The unnatural oligomers that adopt discrete conformations (“foldamers”) have recruited enormous attention because they can serve as excellent model systems to elucidate the fundamental questions in protein folding as well as their therapeutic potential in development of protein-protein interaction inhibitors. We have been especially interested in developing an autonomously folding β-sheet foldamer because it can be an inhibitor of aggregation process by β-sheet formation between the strands of α-peptide segments, which is known to cause Alzheimer diseases. Instead of using peptide-based foldamer, we designed an oligomer of urea that can autonomously folding β-sheet conformation since urea has been widely used as a denaturant of proteins due to its strong hydrogen bonding ability. A helix forming oligourea foldamer has been recently reported, but β-sheet forming oligourea remains unexplored.
To achieve autonomous β-sheet formation of oligourea foldamer, the identification of an appropriate β-turn motif to connect two oligourea strands in juxtaposition is a critical step. Since the prolyl-(1,1-dimethyl)-1,2-diaminoethyl (Pro-DADME) unit has been known as a promoter of parallel sheet formation of α- or β-peptides, we synthesized a series of β-turn units composed of D-Pro with several diamines as well as L-Pro with diamines. It is well documented that the stabilization of particular conformational features by the introduction of particular constraints may be of major interest for the establishment of structure-activity relationship. After examination of the relative stability of the turn units in solution, we could identify a new turn motif derived from D-Pro and cis-1,2-cyclohexyldiamine in solution using various methods such temperature coefficient, NMR, solvent titration etc. We also obtained a single crystal of the desired β-turn which could be a direct proof of the type of natural β-turn, where 10 and 12-member ring exist.
Recently, a class of synthetic peptides that has well defined secondary structure (β-turn) in a hydrophobic medium has been a subject of major interest among chemists. These synthetic peptides that can catalyze eantioselective reactions including acyl transfer have proline as stereogenic center at the key position. The stereoselectivity of the product as well as the rate of the reaction depend on stereochemistry of proline. We envisioned that our turn motif could be a suitable candidate for creating diastereomeric environment into the reaction. Two positions could be mimicked: one is the position of proline and other one is the terminal position which is connected with proline. Recently, we have developed e an efficient, highly enantiopure (>99% ee), facile synthetic route to yield a new constrained β-proline analogue, 3,4-methano-β-proline, which is structurally less flexible and less hydrophilic compare to β-proline. This molecule could be used to mimic proline moity into the turn. Also, we were interested to observe how it controls the secondary structure in solution. Therefore, we prepared oligomer of 3,4-methano-β-proline and investigated structural preference in solution. Unnatural cyclic β-amino acids are well-known building block for the cyclic β-peptide. Oligomer of β-proline folds into helical manner in solution. Besides, the pyrrolidine moiety in β-proline is highly nucleophilic and has many applications in catalysis field since these types of compounds are well-known as catalyst for C-C bond forming reactions. Its analogues have been proved to be a very promising catalyst for anti-Mannich reactions. Therefore, to screen the terminal position of our proposed urea β-turn catalyst, we put an effort to develop new type of proline derivatives and cyclic β-amino acids. Herein, we report our initial investigation. We have successfully synthesized substituted diaryl-2-pyrrolidinemethylamine and its derivative which is 100% enantiomerically pure form, but application is yet to explore. To explore its catalytic activity it could be implemented for C-C bond forming reactions. In case of cyclic β-amino acid, form our preliminary result we found that trans-ACPC could be a good catalyst for aldol reaction which is also known to form twelve- helix in solution. It shows high selectivity (dr 70% and 92% ee) in acetonitrile solvent in the presence of 10 eq $H_2O$ at $+5^\circ C$.
Since the turn motif with D-Pro and 1,2-cis-cyclohexyldiamine is well defined, we envisioned that it could promote formation of stable β-sheet. There have been many reports in literature about formation of anti-parallel β-sheets, but control of parallel β-sheet formation is a challenging task. We applied this linker to extend β-turn into a parallel β-sheet model and obtained a crystal of the incomplete β-sheet model, indicating the core turn motif is strongly stabilized by 10 and 12-membered H-bonding. It proves that urea has strong tendency to form intramolecular H-bonding. With this linker unit, we completed synthesis of few parallel β-sheet models through solid and solution phase synthesis. From the preliminary observation, we could observe one of our extended model has weak intrastrand NOEs which ensure the suitability of cis-1,2-cyclohexane diamine-D-pro as an efficient linker unit. We tried to observe the folding pattern through CD experiments and we found a promising conformation in methanol but NMR study was not fruitful. Finally observing all the data, we could able to prove that D-pro and cis-1,2-cyclohexyldiamine is the best linker among all described here, and also we describe possibilities of formation of β-sheet by different linkers such as D-pro-1,1'-dimethyl, D-pro-L-phe.
