordingly, various methods for lowering the cytotoxicity of cationic polyamines have been studied. In particular, attempts have been made to synthesize polyamines having biocompatible polyether skeletons using novel amine containing monomers.

For example, N, N-dibenzylaminoglycidol, N, N-diallyylglycidylamine, N, N-diethylglycidylamine, epithianohydrin, poly -Glycidyl amines) and polyglycidol-b-polyglycidyl amines have been reported (see Macromolecules 2011, 44 , 4082-4091). However, the conventional polymers are limited to linear polyamines having a polyether skeleton by using aminoglycidyl ether-based monomers. In addition, a method of copolymerizing a monomer having an amine group with another reactive monomer has been developed, but it has disadvantages in that it does not have the simplicity of single polymerization and the size controllability in spite of improvement of physical properties due to copolymerization.

Conventionally, attempts have been made to add a terminal amine group to a shell of a high-molecular-weight polyglycerol. However, in this case, since a high-molecular-weight polyglycerol must first be synthesized and then a terminal amine group must be added by an additional synthetic reaction, And the terminal amine groups added to the shell significantly lowered the biocompatibility, making them unsuitable for use in fields such as biology.

 Meyer, J .; Keul, H .; Moller, M. Macromolecules 2011, 44, 4082-4091

Therefore, the present inventors have made efforts to develop a high-molecular-weight polyether having a useful function in the field of biomedical science. As a result, the present inventors have found that by using a protected aminoethanol glycidyl ether monomer, One-pot synthesis was possible.

Accordingly, an object of the present invention is to provide a method for producing a high-differential polyamino glycerol having an amine functional group therein.

Another object of the present invention is to provide a high-diffusive polyamino glycerol produced by the above method.

It is still another object of the present invention to provide novel monomers for use in the process for preparing the highly branched polyamino glycerol and a process for producing the same.

It is a further object of the present invention to provide the biological / biomedical use of said high-affinity branched polyamino glycerol.

According to the above object, the present invention provides a process for producing a high-diffusing-type polyamino acid, which comprises the steps of adding a polyol initiator to an aminoglycidyl ether-based monomer having a protected secondary amine group and a hydroxyl- A method for producing hyperbranched polyaminoglycerol is provided.

The present invention also provides a homopolymer of an aminoglycidyl ether monomer, wherein the aminoglycidyl ether monomer has a protected secondary amine group and provides a high-diffusible polyamino glycerol which is deprotected after monopolymerization do.

Further, the present invention provides an aminoglycidyl ether-based monomer used in the above-mentioned single polymerization,

[Chemical Formula 1]

In Formula 1, m and n are each an integer of 1 to 4, and PG is a protecting group.

The present invention also provides a biological carrier comprising the high-differential polyamino glycerol.

The high-molecular-weight terpolymer according to the present invention has a polyglycerol skeleton, which is excellent in biocompatibility and can be bonded to various biological molecules by having an amine functional group. In particular, since the amine functional group is contained in the high-molecular-weight polyglycerol skeleton to maintain high biocompatibility and is contained in the form of a secondary amine group, a dense structure and a high amine density Lt; / RTI >

Further, according to the present invention, the novel aminoglycidyl ether-based monomer can be used for one-pot synthesis of a high-differential polyglycerol having an amine functional group, and the molecular weight can be easily controlled by controlling the polymerization reaction An excellent molecular weight distribution can be achieved. In particular, the monomers have a protected amine functional group, which can be deprotected after a long reaction time for high-order polymerization and a severe polymerization process such as a strong base and a high temperature to form a polymer structure having a secondary amine group therein

In addition, the aminoglycidyl ether-based monomer can be synthesized in a simple manner and can be usefully used for the production of a high-molecular-weight type polymer. In particular, asymmetric monomers having different functional groups at both terminals can be single- Simple and easy to adjust the size.

Therefore, the high-differential polyamino glycerol of the present invention can be applied to various biological / biomedical fields such as biological carriers such as DNA, RNA and the like.

Hereinafter, the present invention will be described in more detail with reference to the drawings. The meanings of the abbreviations used in the following are described in detail for the purpose of carrying out the invention.
Figure 1 (a) shows the synthesis of Boc-protected monomers (BAG), (b) shows the anionic ring-opening polymerization of Boc-protected polymers (PBAG) and deprotected polymers (PAG).
2 (a) is a ¹ H-NMR spectrum of a BAG monomer, (b) is a ¹ H-NMR spectrum of a PBAG ₆₆ polymer, and (c) is a ¹ H-NMR spectrum of a deprotected PAG ₆₆ polymer.
3 is an expanded MALDI-ToF mass spectrum ranging from 1600 to 2300 Da of PAG ₁₃ .
4 is a ¹³ C-NMR spectrum of PAG ₆₆ polymer.
Figure 5 is the DSC curve of PBAG ₁₃ and PAG ₁₃ .
Fig. 6 shows the in vitro results of the polymer.
7 is a ¹ H-NMR spectrum of the BAG monomer.
8 is a ¹³ C-NMR spectrum of the BAG monomer.
9 is the COZY spectrum of the BAG monomer.
10 is a ¹ H-NMR spectrum of PBAG ₁₇ (Polymer 2).
11 is a GPC elution curve of PBAG ₁₃ (polymer 1) and PBAG ₆₆ (polymer 5).
12 is a ¹ H-NMR spectrum in which the reaction of deprotecting tert-butyldiethylcarbamate is measured over a period of time.
13 is a ¹³ C-NMR spectrum of PAG ₆₆ polymer (polymer 5).
14 is a DEPT spectrum of PAG ₆₆ polymer (Polymer 5).
Fig. 15 The action of the protecting group in the polymerization process of the BAG monomer is shown.
16 is a ¹ H-NMR spectrum of the Ts-protected monomer.
17 is a < ¹³ > C-NMR spectrum of Ts-protected monomer.
18 is a ¹ H-NMR spectrum of Ts-protected polymer.

