Microwave Assisted Green Synthesis of Polypeptide Conjugated Block Copolymer = 마이크로파를 이용한 그린 환경에서의 합성고분자와 폴리펩티드 중합

국문 초록 (Abstract)

합성 고분자 생물접합, 즉 합성 고분자를 핵산, 올리고펩티드, 단백질, 탄수화물, 바이러스, 세포 등 생물체에 공유결합을 통해 접합시키는 연구는 나노기술(NT)과 생물기술(BT)을 조합하는 것으로 고분자 생물접합체가 재료과학의 여러 새 영역에도 응용될 수 있음이 밝혀지고, 정밀 라디칼 중합, 개환 복분해중합 및 클릭화학과 같은 현대합성법의 개발은 맞춤형(tailor-made) 고분자 생물접합체를 제조할 수 있는 훌륭한 도구로 쓸 수 있어 고분자 생물접합체는 고분자화학에서 중심 토픽의 하나로 부상하고 있다.
본 연구는 전례 없는 성질을 가진 고분자를 제조하기 위해 천연과 합성고분자 각각이 가지는 장점을 최대로 아우르는 접합체를 합성하기 위한 것으로 합성 고분자와 폴리 펩타이드 블록 공중합체의 관습적인 합성방법과 마이크로파를 에너지원으로 하는 합성과 그 분석에 관하여 서술 하고 있다. mPEG-amine를 개시제로 사용하고 모노머로 고리형 아미노산을 개환 중합시켜 폴리펩티드를 블록 중합하는 실험을 진행하고 있다. 합성고분자로 사용되는 PEG는 인체에 무해하고 각종 연구에도 활발히 쓰이며, 여기서는 분자량 2000인 PEG의 -OH 말단기를 보다 반응성이 좋은 -NH2로 치환시켜 기능성을 높이고, 20개의 천연 L-α-아미노산(AA)중, 상대적으로 반응성이 좋고 응용면도 우수한 tyrosine과 lysine 두 가지를 선별해 중합의 활성을 좋게 하기 위해 고리형 아미노산 형태로 만들어 주었다. 실험결과는 좋았으나, 반응시간이 오래 걸리고 유기용매를 사용해야 하는 단점이 있었고 이를 개선하기 위한 대안으로 에너지원을 열에서 마이크로파로 바꾸는 방법을 시도하였다. 기존의 사용하던 용매를 DMF에서 증류수로 바꾸어 'green' 환경에서 실험을 진행할 수 있었고 하루 이상 걸렸던 반응시간을 1~3시간으로 단축하게 되었다.
합성한 고분자의 구성 비 와 분자량 및 구조의 분석은 NMR 같은 분광학적인 분석방법과 겔 침투 크로마토크래피(GPC)를 이용하여 분석하였다. 보호된 기능기를 가진 공중합체의 경우에는 연속적인 탈수 반응(hydrolysis reaction)을 이용하여, 비보호화(deprotection) 시킨 후, 분광학적인 방법을 이용하여 분석 하였다.
블록 고분자의 자가 조립 거동(self assembly behavior)은 그들을 선택적인 용매에 용해시킴으로써 분석할 수 있다. 이러한 자가 조립 거동은 투석(dialysis)이나 써머사이클러(thermal cycler)와 같은 적절한 방법으로 통하여 생성시켰다. 구형의 안정화된 마이셀(micelle)을 용매에 분산 시켜 TEM, NMR 등을 통하여 분석하였다.
Poly(L-tyrosine)-b-Poly ethylene glycol 의 경우에는 단백질이 가지고 있는 알파 헬릭스와 같은 특징적인 구조에 의하여 구형의 마이셀 뿐만 아니라 리본, 로드의 다양한 형태로 자가 조립이 가능하였다
합성고분자와 폴리펩티드를 조합하는 생물접합체 분야에서 생물유기 블록은 폴리펩티드 축조 이론을 따라 (i)아미노산 구조 블록, (ii)올리고 및 고분자형 폴리펩티드, (iii)고분자량 단백질 등 세 가지로 분리할 수 있는데, 펩티드 단량체 서열의 길이(중합도)가 늘어남에 따라 생물부분의 화학적 기능성도 다양화될 수 있어, 최근에는 개시제와 아미노산 양의 비율, 종류를 바꿔 실험하는 등의 시도를 하여 차이가 있음을 확인하였다.

