We synthesized a series of [(l-Ala)_x-co-(l-Thr succinate)_y] (PATs), which are analogous to natural antifreezing glycoprotein with the structure of [l-Ala-l-Ala-l-Thr disaccharide]_n, by varying the composition and degree of succinylation while fixing their molecular weight (M_n) and Ala/Thr ratio at approximately 10–12 kDa and 2:1, respectively. We investigated their ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), dynamic ice shaping (DIS), thermal hysteresis (TH), and protein cryopreservation activities. Both IRI and INI activities were greater for PATs with higher l-Ala content (PATs-3 and PATs-4) than those with lower l-Ala content (PATs-1 and PATs-2). DIS activity with faceted crystal growth was clearly observed in PATs-2 and PATs-4 with a high degree of succinylation. TH was small with <0.1 °C for all PATs and slightly greater for PATs with a high l-Ala content. Except for PATs-1, the protein (lactate dehydrogenase, LDH) stabilization activity was excellent for all PATs studied, maintaining LDH activity as high as that of fresh LDH even after 15 freeze–thaw cycles. To conclude, the cryo-active biomimetic PATs were synthesized by controlling the l-Ala content and degree of succinylation. Our results showed that PATs with an l-Ala content of 65–70% and degree of succinylation of 12–19% exhibited the cryo-activities of IRI, INI, and DIS, and particularly promising properties for the cryoprotection of LDH protein.

中文翻译：

---

聚（L-丙氨酸-L-苏氨酸琥珀酸酯）作为仿生冷冻保护剂

我们合成了一系列[( l -Ala) _x - co -( l -Thr succinate) _y ] (PATs)，其类似于天然抗冻糖蛋白，其结构为[ l -Ala- l -Ala- l -Thr二糖] _n，通过改变组成和琥珀酰化程度，同时将其分子量 ( M _n ) 和 Ala/Thr 比率分别固定在约 10-12 kDa 和 2:1。我们研究了它们的冰重结晶抑制（IRI）、冰成核抑制（INI）、动态冰成型（DIS）、热滞后（TH）和蛋白质冷冻保存活性。L- Ala 含量较高的 PAT （PATs-3 和 PATs-4）的 IRI 和 INI 活性均高于L -Ala 含量较低的 PAT（PATs-1 和 PATs-2）。在具有高度琥珀酰化的 PATs-2 和 PATs-4 中可以清楚地观察到 DIS 活性和多面晶体生长。对于所有 PAT，TH 都很小，<0.1 °C，而对于具有高l -Ala 含量的 PAT，TH 稍大。除 PATs-1 外，所有研究的 PAT 的蛋白质（乳酸脱氢酶，LDH）稳定活性均优异，即使在 15 个冻融循环后仍保持与新鲜 LDH 一样高的 LDH 活性。总之，通过控制l -Ala含量和琥珀酰化程度合成了低温活性仿生PAT。我们的结果表明，l -Ala 含量为 65-70%、琥珀酰化程度为 12-19% 的 PAT 表现出 IRI、INI 和 DIS 的冷冻活性，特别是对 LDH 蛋白的冷冻保护具有特别有前景的特性。