功能性高分子期末報告
題目: Interaction of poly(N-acryloyl-amino acids) with
saccharides in aqueous media
聚(N-丙烯 氨基酸)在水中介質的醣類相互作用
Akihito Hashidzume, Atsushi Tanaka, Takahiro Sato
Department of Macromolecular Science, Graduate
School of Science, Osaka University, 1-1
Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
姓名:宋昱杰
學號:49740030
班級:化材四甲
abstract
The interaction of a series of poly(N-acryloyl-amino acids) (pAXaa) with saccharides
has been investigated by 1H NMR. 1H NMR for methyl-b-D-galactopyranoside
(MbGal) in the presence of pAXaa indicatedthat hydrophobic interaction or hydrogen
bonding was not considerable in the interaction of the polymerswith MbGal in
aqueous media whereas CH–p interaction was relatively important. 1H NMR for
several saccharides in the presence of poly(N-acryloyltryptophan) (pATrp) indicated
that pATrp interactedmore strongly with the b-anomers than with the a-anomers
presumably because of the triple CH–p interactions of the three axial protons in the
b-anomers. In the interaction of pATrp with MbGal, MbGalinteracted with two or
more Trp residues because Trp residues were localized on the polymer chain.
內容摘要
經 1H NMR一系列聚（的N -丙烯酰氨基酸）（pAXaa）與醣類相互作用已展開研
究。pAXaa存在甲基- BD-半乳糖苷的1H NMR（MbGal）表示沒有相當的聚合物的
相互作用，疏水相互作用或氫鍵MbGal在水介質中，而CH- P的相互作用是比較重
要的。 1H核磁共振聚（N - acryloyltryptophan）
（pATrp）存在的一些醣類表示pATrp
互動更強烈，比anomers A - B - anomers大概是因為三聯的CH-在B- anomers三個軸
向質子 p相互作用。在MbGal，MbGal pATrp與互動因為本地化的高分子鏈上的
Trp殘基與兩個或兩個以上的Trp殘基相互作用。
Introduction
Saccharides are one of important classes of compounds in biological systems, not
only because saccharides are the energy source of living organisms but also because
saccharides are the recognition sites on peptides, cells, and viruses [1,2]. In biological
systems, saccharides interact with proteins, i.e., enzymes, antibodies, and lectins, to
form complexes, resulting in expression of various functions [1,2]. Detail studies on
binding sites for saccharides have indicated that complexes of saccharides with
proteins are formed via hydrogen bonding, hydrophobic interaction, and CH–p
interaction [3]. Among these interactions, CH–p interaction has been studied actively
in recent years and these studies have indicated that CH–p interaction is important in
the complexation of saccharides with proteins [4–8]. Recently, we have synthesized
(co)polymers of N-(meth)-acryloyl-amino acids and studied their solution properties
and their interaction with other chemical species [9–11]. Our preliminary
1HNMRstudy exhibited that a relatively large upfield shift upon mixing
poly(N-acryloyltryptophan) (pATrp) with methyl-b-D-galactopyranoside
(MbGal), indicative of a considerable interaction of pATrp
with MbGal. This preliminary result motivated us to study the
interaction of a series of poly(N-acryloyl-amino acids) (pAXaa) with
saccharides not only because there have been only a few examples on the interaction
of synthetic water soluble polymers with saccharides [12] but also because detail
understanding on the interaction of pAXaa with saccharides may allow ones to
develop new types of separation materials or chemical sensors for saccharides. In this
study, we have thus investigated the interaction of a series of pAXaa shown in
Scheme 1 with several saccharides as studied by1HNMRand elucidated that the
interaction of saccharides with pAXaa of aromatic amino acidswas relatively strong,
indicative of the importance of CH–p interaction.