다국어 초록 (Multilingual Abstract)

CHAPTER 1. Synthesis and Application of Polymer Conjugated Polypeptide Block Copolymer

Protein–polymer conjugates undoubtedly are an important biomaterial for a wide range of applications in the areas of medicine and biotechnology. With the advent of new controlled/‘living’ polymerization techniques, the use of protein-reactive initiators to form well-defined polymers is now possible. The preparation of well-defined materials from synthetic polypeptides is now feasible due to the development of initiators that allow the ring-opening polymerization of α-amino acid-N-carboxyanhydrides (NCAs). The versatility of this polymerization reaction, in particular the ability to incorporate a large variety of amino acid monomers, both natural and synthetic, results in unprecedented flexibility of molecular design. Recently the use of protein macroinitiators to prepare protein–polymer conjugates in situ has also been described. This route eliminates the necessity to form a reactive polymer-chain altogether. Here we report the design and synthesis of a series of synthetic polymer polypeptide materials and their self-assembly behavior to form diverse nanomaterials.
Polypeptides are interesting polymers because their structural complexity imparts these materials with specific biological functions. Synthesis and polymerization of α-amino acid-N-carboxyanhydrides (NCAs) were firstly discovered by Hermann Leuchs in the beginning of 20th century and this method was used for the stepwise peptide synthesis mainly for the formation of polypeptides by ring-opening polymerizations.[3-5] There are a variety of strategies for obtaining NCA molecules from amino acids according to the nature of required polypeptides.[6] This polymerization method will allows the preparation of high molecular weight polypeptides, which cannot be obtained by stepwise solid-phase synthesis.
Direct addition of phosgene to the α-amino acids has been the preferred route, principally because facile, rapid reaction prevents racemization of the resulting NCA.[7] While considering the harmful nature of the phosgene, an alternative was developed and later NCAs was prepared, typically in a single step from commercially available α-amino acids by using a less harmful triphosgene as the reagent which acts as a substitute for phosgene gas.[8] Triphosgene is a crystalline solid which can be safely handled and stored; no catalyst is required to deliver three equivalents of phosgene in situ. Moreover, due to its high solubility in reaction solvents like tetrahydrofuran (THF), hexane etc. removal of any residual triphosgene from NCA is accomplished by recystallization of the product. General schematic representation of NCA synthesis is depicted in Scheme 1-1.
And we report a new strategy that uses N-trimethylsilyl (N-TMS) amine to mediate controlled ring-opening polymerization of amino acid N-carboxyanhydrides (NCAs)[]. This polymerization proceeds via a unique, trimethylsilyl carbamate (TMS-CBM) propagating group that results from the cleavage of the Si−N bond of N-TMS amine followed by NCA ring opening. Propagation of the polypeptide chains proceeds through the transfer of the TMS group from the terminal TMS-CBM to the incoming monomer followed by the formation of a new TMS-CBM propagating group. Formation of the TMS-CBM was confirmed by NMR (scheme1-2). Polypeptides formed by the N-TMS amine-mediated polymerization have the expected molecular weights, narrow molecular weight distributions, and controlled functional groups at the C-termini of the polypeptides.

CHAPTER 2. Microwave Assisted Green Synthesis of Polypeptide Conjugated Block Copolymer
Today, microwave (MW) technology is often used in pharmaceutical laboratories and has also become increasingly important for polymer synthesis and reactions on polymers. Up to now, higher yields, acceleration of reaction rates, and greater purity of the products are the main advantages of the thermal, specific, or non-thermal MW effects. However, the aspect of selectivity control of chemical reactions by use of MW activation in closed or open systems is a fascinating area that is not yet fully understood. The main reason for the better progress of chemical reactions is a simple thermal/kinetic effect, a consequence of quickly achieved high temperature and higher pressure (in sealed vessels). In this case, the reaction follows the Arrhenius law. The dielectric heating mechanism causes specific MW effects that differ from conventional heating. A high MW absorbing solvent can be superheated above its boiling point by using a sealed vessel.[1] If high absorbing reagents are used, microscopic hotspots will be formed.[2] Heterogeneous polar catalysts or reagents in a less polar medium often lead to acceleration of the reaction.[2–4] The MW field causes an inverted temperature gradient without wall effects. In some cases, scientists discuss the non-thermal MW effect, which results from a direct interaction between the electric field with specific molecules in the reaction medium. If the polarity in the reactants is increased during the transfer from a non-polar ground state to a polar transition state, the activation energy could be reduced.[5] The new topics of investigations specifically ring-opening polymerization, peptide synthesis, and controlled radical polymerization are more and more interest in MW-assisted polymer chemistry.

목차 (Table of Contents)