최근 foldamer 형성에 쓰이는 합성 oligomer에 대한 연구에 많은 관심이 모아지고 있는데, 이는 그러한 연구를 통해 단백질의 접힘현상(protein folding)뿐만 아니라 단백질-단백질간 상호작용 제어에 대해 많은 정보를 얻을 수 있기 때문이다. 특별히 우리는 자발적으로 접히는 β-병풍구조의 foldamer에 대해서 관심을 가지고 연구를 진행해왔는데, 이는 자발적으로 접히는 β-병풍구조의 foldamer가 알츠하이머의 치료 연구에 쓰일 수 있기 때문이다. 보통 알츠하이머 병은 α-펩티드 가닥들이 β-병풍구조를 형성할 때 불필요하게 엉김현상이 일어나면서 생기게 되는데, 자발적으로 접히는 β-병풍구조의 foldamer가 이를 방지해줄 수 있다. 본 연구에서는 펩티드-기반의 foldamer 대신에 강한 수소결합을 갖는 요소(urea) oligomer (oligourea)를 이용하여 자발적으로 접히는 β-병풍구조를 만들고자 하였다. 현재까지 보고된 바로는 helix를 형성하는 oligourea foldamer에 대해서는 보고가 되었지만, β-병풍구조를 형성하는 oligourea에 대해서는 보고가 되어있지 않기 때문에 중요한 연구분야라고 할 수 있다.
Oligourea를 이용하여 자발적으로 접히는 β-병풍구조를 형성하기 위해 여러가지 turn motif를 이용하였으며, D-Pro와 여러가지 아민으로 구성된 β-turn unit을 합성하였다. 합성한 β-turn unit에 대해서 용액상에서 안정성을 확인해본 결과 D-Pro와 cis-1,2-cyclohexyldiamine으로부터 합성한 trun motif가 가장 안정함을 알 수 있었다. 온도 계수, NOEs, 결정 구조 연구를 통해 확인한 결과 앞에서 합성한 motif는 용액상과 고체상에서 분자내 수소결합을 갖고 있으며 이를 이용하여 잘 갖춰진 β-turn 구조를 형성하고 있음을 확인할 수 있었다.
한편 이렇게 합성한 turn motif는 그것이 가지는 구조적 성질로 인하여 거울상 이성질체의 분리 등에 효과적으로 사용될 수 있을 것으로 예상된다. 즉, 촉매로서 사용되어질 수 있는데, 현재까지 보고된 바에 따르면 펩티드를 기반으로 하는 β-turn motif가 촉매로 사용되어질 경우 product의 입체성질과 반응 속도에 영향을 미치는 것으로 알려져 있다. 또한 이러한 촉매로서의 효과는 그 물질에 포함되어 있는 proline의 입체적 성질에 의해 영향을 받는 것으로 알려져 있는데, 본 연구에서는 지금까지 보고된 바와 다른 구조를 갖는 proline을 합성함으로써 그 효과를 보고자 하였다. 먼저 α-proline에 비해서 구조적으로 좀 더 견고하며 친수성이 적은 물질인 3,4-methano-β-proline을 아주 고순도로 합성하였고 이를 이용하여 연구를 진행하였다. (>99 % ee) 또한 그와 더불어 turn motif에 포함되어 있는 말단의 chiral center를 찾기 위한 연구도 함께 진행했는데, chiral 촉매로서 잘 알려져 있는 β-proline 유도체와 cyclic β-amino acids를 이용하여 연구를 수행하였다. β-proline 유도체로는 diaryl-2-pyrrolidinemethylamine (100 % enantiomerically pure) 화합물을 합성하였으며, cyclic β-amino acids로는 효과적인 chiral 촉매로 사용될 수 있는 trans-ACPC (trans-aminocyclopentane carboxylic acid)를 합성하여 연구를 진행하였다. 실제로 trans-ACPC는 알돌 반응에 대해서 아주 놓은 선택성을 보였다. (dr 70 %, 92 % ee)
D-Pro와 cis-1,2-cyclohexyldiamine을 이용하여 만든 turn motif가 잘 규명된 구조를 갖기 때문에, 이를 이용하면 안정한 병렬식의 β-병풍구조를 만들 수 있을 것으로 예상된다. 따라서 우리는 D-Pro, cis-1,2-cyclohexyldiamine를 이용하여 β-병풍구조를 만들어보았으며 뿐만 아니라 D-pro-1,1’-dimethyl, D-pro-L-phe, D-phe-L-phe 등을 이용해서도 몇가지의 β-병풍구조를 만들었다. 여러가지 β-병풍구조를 만든 후 NOE 분석을 통해 확인한 결과, D-Pro와 cis-1,2-cyclohexyldiamine가 포함되어 있을때에만 형태가 잘 조직된 β-병풍구조가 만들어짐을 알 수 있었다.