Hereinafter, the present invention will be described more specifically.

According to one aspect of the present invention, there is provided a process for preparing a high-diffusing-type polyamino acid, comprising the steps of adding a polyol initiator to an aminoglycidyl ether-based monomer having a protected secondary amine group and a hydroxyl- A process for preparing hyperbranched polyaminoglycerol is provided.

Preferably, the aminoglycidyl ether-based monomer is an asymmetric monomer having different functional groups at both ends in chemical structure. Specifically, the aminoglycidyl ether-based monomer is an asymmetric monomer having an epoxy group at one end and a hydroxyl group at the other end.

In the asymmetric monomer used in the present invention, the epoxy group at one end of the monomer reacts with the hydroxyl group at the other end of the monomer to cause a ring-opening polymerization reaction. The hydroxy group newly formed by the ring opening of the epoxy group functions as a new polymerization initiation site By causing a ring-opening polymerization reaction with the epoxy group of another monomer, the chain can be extended chain by chain. As a result, single polymerization of the high-differential polyglycerol from one kind of monomer of the present invention is possible.

On the other hand, symmetric monomers having the same functional groups at both ends can not be polymerized by itself, and can be polymerized only by copolymerizing with other types of monomers to extend the chain or crosslinking other types of compounds.

Even if an asymmetric monomer is present, even if it has a functional group at only one end of the monomer and a functional group protected at the other end so as not to have a functional group or to have no reactivity, there is also a disadvantage in that single polymerization can not be performed by itself.

The monomer also has a protected secondary amine group, which can withstand a severe polymerization process such as a long reaction time, a strong base and a high temperature (see FIG. 15), and is subsequently deprotected to form a polymer structure having a secondary amine group therein .

As described above, according to the present invention, a chain can be easily grown by using only one type of monomer.

Further, the high-branched polyglycerol of the present invention can grow a polymer using only one kind of monomer, so that the size and molecular weight can be made very small as needed. Or conversely, when the chain is grown by continuing the polymerization reaction, it can be also produced as a polymer having a very large size and molecular weight as required. Therefore, the highly branched polyglycerol of the present invention can be much more selective in terms of size and molecular weight controllability.

As an example, the aminoglycidyl ether-based monomer may be represented by the following general formula (1).

[Chemical Formula 1]

In Formula 1, m and n are each an integer of 1 to 4, and PG is a protecting group.

Preferably, m and n in formula (1) may each be an integer of 1 to 3, more preferably 2, respectively.

The PG may also be a protecting group selected from the group consisting of t-butyloxycarbonyl (Boc), p-toluenesulfonyl (Ts), fluorenylmethyloxycarbonyl, benzyl, triphenylmethyl and carboxybenzyl .

As a preferred example, m and n are each 2 and PG is t-butyloxycarbonyl.

The method of producing the homopolymer is schematically shown in Fig. 1 (b).

According to the present invention, an aminoglycidyl ether-based monomer having a protecting group is used in the preparation of polyglycerol and deprotected after single polymerization using a polyol initiator, whereby a compound having a secondary amine group in a one- It is possible to prepare a branched polyamino glycerol.

The polyol initiator may be, for example, a compound having three or more alcohol groups. As a specific example, the polyol initiator may be trimethylol propane (TMP).

Also, the single polymerization may be anionic ring-opening multibranching polymerization.

According to a preferred embodiment, the polyol initiator is trimethylolpropane, and the monopolymerization reaction may be an anionic ring-opening multi-branched polymerization reaction.

The deprotection may be carried out in a conventional manner, for example, by adding an acid such as hydrochloric acid (HCl), bromic acid (HBr) or the like.

According to another aspect of the present invention, there is provided a homopolymer of an aminoglycidyl ether-based monomer, wherein the aminoglycidyl ether-based monomer has a protected secondary amine group and is a high- Glycerol is provided.

The homopolymer has a unit derived from the aminoglycidyl ether-based monomer as a repeating unit constituting the homopolymer.

For example, the aminoglycidyl ether-based monomer may be represented by the formula (1). As an example, m and n in the formula (1) may each be 2, and PG may be t-butyloxycarbonyl.

The high-differential polyamino glycerol may have a dendritic structure, and thus may have a spherical shape as a whole, wherein a shell and a core may be distinguished from each other.

Specifically, the high-differential polyamino glycerol comprises a plurality of branched chains extending from the core. Wherein the core may comprise units derived from the polyol used as initiator. In addition, the branched chain may be a polyether chain having a hydroxyl group at the terminal. Accordingly, a hydroxyl group is present in the shell of the highly branched polyaminoglycerol.