簡介
醣類是其中一個重要的類化合物在生物系統中，不僅因為醣類是生物體的能量來
源，但也因為糖肽，細胞和病毒[1,2]上的識別位點。在生物系統中，醣類，即蛋
白質，酶，抗體，和凝集素相互作用，形成複合物，導致各種功能[1,2]的表達。
醣類的結合位點的詳細研究表明，形成的醣類與蛋白質的複合物通過氫鍵，疏水
相互作用，和CH- P的相互作用[3]。在這些相互作用中，CH- P的相互作用一直積
極研究在近年來，這些研究表明，CH- P的相互作用是重要的醣類與蛋白質絡合
[4-8]。最近，我們已合成（CO）聚合物的N -（甲基）丙烯酰氨基酸和研究與其
他化學物質[9-11]的溶液性質及其相互作用。我們的初步1HNMRstudy展出一個比
較大的高場移後混合聚（N - acryloyltryptophan）（pATrp）與甲基- BD-半乳糖苷
（MbGal），表明相當互動的pATrp MbGal。這一初步結果促使我們研究互動的一
系列聚（的N -丙烯酰氨基酸）（pAXaa）糖不只是因為曾經有幾個例子合成水與
水溶性聚合物的相互作用醣類[12]，但也因為細節上的了解pAXaa與醣類相互作
用可能允許的開發醣類的分離材料或化學傳感器的新類型。在這項研究中，因
此，我們已經調查了幾個糖計劃1所示研究by1HNMRand鑑定 pAXaa醣類與互動
芳香族氨基酸acidswas比較強，指示的pAXaa一系列的互動CH- P的相互作用的重
要性。糖不只是因為曾經有幾個例子合成水與水溶性聚合物的相互作用醣類
[12]，但也因為細節上的了解pAXaa與醣類相互作用可能允許的開發醣類的分離
材料或化學傳感器的新類型。在這項研究中，因此，我們已經調查了幾個糖計劃
1所示研究by1HNMRand鑑定 pAXaa醣類與互動芳香族氨基酸acidswas比較強，指
示的pAXaa一系列的互動CH- P的相互作用的重要性。
2. Experimental
2.1. Materials
DL-Aspartic acid, DL-asparagine, DL-tyrosine, DL-phenylalanine,DL-tryptophan,
and acryloyl chloride were purchased from TCI [13].D-Galactose (Gal) and
D-maltose (Mal) were purchased from Sigma–Aldrich. Sucrose (Suc) and treharose
(Tre) were purchased from Wako. Methyl-b-D-galactopyranoside (MbGal) was
purchased from NBS Biologicals. D-Fucose (Fuc)was purchased from Kanto
Chemical.These reagents were used as received. 2,20-Azobis(isobutyronitrile)
(AIBN) was purchased from Wako and purified by recrystallization using ethanol.
N,N-Dimethylformamide (DMF) used as solvent for polymerization was distilled
under reduced pressure. Water was purified with a Millipore Milli-Q system. Other
reagents were used without further purification.Sodium salt of DL-tryptophan (Trp),
used as a model compound in this study, was prepared by neutralization with an
equimolar amount of NaOH and then recovered by freeze–drying.
2.實驗
2.1材料
DL-天門冬氨酸，DL-天冬酰胺，DL-酪氨酸，DL-苯丙氨酸，DL -色氨酸，丙烯酰
氯均購自TCI的[13]。D -半乳糖（GAL）和D -麥芽糖（MAL）購適馬Aldrich公司。
蔗糖（SUC）和treharose（TRE）購和光。甲基- BD -半乳糖苷（MbGal）購自國
家統計局生物。 D -岩藻糖（FUC）從關東 Chemical.These試劑購買，使用了收
到的。 2,20 - 偶氮雙（異丁腈）丁腈（AIBN）從瓦科購買和使用乙醇重結晶純
化。 N -二甲基甲酰胺（DMF）為聚合溶劑減壓蒸餾。純化水與 Millipore公司的
Milli - Q系統。其他試劑均為無 DL -色氨酸（TRP）作為模型化合物，在這項研
究中，進一步 purification.Sodium鹽，是摩爾量的NaOH中和準備，然後冷凍乾燥
收回。
2.2. Monomers
N-Acryloylaspartic acid (AAsp), N-acryloylasparagine
(pAAsn),N-acryloylphenylalanine (APhe), N-acryloyltyrosine (ATyr), and
N-acryloyltryptophan (ATrp) were prepared from acryloyl chloride
and the corresponding amino acids, respectively, according to the
procedure of Kulkarni and Morawetz [14]. AAsp, AAsn, APhe, ATyr,
and ATrp were purified by recrystallization using ethyl acetate or
a mixed solvent of ethyl acetate and hexane.