• CHAPTER 1. Synthesis and Application of Polymer Conjugated Polypeptide Block Copolymer 8
• 1-1. INTRODUCTION 8
• 1-2. EXPERIMENTAL 10
• 1-2-1. Materials and Measurements 10
• 1-2-2. Synthesis of L-tyrosine NCA 11
• 1-2-3. Synthesis of N?-CBZ-ʟ-lysine 12
• 1-2-4. Synthesis of N-TMS mPEG2000-amine as an initiator 12
• 1-2-5. Synthesis of Poly (L-tyrosine)-block-mPEG-NH-TMS 13
• 1-2-6. Self assembly of Poly (L-tyrosine)-block- mPEG-NH-TMS 13
• 1-2-7. Synthesis of Poly (L-lysine)-block-PEG 14
• 1-2-8. Synthesis of Poly mPEG-block-poly (L-lysine)-block-poly (isoleucine) 14
• 1-3. RESULT AND DISCUSSION 15
• 1-3-1. Synthesis of ?-amino acid-N-carboxyanhydrides 15
• 1-3-2. Synthesis of N-TMS mPEG2000-amine as an initiator 16
• 1-3-3. Synthesis of Poly (L-tyrosine)-block-PEG 16
• 1-3-4. Synthesis of Poly (L- lysine)-block-PEG 17
• 1-3-5. Synthesis of Poly mPEG-block-poly (L-lysine)-block-poly (isoleucine) 17
• 1-3-6. Application of Poly (L-tyrosine)-block-PEG 18
• 1-4. CONCLUSIONS 19
• 1-5. REFERENCES 20
• CHAPTER 2. Microwave Assisted Green Synthesis of Polypeptide Conjugated Block Copolymer 37
• 2-1. INTRODUCTION 37
• 2-2. EXPERIMENTAL 38
• 2-2-1. MW assisted synthesis of Poly (L-tyrosine)-block-PEG 38
• 2-2-2. MW assisted synthesis of L-tyrosine polymer by n-butyl amine initiator 39
• 2-2-3. MW assisted synthesis of L-tyrosine polymer by several amine initiators 39
• 2-2-4. MW assisted synthesis of L-tyrosine-block-poly (L-lysine) copolymer by using n-butyl amine as an initiator 40
• 2-2-5. Self assembly of Poly (L-tyrosine)-block-PEG 41
• 2-3. RESULT AND DISCUSION 41
• 2-4. CONCLUSIONS 45
• 2-5. REFERENCES 47
• LIST OF SCHEME
• Scheme 1-1. Synthesis of ?-amino acid NCAs and conventional NCA polymerization
• Scheme 1-2. Synthesis of N-TMS mPEG amine2000
• Scheme 1-3. Synthesis of Poly (L-tyrosine)-block-mPEG-NH-TMS
• Scheme 2-1. Synthesis of Poly (L-tyrosine)-block-mPEG-NH2 using MW oven
• Scheme 2-2. Synthesis of Poly (L-tyrosine) using several amines as initiator in ehtanol
• LIST OF TABLE
• Table 1-1. GPC curves and their values of Poly (L-tyrosine) using n-butyl amine as an initiator in ETOH
• Table 2-1. Physical properties of common solvents
• LIST OF FIGURE
• Fugure 1-1. 1H NMR spectrum of N-TMS mPEG amine2000
• Figure 1-2. 1H NMR spectrum of L-tyrosine NCA
• Figure 1-3. 13C NMR spectrum of L-tyrosine NCA
• Figure 1-4. 1H NMR spectrum of L-lysine NCA
• Figure 1-5. 1H NMR spectrum of Poly (L-tyrosine)-block-mPEG-NH-TMS 3days and 10days
• Figure 1-6. GPC curves of Poly (L-tyrosine)-block-mPEG-NH-TMS 3days and 10days
• Figure 1-7. 1H NMR spectrum of Poly (L-lysine)-block-mPEG-NH-TMS
• Figure 1-8. 1H NMR of a series of poly (L-lysine)-b-poly (L-isoleucine)
• Figure 1-9. GPC curves of mPEG-b-poly (L-lysine) and mPEG-b-poly (L-lysine)-b-poly (L-isoleucine) tri block copolymer
• Figure 1-10. TEM image of ribbon shape self assembly from Poly (L-tyrosine)-block-mPEG-NH-TMS in DMF
• Figure 2-1. Comparison of conventional (a) and microwave heating (b).
• Figure 2-2. 1H NMR of a series of poly (L-tyrosine)-b-poly (L-lysine) by n-butyl amine as an initiator
• Figure 2-2. Temperature (T), pressure ( p), and power (P) profile for a 3mL sample of methanol heated under sealed-vessel microwave irradiation conditions.
• Figure 2-3. Microwave heating (single mode reactor) of ethanol under open-vessel conditions.
• Figure 2-4. Approximate vapor pressures attained by solvents at various temperatures
• Figure 2-5. 1H NMR spectrum of poly (L-tyrosine)-b-mPEG amine using MW oven
• Figure 2-6. GPC curves microwave assisted poly (L-tyrosine)-b-mPEG amine synthesis
• Figure 2-7. 1H NMR of a series of poly (L-tyrosine)-b-poly (L-lysine) by n-butyl amine as an initiator
• Figure 2-8. GPC curves of poly (L-tyrosine)-b-poly (L-lysine) copolymer by n-butyl amine as an initiator
• Figure 2-9. 1H NMR of poly (L-tyrosine) by using several amines as initiator
• Figure 2-10. GPC curves and their values of Poly (L-tyrosine) using n-butyl amine as an initiator in ethanol
• Figure 2-11. TEM image of ribbon shape self assembly from Poly (L-tyrosine)-block-mPEG (1mg in 10 mL water only)
• Figure 2-11. TEM image of ribbon shape self assembly from Poly (L-tyrosine)-block-mPEG (1mg in 10 mL water only)