Specifically, the branched chain diffuses from the core to a dendritic, and is divided into a plurality of branches, and each branch is terminated with a hydroxyl group. Wherein the high-differential polyamino glycerol comprises a secondary amine group in the middle of the polyether chain constituting the polyamine glycerol.

That is, the homopolymer includes a plurality of branched chains having a polyether skeleton, and the branched chain may include a secondary amine group in the middle and a hydroxyl group at the end. More specifically, the branched chain may include a group of the following formula (2).

(2)

In Formula 2, m and n are each an integer of 1 to 4.

Accordingly, the amine functional group may not be present in the shell of the highly branched polyaminoglycerol. That is, since the amine functional group is concealed inside the highly branched polyglycerol skeleton, high biocompatibility can be maintained.

In addition, the highly branched polyaminoglycerol has a secondary amine group therein, so that it can have a denser structure as compared with the case of having a primary amine group and the like.

The high-differential polyamino glycerol may have a number average molecular weight (Mn) in the range of 1,000 to 50,000 g / mol, in the range of 2,000 to 30,000 g / mol, or in the range of 3,000 to 20,000 g / mol.

In addition, the homopolymer may have a polydispersity index (Mw / Mn) in the range of 1 to 3, in the range of 1 to 2, or in the range of 1 to 1.7.

The homopolymer may also have a zeta potential in the range of 10 to 50 mV, in the range of 10 to 40 mV, or in the range of 10 to 30 mV. When the zeta potential is within the preferred range, bonding by biological molecules and electrical attraction may be more advantageous.

The homopolymer may also have a glass transition temperature (Tg) in the range of -60 캜 to -10 캜, in the range of -50 캜 to -20 캜, or in the range of -40 캜 to -30 캜.

As a specific example, the homopolymer may have a number average molecular weight (Mn) of 3,000 to 20,000 g / mol, a zeta potential of 10 to 50 mV, and a glass transition temperature (Tg) of -50 ° C to -20 ° C.

According to another aspect of the present invention, there is provided a compound represented by the following formula (1), which can be used as a monomer in the production of the highly branched polyamino glycerol:

[Chemical Formula 1]

In Formula 1, m and n are each an integer of 1 to 4, and PG is a protecting group.

Preferably, m and n in formula (1) may each be an integer of 1 to 3, more preferably 2.

The PG may also be a protecting group selected from the group consisting of t-butyloxycarbonyl (Boc), p-toluenesulfonyl (Ts), fluorenylmethyloxycarbonyl, benzyl, triphenylmethyl and carboxybenzyl , Preferably t-butyloxycarbonyl.

According to a preferred example, m and n are each 2, and PG may be t-butyloxycarbonyl, which may be represented by the following formula (1a).

[Formula 1a]

The compound of formula (Ia) is referred to as Boc-protected aminoethanol glycidyl ether or t-butyl (2-hydroxyethyl) (2- (oxiran- Methoxy) < / RTI > ethyl carbamate.

The compound of formula (Ia) may be prepared by, for example, (1) reacting diethanolamine with di-t-butyl dicarbonate (Boc ₂ O) to prepare t-butyl bis (2-hydroxyethyl) carbamate; And (2) reacting the t-butyl bis (2-hydroxyethyl) carbamate with epichlorohydrin.

The method for preparing the compound of formula (1a) is illustrated in FIG. 1 (a).

An amine compound such as triethylamine (TEA) may be further added during the reaction of step (1).

The reaction of step (2) may be carried out at a temperature of, for example, room temperature to 30 ° C for 10 to 30 hours. The reaction may also be carried out in the presence of a phase transfer catalyst such as tetrabutyl ammonium hydrogensulfate (TBAHS). Further, the epichlorohydrin may be used in the same equivalent amount to excess amount, for example, 1.5 to 10 times equivalent amount, or 2 to 5 times equivalent amount to the t-butyl bis (2-hydroxyethyl) carbamate.

The compound wherein m and n are each 1, 3, 4 or 5 in the above formula (1) can be prepared by reacting a compound represented by the formula (1) in the presence of a base such as dimethanolamine, Butanol amine, or dipentaanolamine.

In the above formula (1), PG is a protecting group other than Boc can be prepared by using a suitable reaction compound instead of t-butylbis (2-hydroxyethyl) carbamate in the step (1) ≪ / RTI >

According to another aspect of the present invention, there is provided a biological carrier comprising the high-differential polyamino glycerol.

The high-differential polyamino glycerol can be conjugated to biological molecules by an electrical attraction due to the amine functional group to transport the biological molecules to the destination. In particular, the amine group is concealed inside the highly branched polyglycerol skeleton, so that high biocompatibility can be maintained.

For example, the biological carrier can be one that carries DNA or RNA.

The carrier containing the high-differential type polyamino glycerol can be used as a powerful carrier in biological and biomedical fields since it can be conjugated with a biological molecule and be introduced into cells by intracellular ingestion or the like.

Hereinafter, the present invention will be described in more detail by way of examples. The following examples are illustrative of the present invention, but the present invention is not limited to the following examples.