2.2單體
N - Acryloylaspartic酸（AASP）N - acryloylasparagine（pAAsn），N acryloylphenylalanine（APhe）N - acryloyltyrosine（ATyr），和N - acryloyltryptophan
（ATRP）製備丙烯酰氯相應的氨基酸，分別根據庫卡尼和Morawetz過程[14]。
AASP，AAsn，APhe，ATyr和ATRP使用乙酸乙酯重結晶純化
乙酸乙酯和正己烷的混合溶劑。
2.3. Polymers
An N-acryloyl-amino acid (i.e., AAsp, AAsn, APhe, ATyr, or ATrp)and AIBN were
dissolved in DMF in a flask equipped with a three way stop-cock under an argon
atmosphere. The sealed flask was immersed in an oil bath thermostated at 70 _C.
After 24 h, the reaction mixture was poured into a large excess of ethyl acetate to
precipitate polymer. The polymer obtained was purified by reprecipitation from
methanol into a large excess of ethyl acetate or water and dissolved in an aqueous
solution of NaOH. The aqueous solution was dialyzed against water for a week. The
polymer (i.e., pAAsp,pAAsn, pAPhe, pATyr, or pATrp) was recovered by
freeze–drying.Poly(N-acryloylglycine) (pAGly), poly(N-acryloylvaline) (pAVal),
and poly(N-acryloylisoleusine) (pAIle) were the same as those used
in our previous work [11].The weight-average molecular weights (Mw) and the ratio
of z-average to weight-average molecular weight (Mz/Mw) for the polymers used
were (1.51–9.69) _104 and 1.28–2.78, respectively.
2.3.聚合物
一個 N-丙烯酰氨基酸（即AASP，AAsn，APhe，ATyr，或ATRP）和AIBN溶解在
DMF在配備三路氬氣氣氛下停止公雞燒瓶。密封瓶中浸泡在70度C恆溫油浴。 24
h後，反應混合物倒入大量過剩的乙酸乙酯沉澱聚合物。從甲醇中再沉澱成乙酸
乙酯或水的大量過剩和聚合物獲得純化溶解於 NaOH水溶液。水溶液對水透析一
個星期。冷凍乾燥的聚合物（即pAAsp，pAAsn，pAPhe，pATyr，或pATrp）被收
回。聚（N - acryloylglycine）（pAGly），聚（N - acryloylvaline）（pAVal）和聚
（N - acryloylisoleusine）（pAIle）使用的相同在我們以前的工作[11]。重均分子量
（MW）和Z -平均體重均分子量的聚合物（MZ/ MW）的比例分別為（1.51-9.69）
_104和1.28-2.78分別。
2.4. Measurements
One-dimensional 1H NMR spectra were measured on a JEOL JNMLA500
spectrometer in D2O at 30 _C using sodium 2,2-dimethyl-2-silapentane-5-sulfonate
(DSS) as an internal standard (0 ppm).Two-dimensional nuclear Overhauser effect
spectroscopy (NOESY)measurements were carried out with a Varian Unity INOVA
600spectrometer in D2O. Mixing time before the acquisition of free induction decay
was carefully varied and fixed at 150 ms to obtain a genuine NOE and to avoid the
effect of spin diffusion. In this study,the concentrations of saccharides were fixed at 1
mM, and the monomer unit concentrations of pAXaa were fixed at 30 and 5 mM
for the one-dimensional 1H NMR and NOESY measurements,respectively.