The abbreviations used in the following Examples and Test Examples have the following meanings:

- Boc: t-Butyloxycarbonyl

- BAG: Boc-protected aminoethanol glycidyl ether, i.e. t-butyl (2-hydroxyethyl) (2- (oxiran-2-yl) methoxy) ethylcarbamate

- PBAG: homopolymer of BAG

- PAG: homopolymer obtained by deprotection of PBAG

- PBAG _n or PAG _n : a homopolymer having n number of BAG units, or a homopolymer obtained by deprotecting it

- Ts: p-toluenesulfonyl (tosyl).

The materials and measurement methods used in the following Examples and Test Examples are as follows:

The reagents and solvents used in the examples below were purchased from Sigma-Aldrich and Acros unless otherwise noted.

Deuterated NMR solvents such as CDCl ₃ , D ₂ O, and DMSO-d ₆ were purchased from Cambridge Isotope Laboratory.

- ¹ H-NMR and ¹³ C-NMR spectra were obtained with CDCl ₃ and D ₂ O solvents using a 400-MR DD2 (400 MHz) spectrometer and the chemical shift values were recorded in ppm with TMS as standard.

- The weight average molecular weight (M _w ) and the molecular weight distribution (M _w / M _n ) can be measured by using polymethyl methacrylate (PMMA) as a standard substance using gel permeation chromatography (GPC, Agilent Technologies 1200 series) (DMF) as an eluent at a flow rate of 1.00 mL / min at 30 캜.

- Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF) analysis was performed using? -Cyano-4-hydroxycinnamic acid (CCA) as a matrix using an Ultraflex III MALDI mass spectrometer .

- Differential scanning calorimetry was carried out under a nitrogen atmosphere at a temperature of -80 ° C to 20 ° C at a heating rate of 10 K / min using a DSC (Q200 model, TA Instruments).

Zeta potential was measured using a Malvern Zetasizer Nano-ZS (ZEN3600, Malvern, UK).

Example 1-1 Synthesis of Boc-Protected Monomer

Step (1) Synthesis of Boc-protected diethanolamine

t-Butyl bis (2-hydroxyethyl) carbamate was synthesized by a route similar to that known in the art (see J. Med. Chem. 2006 , 49 , 4183-4195, etc.). First, a solution of diethanolamine (10 g, 95.1 mmol) and triethylamine (13.9 mL, 99.9 mmol) in di-tert-butyl dicarbonate (22.9 mL, 99.9 mmol) in CH ₂ Cl ₂ (50 mL) To the CH ₂ Cl ₂ (30 mL) solution was added dropwise at room temperature for 1 hour using a funnel. The reaction mixture was stirred at room temperature for 6 hours, dissolved in CH ₂ Cl ₂ and extracted with water and brine. The organic layer is dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography using 10% hexane / ethyl acetate (EA) as an eluent to give pure t-butyl bis (2-hydroxyethyl) carbamate as a pale yellow oil (13.67 g, 70%).

¹ H-NMR (400 MHz, CDCl ₃ ):? Ppm 3.85 (d, 6H, J = 55.9 Hz), 3.42 (s, 4H), 1.46 (s, 9H).

Step (2) Synthesis of Boc-protected monomers

An aqueous solution of sodium hydroxide (3.90 g, 50 wt%), epichlorohydrin (10.7 g, 116 mmol) and tetrabutylammonium hydrogensulfate (TBAHS, 1.65 g, 4.87 mmol) Respectively. Thereto was slowly added dropwise a solution of t-butyl bis (2-hydroxyethyl) carbamate (10 g, 48.7 mmol) in THF (30 mL) for 30 minutes and further stirred at room temperature for 15 hours. To the resulting reaction mixture was added CH ₂ Cl ₂ and the extracted product was neutralized by washing with water and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained residue was purified by flash column chromatography using 17% hexane / ethyl acetate as an eluent to obtain t-butyl (2-hydroxyethyl) (2- (oxiran- Ylmethoxy) ethylcarbamate (4.1 g, 32%).

The synthesized Boc-protected monomers (i.e., BAG) were identified by various spectroscopic / mass spectrometry such as ¹ H-NMR, ¹³ C-NMR, COZY, DEPT and ESI-MS.

^{1 H-NMR (400 MHz,} CDCl 3): δ ppm 3.88-3.21 (m, 11H), 3.21-3.06 (m, 1H), 2.88-2.73 (m, 1H), 2.62 (s, 1H), 1.47 ( s, 9H).

¹³ C-NMR (101 MHz, CDCl ₃ ):? Ppm 156.03, 80.06, 77.11, 71.69, 70.34, 62.20, 52.21, 50.55, 48.97, 43.92, 28.32.

^{MS (m / z + Na +} , ESI +) calcd for C 12 H 23 NO 2 284.3, found 283.9

Example 1-2: Synthesis of Ts-protected monomers

Step (1) Synthesis of Ts-protected diethanolamine

A solution of 4-toluenesulfonyl chloride (9.14 g, 48 mmol) in CH ₂ Cl ₂ (40 mL) was added to a solution of diethanolamine (5.46 mL, 58 mmol) and triethylamine (20.2 mL, At 0 < 0 > C for 30 minutes. The reaction mixture was stirred at room temperature overnight, dissolved in CH ₂ Cl ₂ and extracted with water and brine. The organic layer is dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting residue was purified by precipitation with a small amount of CH ₂ Cl ₂ and excess hexane to obtain pure N, N-bis (2-hydroxyethyl) -4-methylbenzenesulfonamide in the form of a white powder 9.5 g, 76%).

^{1 H NMR (600 MHz, CDCl} 3): δ 7.72-7.64 (m, 2H), 7.31 (d, J = 8.1 Hz, 2H), 3.95-3.74 (m, 4H), 3.32-3.19 (m, 4H) , ≪ / RTI > 2.42 (s, 3H).

¹³ C NMR (151 MHz, CDCl ₃ ):? 143.71, 135.21, 129.82, 127.26, 77.21, 62.23, 52.95, 21.48.

Step (2) Synthesis of Ts-protected monomers

N, N-bis (2-hydroxyethyl) -4-methylbenzenesulfonamide (2.59 g, 10 mmol) was dissolved in t-butanol (30 mL) With stirring. To this was slowly added dropwise a solution of epichlorohydrin (3.91 mL, 50 mmol) in THF (30 mL) and further stirred at room temperature for 4 hours. To the resulting reaction mixture was added CH ₂ Cl ₂ and the extracted product was neutralized by washing with water and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained residue was purified by flash column chromatography using 33% hexane / ethyl acetate (EA) as an eluent to give a Ts-protected monomer in the form of a pale yellow oil (1.49 g, 47%).

The synthesized Ts-protected monomers were identified by spectroscopic / mass spectrometry such as ¹ H-NMR, ¹³ C-NMR and ESI-MS.

¹ H NMR (400 MHz, CDCl ₃ ):? 7.77-7.63 (m, 2H), 7.37-7.28 (m, 2H), 3.86-3.72 (m, 5H), 3.47-3.18 ddt, J = 5.8, 4.2, 2.7 Hz, 1H), 2.81 (dd, J = 4.9, 4.1 Hz, 1H), 2.62 (dd, J = 4.9, 2.7 Hz, 1H), 2.43 (s, 3H).

¹³ C NMR (151 MHz, CDCl ₃ ):? 143.58, 135.59, 129.75, 127.25, 77.27, 71.76, 71.06, 61.73, 53.11, 50.40, 49.86, 44.16, 21.45.

^{MS (m / z + Na +} , ESI +) calcd for C 14 H 21 NO 5 S 315.38, found 338.11.

Example 2-1: Synthesis of PAG (using Boc-protected monomer)

Step (1) Synthesis of PBAG

1,1,1-Trimethylolpropane (TMP, 26.8 mg, 0.2 mmol) was placed in a one neck round bottom flask. Methanol solution of potassium methoxide (25.0 wt%, 22.4 μL, 0.0758 mmol) was diluted with 0.70 mL of methanol, added to the flask, and stirred at room temperature under argon atmosphere for 30 minutes. A high vacuum was applied at 60 캜 for 4 hours to remove methanol, whereby a white salt of an initiator was obtained. The flask was purged with argon and heated at 90 < 0 > C. The monomer of t-butyl (2-hydroxyethyl) (2- (oxiran-2-ylmethoxy) ethylcarbamate (BAG, 1.0 g, 19.1 mmol) prepared in Example 1-1 was dissolved in 12 After the monomer injection was completed, the solution was further stirred for 36 hours. To the obtained homopolymer, 1.0 mL of methanol was added to terminate the reaction, the polymer solution was precipitated in cold diethyl ether, Was washed twice with diethyl ether. The obtained polymer (PBAG ₁₇ ) was dried under vacuum at 60 ° C for 1 day.

NMR data (see Fig. 10) was a PBAG ₁₇ of the M _n was 4524 g / mol calculated using the equation below from, considering the margin of error of NMR integrals, the M _n value calculated from the NMR to 4500 g / mol Lt; / RTI >

Number of repeating units (BAG) = 218.39 (integral value of polyether skeleton) / 13 (number of quantities of polyether skeleton) = 16.80; M _n = 261.32 (molecular weight of BAG monomer) x 16.80 + 134.17 (molecular weight of TMP) = 4524.35 g / mol.

Step (2) Removal of protecting group

The previously obtained Boc-protected polyamino glycerol (PBAG ₁₇ ) was dissolved in 1.0 mL of 1.0 M HCl in CH ₂ Cl ₂ and stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a deprotected polymer, which was dissolved in 1.0 mL of methanol. The resultant PAG ₁₇ polymer was dried under vacuum at 60 ° C for 1 day.

The same procedure as in Example 2-1 was repeated except that the degree of polymerization during the reaction was changed to synthesize PAGs of various molecular weights. NMR and GPC data were obtained for each synthesized polymer, and the polymer composition and Mn were calculated from the NMR and GPC data.

The physical properties of various PAG polymers thus synthesized are summarized in Table 1 below.