2.4測量
一維核磁共振譜上JEOL JNMLA500光譜儀在D2O在30度C鈉2,2 - 二甲基- 2silapentane- 5-磺酸（0 PPM）作為內部標準（DSS）的測定。二維核奧佛效果光譜
（NOESY）測量進行了統一在Varian INOVA的600spectrometer在D2O。自由感應衰
減收購前攪拌時間進行了仔細的變化，並固定在150毫秒，獲得真正的NOE和避
免自旋擴散的效果。在這項研究中，糖的濃度被固定在1毫米，分別在pAXaa單
體單元的濃度固定在30和5 mm一維1H NMR和NOESY譜的測量。
3. Results and discussion
Fig.1 shows expanded 1H NMR spectra for the C3 and C5 protons in MbGal in the
absence and presence of pAXaa (30 mM monomer units). In the presence of pAGly,
the 1H NMR spectrum for MbGal does not exhibit any significant changes (Fig. 1b),
indicating that pAGly does not interact or interacts only weakly with MbGal. In the
presence of pAVal or pAIle, which is expected to show hydrophobic interaction, the
1H NMR spectrum for MbGal exhibits no significant shifts (Fig. 1c and d), indicating
that hydrophobic interaction does not contribute significantly in the interaction of
these polymers with MbGal. In the presence of pAAsp or pAAsn, which is considered
to act as hydrogen bonding sites, the 1H NMR spectrum for MbGal does not indicate
significant shifts, neither (Fig. 1e and f).This observation indicates that hydrogen
bonding is not considerable in the interaction of these polymers with MbGal because
both the components are strongly hydrated in aqueous media. In the presence of
pAPhe, pATyr, or pATrp, on the other hand, the 1H NMR spectrum for MbGal
exhibits upfield shifted signals because of the ring current of the aromatic amino acid
residues (Fig. 1g–i), indicative of the interaction of these polymers with MbGal
through the CH–p interaction [15]. It should be noted here that the largest shifts were
observed in the presence of pATrp. This may be because the indolyl ring, the largest
aromatic ring, causes the strongest CH–pinteraction and the strongest effect of the
ring current. NOESY experiments confirmed these observations: NOESY data did not
exhibit significant correlation signals for a mixture of pAGly, pAVal,pAIle, pAAsp,
or pAAsn with MbGal, whereas NOESY data showed weak but clear correlation
signals for a mixtures of pAPhe, pATyr,or pATrp with MbGal (see Fig. S1 in
Electronic SupplementaryMaterial).Since pATrp caused the largest upfield shift in
1HNMR for MbGal,the interactions of pATrp with five other saccharides (i.e., Gal,
Fuc,Tre, Suc, and Mal) were also investigated by 1H NMR spectroscopy.1H NMR
spectra indicated upfield shifted signals of all the saccharides examined in the
presence of pATrp (30 mM Trp units), indicative of interaction. From the spectra,
peak shifts for the C1 protons (DdC1H) were determined as listed in Table 1. Table 1
also includes DdC1H forMbGal. The data inTable 1 indicate thatDdC1H values for
the b-anomers (2.97–5.49 Hz) are significantly larger than those for the a-anomers
(0.38–1.30 Hz), indicating that pATrp interacts with the b-anomers more strongly
than with the a anomers. This may be because all the b-anomers examined bear three
axial protons and the indolyl ring in pATrp interacts with the three axial protons
Fig. 3. A conceptual illustration for the interaction of pMTrp with MbGal.
through the triple CH–p interactions. The noteworthy is that MbGal exhibits the
largest DdC1H among the saccharides examined. This is because the methyl group
also interacts with a neighboring Trp residue in pATrp as discussed later. Interaction
of a reference system containing Trp and MbGal was also investigated by 1HNMR.