Polymer
number Polymer composition
(NMR) ^a M _n (NMR) Mw ^b M _w / M _n ^b Tg (占 폚) (DSC) Zeta potential
(mV) PBAG PAG PBAG PBAG PBAG PAG One PAG ₁₃ 3500 2300 3400 1.19 -28.4 -33.5 14.1 ± 1.0 2 PAG ₁₇ 4500 2800 4100 1.25 -15.4 -37.5 16.7 ± 0.7 3 PAG ₃₃ 8700 5300 8200 1.39 -14.0 -37.5 19.1 ± 2.2 4 PAG ₅₀ 13300 7700 9400 1.67 -21.0 -31.2 17.0 ± 0.9 5 PAG ₆₆ 17400 10800 10600 1.83 -25.6 -43.8 23.7 ± 1.4 ^a < ¹ > H-NMR spectroscopy.
^b Measured using GPC-RI and PMMA standards in DMF.

Example 2-2: Synthesis of PAG (using Ts-protected monomer)

1,1,1-Trimethylolpropane (TMP, 22.54 mg, 0.168 mmol) was placed in a one neck round bottom flask. A methanol solution (25.0 wt%, 49.6 μL, 0.168 mmol) of potassium methoxide was diluted with 0.70 mL of methanol, and the mixture was added to a flask, followed by stirring at room temperature under an argon atmosphere for 30 minutes. A high vacuum was applied at 60 캜 for 4 hours to remove methanol, whereby a white salt of an initiator was obtained. The flask was purged with argon and heated at 90 < 0 > C. (1.0 g, 3.86 mmol), which was prepared in Example 1-2, was added to a solution of N- (2-hydroxyethyl-4-methyl-N- After the completion of the monomer injection, the solution was further stirred for 24 hours. To the obtained homopolymer, 1.0 mL of methanol was added to terminate the reaction, the polymer solution was precipitated in cold diethyl ether, The precipitate was washed twice with diethyl ether. The obtained Ts-protected polymer was dried under vacuum at 60 DEG C for 1 day.

NMR data (see Fig. 18) had the following formula The Ts- is 5821 g / mol M _n of the protected polymer with from calculation, considering a margin of error of NMR integrals, 5800 g of an M _n value calculated from NMR / mol:

Number of repeating units = 285.13 (integral value of polyether skeleton) / 13 (number of quantities of polyether skeleton) = 21.93; M _n = 259.32 (molecular weight of Ts-protected monomer) x 21.93 + 134.17 (molecular weight of TMP) = 5821.06 g / mol.

The protecting group was removed in a similar manner to the step (2) of Example 2-1 above using HBr from the Ts-protected polymer obtained above.

Example 3: Cytotoxicity analysis

Mouse murine macrophage cell line RAW264.7 was purchased from Korean Cell Line Bank (Seoul, Korea). Cytotoxicity assays were performed by routine WST-1 assay. Cells were seeded at a density of 1 x 10 ⁵ cells / well in 96-well plates and cultured for 24 hours at 5% CO ₂ and 37 ° C. RAW 264.7 cells were cultured in medium containing 10% fetal bovine serum (FPS) and 1% penicillin-streptomycin (RPMI, WELGENE). Each well was treated with various concentrations of PAG solution (Polymers 2 and 4) and further cultured for 24 hours. For WST-1 analysis, each well was filled with 10 μL of assay reagent (EZ-Cytox, EZ-3000; Dogen bio). After incubation for 1 hour, the plate was gently shaken for 1 minute and the absorbance was measured. The absorbance of the solution was measured at 450 nm and the reference wavelength was 600 to 650 nm.

Test results and analysis

Fig. 1 (a) shows the synthesis of BAG monomer, and (b) shows the synthesis of PAG by anionic ring-opening polymerization and deprotection of PBAG. In Examples 1-1 and 2-1, the BAG monomer and the PAG polymer were synthesized in the same manner as shown in Figs. 1 (a) and 1 (b), respectively. First, a protecting group was added to the diethanolamine using a CH ₂ Cl ₂ solution of di-t-butyl dicarbonate (Boc ₂ O) and triethylamine (TEA). The obtained t-butylbis (2-hydroxyethyl) carbamate was reacted with epichlorohydrin to obtain Boc-protected aminoethanol glycidol (BAG), namely t-butyl (2-hydroxyethyl) - (oxiran-2-ylmethoxy) ethylcarbamate). The synthesized BAG monomer was confirmed by various spectroscopic / mass spectrometry such as ¹ H-NMR, ¹³ C-NMR, COZY, DEPT and ESI-MS (see FIGS. 2, 7 to 9).

After the synthesis of the BAG monomer, anionic ring opening multistage polymerization using a potassium alkoxide initiator formed by trimethylol propane (TMP) and potassium methoxide solution was performed. Then, BAG monomer was added to the deprotonated TMP initiator and polymerization was carried out at 90 DEG C for 48 hours. The polymerization structure of the thus-prepared PBAG polymer was confirmed by ¹ H-NMR and GPC measurements.

2B is a ¹ H-NMR spectrum (solvent D ₂ O) of a PBAG ₆₆ polymer (Polymer 5), and FIG. 2C is a ¹ H-NMR spectrum of a BAG monomer (solvent CDCl ₃ ) Is a ¹ H-NMR spectrum (solvent D ₂ O) of the deprotected PAG ₆₆ polymer (Polymer 5). As shown in FIG. 2, the ¹ H-NMR spectrum of the BAG monomer and the polymer corresponded to characteristic quantum peaks. The M _n value was also calculated by the ratio between the peak integral value (0.75 and 1.25 ppm) of the methyl and methylene groups of the TMP initiator and the peak integral value (3.0 to 4.0 ppm) of the polyether skeleton.

The Boc-protected PBAG was then treated with hydrochloric acid (HCl) for 2 hours to obtain the final PAG. The deprotection of the Boc residue was confirmed by disappearance of the strong t-butyl group at 1.34 ppm in ¹ H-NMR spectrum (see Fig. 2 (c)). The synthesized PBAG and the deprotected PAG polymer were very well dissolved in water as well as organic polar solvents such as methanol, DMSO and DMF. In particular, after deprotection, the peak of the PAG skeleton (3.0-4.0 ppm) became sharper than the peak of the PBAG skeleton. It can be seen that the removal of the bulky hydrophobic Boc protecting group improves the aqueous solubility of PAG.

The polymer synthesized by GPC was further analyzed. PBAG was used instead of PAG because the secondary amine group easily reacts with the column-packed particles. The GPC results showed a molecular weight controlled by a single distribution (see Table 1 and FIG. 11), which means that there are few impurities or side products in the synthesized polymer. Specifically, the M _w of the PBAG polymer was measured at 3400 ~ 10600 g / mol, and the polydispersity index (M _w / M _n ) using GPC and PMMA standards was measured to be 1.19 ~ 1.83, . The molecular weight of the polymer was generally in agreement with the molecular weight calculated from ¹ H-NMR, but slightly differed in the case of the polymer having a high molecular weight such as PBAG ₅₀ and PBAG ₆₆ (Polymers 4 and 5).

In order to synthesize a polymer having a large molecular weight, a long reaction time and a severe reaction condition such as a strong base and a high temperature are required, so that the polymerization can be protected by using a Boc protecting group. That is, by protecting the amine group in the polymerization process, the secondary amine group may be contained in the final polymer, whereas if the amine group is not protected during the polymerization, a tertiary amine may be formed by reacting with other monomers and the like (see FIG. 15 ) . The formation of such a tertiary amine may modify the structure of the desired polymer and lower the physical properties of the polymer such as solubility.

On the other hand, the ratio of deprotection occurring using tert-butyldiethylcarbamate was tested (see FIG. 12). As a result, it was presumed that the ratio of the Boc group to be deprotected under the same reaction conditions as the polymeric polymerization was found to be about 5% or less, so that the ratio of side reactions in the polymerization process would be small.

MALDI-TOF spectroscopy was performed to confirm the presence of a TMP initiator and the presence of a functional monomer segment in the PAG polymer. 3 is an expanded MALDI-ToF mass spectrum ranging from 1600 to 2300 Da of PAG ₁₃ (Polymer 1). The inter-signal spacing corresponds to the mass of each monomer (AG: 161.2 g / mol). As shown in FIG. 3, the inter-signal spacings corresponded to the mass of each monomer in the PAG polymer, so that the polymerization of the PAG was clearly confirmed. For example, a mass peak of 1946.56 g / mol corresponds to a polymer with initiator TMP, 11 monomer units, and a counter ion K ⁺ (TMP (134.17) + PAG monomer (161.2) x 11 + K ⁺ (39.1).

Further, the degree of branching (DB) of the PAG polymer was calculated by a known calculation formula through analysis of the ¹³ C-NMR spectrum (see FIGS. 4, 13, 14 and Table 2) Respectively. As a result, the ratio between the segmented segments in the PAG skeleton was confirmed.

For example, Table 2 below shows the distribution of PAG ₆₆ polymer (polymer 5) through ¹³ C-NMR spectrum. The degree of branching (DB) of the polymer was calculated according to the AB ₂ equation using the relative intrinsic value of ¹³ C-NMR in the following Table 2, and the fraction of PAG ₆₆ was calculated to be 0.37.

domain Chemical shift (ppm) Relative integral value PAG ₆₆ L _(1,10) 71.49 to 72.43 0.89 end 69.33 ~ 70.09 0.92 D 70.24 to 70.96 0.55 L _(1,3) 62.87 to 63.39 1.0

FIG. 4 is a ¹³ C-NMR spectrum (solvent D ₂ O) of PAG ₆₆ polymer (Polymer 5), confirming linear, dendrimer, and terminal groups within the polymer structure. The DB of PBAG ₆₆ polymer was about 0.37, which was slightly lower than that of DB (0.4 ~ 0.6) of the conventional high molecular weight terpolymer. This appears to be due to the long spacer unit in the BAG monomer, as compared to a simple glycidol, limiting the branching of the terminal hydroxyl groups.

The thermal properties of synthesized PBAG and PAG polymers were measured by differential scanning calorimetry (DSC). As a result of DSC, the glass transition temperature (Tg) of the PAG polymer was measured in the range of -43.8 캜 to -31.2 캜. In addition, the DSC results of the PBAG polymer showed higher Tg, which was consistent with decreasing the degree of rotational freedom of the polymer chain due to bulky protective groups.

Figure 5 is the DSC curve of PBAG ₁₃ and PAG ₁₃ . The Tg (-28.4 ° C) of PBAG ₁₃ was higher than the Tg (-33.5 ° C) of PAG ₁₃ after deprotection. Further, when the free amine group in the polymer was deprotected, the charge density was confirmed by zeta potential measurement. As a result, it was confirmed that the synthesized PAG polymer exhibited a high positive charge between 14.1 and 23.7 mV as expected, so that it is useful as a gene transfer carrier (see Table 1).