Fig. 2 compares 1HNMRspectra for the C1, C2, and methoxy protons in MbGal in the
absence and presence of Trp orpATrp (30 mMTrp units). In both the spectra in the
presence of pATrp or Trp, the signal ascribed to the C1 proton exhibits an upfield
shift, indicating the interaction of Trp or pATrp with MbGal (Fig. 2b and c). It should
be noted here that the signals of the C2 and methoxy protons in MbGal exhibit no
significant shifts in the presence of Trp(Fig. 2b) whereas those indicate upfield shifts
in the presence of pATrp (Fig. 2c). These spectra indicate that both Trp and pATrp
interact with MbGal, but there are some differences in how to interact with MbGal. In
the case of Trp, Trp and MbGal form mainly a one-to-one complex at the Trp
concentration of 30 mM. On the other hand, in the case of pATrp, two or more Trp
residues in pATrp interact with anMbGal molecule because Trp residues are localized
on the polymer chain as shown in Fig. 3.In the case of the protein–saccharide
interaction in biological systems, proteins bear well-defined three-dimensional
arrangements of amino acid residues appropriate for certain saccharides,and realize
specific binding utilizing combinations of different noncovalent bonds, e.g.,
hydrophobic interaction, hydrogen bonding, and CH–p interaction. In the case of the
pAXaa–saccharide interaction, on the other hand, pAXaa polymers used in this study
are practically atactic and do not have any defined three-dimensional arrangements,
but pAPhe, pATyr, and pATrp interact with saccharides through plural aromatic
groups on the polymer chain. If pAXaa copolymers bearing suitable
three-dimensional arrangements of amino acid residues are prepared from different
AXaa monomers, it may provide a new approach towards artificial lectins.
3.結果與討論
圖 1顯示了擴大 1H NMR譜為 C3和C5在質子 pAXaa存在（30毫米單體）在沒有
MbGal。 1H NMR譜 MbGal pAGly存在，並沒有表現出任何顯著變化（圖 1b），
這表明，pAGly不互動，或只有弱相互作用與 MbGal。在pAVal或pAIle的存在，
這是預計將顯示疏水相互作用的1H NMR譜 MbGal展品表明，疏水相互作用並不
在這些聚合物的相互作用顯著貢獻 MbGal沒有顯著的變化（圖 1C和D）。 1H
NMR譜 MbGal pAAsp或pAAsn的存在，認為這是作為氫鍵網站，不表明顯著的變
化，既不（圖 1E，F）的觀察表明，氫鍵不在相當與 MbGal這些聚合物的相互
作用，因為這兩個組件是在水介質中強烈水合。在pAPhe，pATyr，或pATrp，另
一方面，MbGal展品高場的1H NMR譜轉移，因為環的芳香氨基酸殘基（圖 1G I），互動的指示信號MbGal這些聚合物通過 CH - P的相互作用[15]。應該指出，
這裡最大的變化是在pATrp存在觀察。這可能是因為吲哚環，最大的芳香環，使
最強的CH - pinteraction和環電流的作用最強。 NOESY實驗證實了這些意見：
NOESY數據沒有展覽為顯著相關 pAGly，pAVal，pAIle，pAAsp，或與 MbGal
pAAsn，混合信號，而NOESY數據顯示 pAPhe，pATyr，或pATrp與 MbGal一個混
合物弱，但明顯的相關性信號（見圖。S1在電子 SupplementaryMaterial ）。pATrp
造成最大的高場移1HNMR MbGal，與五個其他醣類（即GAL，FUC，TRE，SUC，
和Mal）的相互作用pATrp還研究通過 1H NMR spectroscopy.1H核磁共振光譜表明
高場轉移的所有檢查中pATrp（30毫米色氨酸單位），互動的指示存在的醣類的
信號。從光譜峰位移確定為 C1的質子（DdC1H）如表1所列。表1還包括DdC1H
forMbGal。 inTable1的數據表明B - anomers（2.97-5.49赫茲）的thatDdC1H值顯著
高於 A - anomers（0.38-1.30赫茲）的大，表明pATrp交互與 A與 B- anomers更強
烈anomers。這可能是因為所有的B- anomers審查了三個軸向質子承擔三個軸向質
子和pATrp吲哚環交互通過三重CH- P的相互作用。值得一提的是，最大的糖
DdC1H研究MbGal展品。這是因為甲基組還與鄰近 TRP在pATrp討論後殘留物交
互。參考含有色氨酸和MbGal系統之間的相互作用也通過 1HNMR調查。圖2比較
的C1，C2，和甲氧基的質子和色氨酸orpATrp（30 mMTrp單位）存在的情況下
inMbGal核磁共振光譜。兩者在光譜 pATrp或色氨酸的存在，歸因於 C1的質子信
號出現一個高場轉變，表明色氨酸或pATrp MbGal（圖 2b和c）的相互作用。這
裡應該指出，MbGal展出的C2和甲氧基質子的信號色氨酸（圖 2b），而這些表
明在pATrp（圖 2c）存在高場轉移的存在沒有顯著變化。這些光譜表明，色氨酸
和pATrp與 MbGal互動，但也有一些分歧在如何與 MbGal。色氨酸，色氨酸和
MbGal形式主要是色氨酸濃度在30毫米到一個複雜的案件。另一方面，在pATrp
的情況下，兩個或兩個以上的色氨酸pATrp殘留互動 MbGal分子，因為 Trp殘基
的高分子鏈上圖所示的本地化。 3.In在生物系統中的蛋白質，醣類相互作用的情
況下，蛋白質承擔定義安排適當對某些醣類，和實現特定約束力的利用組合不同
的非共價鍵，例如，疏水相互作用，氫鍵，和CH- P的相互作用的氨基酸殘基。
pAXaa醣類相互作用的情況下，另一方面，在這項研究中使用的pAXaa聚合物幾
乎無規並沒有任何定義的三維安排，但pAPhe，pATyr，並 pATrp交互通過複數芳
香組與糖高分子鏈。如果準備從不同AXaa單體 pAXaa共聚物軸承合適的三維氨
基酸殘基的安排，它可能提供一個對人工凝集素的新方法。
4. Conclusion
The interaction of pAXaa with saccharides has been investigated by 1H NMR. MbGal
did not interact significantly with pAGly, pAVal,pAIle, pAAsp, or pAAsn, indicating
that hydrophobic interaction or hydrogen bonding was not considerable in the
interaction of pAXaa with MbGal in aqueous media. On the other hand,MbGal
interacted with pAPhe, pATyr, or pATrp, indicative of the importance of CH–p
interaction. The interaction of pATrp with several saccharides indicated that pATrp
interacted more strongly with the b-anomers than with the a-anomers. These
observations indicated that the three axial protons in the b-anomers interacted with the
Trpresidue in pATrp. In the interaction of pATrp with MbGal, MbGal interacted with
two or more Trp residues because Trp residues were localized on the polymer chain.
Details on the formation of complexes of pATrp with MbGal are being investigated.
4.結論
pAXaa與醣類相互作用已經 1H NMR考察。 MbGal沒有顯著互動 pAGly，
pAVal，pAIle，pAAsp，或pAAsn，表明疏水相互作用或氫鍵與 MbGal互動，在
水介質中的pAXaa相當。另一方面，MbGal pAPhe，pATyr，或pATrp相互作用，
表明CH- P的重要性互動。幾個醣類 pATrp與互動表示，pATrp互動與 B- anomers
更強烈，比A - anomers。這些觀測結果表明，在B- anomers三個軸向質子與 pATrp
Trpresidue互動。 MbGal pATrp與互動，MbGal與兩個或兩個以上的Trp殘基相互
作用，因為 Trp殘基的高分子鏈上的本地化。 MbGal pATrp配合物的形成詳情正
在調查之中。
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