The cytotoxicity of the polymers (PAG ₁₇ and PAG ₅₀ ), which have been confirmed to have high charge densities as described above, was evaluated for their potential in the biomedical field. First, the individual macromolecules were treated with the normal mouse macrophage cell line RAW264.7. The cytotoxicity of the polymer was then evaluated by WST-1 analysis, which is commonly used to test the cytotoxicity of polymers and nanomaterials. Unlike MTT analysis, which requires a dissolution step, WST-1 analysis allows for higher sensitivity and a wider range of measurement.

Fig. 6 shows the in vitro results of the polymer. The black bar is the result of PAG ₁₇ (Polymer 2) and the white bar is the result of PAG ₅₀ (Polymer 4), which was obtained by WST-1 analysis using RAW264.7 cell line. As shown in FIG. 6, the cell viability of each cell line treated with various concentrations of PAG17 exceeded 90% even at a concentration of 500 μg / mL. In the case of PAG ₅₀ having a high content of amine residues, it showed some toxicity due to free amine groups involved in rigid cell binding, Thus, the cell viability tended to decrease gradually to 250 μg / mL. Although many polyamines are known to have high cytotoxicity due to the free amine groups involved in tight cell binding, the PAG polymers according to the present invention exhibit relatively low cytotoxicity, which is concealed by shells of high-differential polyglycerols This is because the cytotoxicity is mitigated by the amine group.

According to the above Examples and Test Examples, it was confirmed that one-pot synthesis of high-molecular weight polyglycerol having an amine functional group is possible. That is, it was possible to synthesize protected aminoethanol glycidyl ether (BAG), which is a novel monomer, and to polymerize it through anionic ring opening multibranched polymerization to prepare PBAG which can control the molecular weight and has a low molecular weight distribution. Subsequently, PBAG was deprotected to obtain a desired high-differential polyamino glycerol (PAG). Also, it was confirmed that the polymer was successfully polymerized through ¹ H-NMR, ¹³ C-NMR, GPC, MALDI-TOF, and DSC. The PAG has a great potential in the biological and biomedical fields because of its high zeta-potential and good biocompatibility, and can have the advantage of free amine groups hidden within high-differential polyglycerols. Accordingly, the novel functional epoxide monomers and polymers according to the present invention can be used as polyglycerol-based polymers useful in biomedical fields.

Claims (15)

A step of adding a polyol initiator to an aminoglycidyl ether-based monomer having a protected secondary amine group and a hydroxyl end to perform a single polymerization reaction and then deprotecting the polyamine glycerol.
The method according to claim 1,
Wherein the aminoglycidyl ether-based monomer is an asymmetric monomer having an epoxy group at one end and a hydroxyl group at the other end.
3. The method of claim 2,
Wherein the aminoglycidyl ether-based monomer is represented by the following formula (1): < EMI ID =
[Chemical Formula 1]

In Formula 1, m and n are each an integer of 1 to 4, and PG is a protecting group.
The method of claim 3,
Wherein PG is a protecting group selected from the group consisting of t-butyloxycarbonyl, p-toluenesulfonyl, fluorenylmethyloxycarbonyl, benzyl, triphenylmethyl and carboxybenzyl, Method of producing glycerol.
The method of claim 3,
Wherein m and n are each 2 and PG is t-butyloxycarbonyl in the above formula (1).
The method according to claim 1,
The polyol initiator is trimethylol propane (TMP)
Wherein the single polymerization reaction is anionic ring-opening multibranching polymerization.
As the homopolymer of the aminoglycidyl ether-based monomer,
The aminoglycidyl ether-based monomer has a protected secondary amine group,
Highly branched polyamino glycerol deprotected after monopolymerization.
8. The method of claim 7,
The aminoglycidyl ether-based monomer is a high-differential polyamino glycerol:
[Chemical Formula 1]

In Formula 1, m and n are each an integer of 1 to 4, and PG is a protecting group.
9. The method of claim 8,
Wherein m and n are each 2 in Formula 1, and PG is t-butyloxycarbonyl.
8. The method of claim 7,
Wherein the homopolymer comprises a plurality of branched chains having a polyether skeleton, wherein the branched chain comprises a secondary amine group in the middle and a hydroxyl group at the end, wherein the branched polyamino glycerol has a polyether skeleton.
11. The method of claim 10,
Wherein said branched chain comprises a group of the formula (2): < EMI ID =
(2)

In Formula 2, m and n are each an integer of 1 to 4.
8. The method of claim 7,
Said homopolymer having a number average molecular weight (Mn) of 3,000 to 20,000 g / mol, a zeta potential of 10 to 50 mV, and a glass transition temperature (Tg) of from -50 DEG C to -20 DEG C, .
A compound represented by the following formula (1):
[Chemical Formula 1]

In Formula 1, m and n are each an integer of 1 to 4, and PG is a protecting group.
14. The method of claim 13,
Wherein m and n are each 2, and PG is t-butyloxycarbonyl.
13. A biological carrier comprising a high-differential topo-polyamino glycerol according to any one of claims 7 to 12.

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