Design,Synthesis, and Biochemical Evaluation of Alpha-Amanitin Derivatives Containing Analogs of the trans-Hydroxyproline Residue for Potential Use in Antibody-Drug Conjugates

Alpha-amanitin, an extremely toxic bicyclic octapeptide extracted from the death-cap mushroom, Amanita phalloides, is a highly selective allosteric inhibitor of RNA polymerase II. Following on growing interest in using this toxin as a payload in antibody-drug conjugates, herein we report the synthesis and biochemical evaluation of several new derivatives of this toxin to probe the role of the trans-hydroxyproline (Hyp), which is known to be critical for toxicity. This structure activity relationship (SAR) study represents the first of its kind to use various Hyp-analogs to alter the conformational and H-bonding properties of Hyp in amanitin.     

亲水相互作用色谱-四极杆/静电场轨道阱高分辨质谱法测定肝组织中羟脯氨酸

采用亲水相互作用色谱-四极杆/静电场轨道阱高分辨质谱系统,建立了肝组织中胶原蛋白水解物羟脯氨酸(Hyp)的快速定量检测方法。将正常及四氯化碳肝纤维化模型小鼠的肝组织样品酸水解,经过滤、稀释后,采用Hypersil GOLD HILIC色谱柱(100 mm×2.1 mm,3μm)分离,以水-乙腈(28∶72,v/v)为流动相进行等度洗脱,最后用配有电喷雾离子源的四极杆/静电场轨道阱高分辨质谱在正离子模式下进行检测。结果表明,Hyp在0.78~100.00μg/L范围内呈良好的线性关系,相关系数(R2)为0.998 3,方法的定量限为0.78μg/L,样品加标回收率为97.4%~100.9%,相对标准偏差为1.4%~2.0%(n=6)。此外,该方法与传统的氯胺T法进行比较,发现两种方法的检测结果相关性良好,Pearson相关系数为0.927;较氯胺T法,该法具有操作简便、准确度高的优点。该方法可用于肝组织中Hyp的快速定量分析。     

Synthesis of collagen-like sequential polypeptides containing O-phospho-L-hydroxyproline and preparation of electrospun composite fibers for possible dental application

A synthetic route is described for collagen-like polypeptides constructed from O-phospho-L-hydroxyproline [Hyp(PO(3)H(2))] residues. Using the synthetic polypeptides and a natural protein, gelatin, fine fibers and their network structures (ESNWs) were prepared via electrospinning. The composite ESNWs can induce the mineralization of calcium phosphate. The phosphoryl groups of the Hyp(PO(3)H(2)) residues affect both the crystalline phase and amount of the calcium phosphate, depending on the chemical structure in the repeating sequence. The composite ESNWs can be developed as a biocompatible replacement of the extracellular matrix of hard tissues, and thus can be applied as dental materials for restoration of dental cavities or as a sealant for pits and fissures.     

The effect of topical ethanol extract of Cotinus coggygria Scop. on cutaneous wound healing in rats

The aim of this study is to determine the cutaneous wound healing effects of the ethanol extract of Cotinus coggygria leaves in rats by excision wound model to provide scientific evidence for the traditional use of C. coggygria Scop. The levels of malondialdehyde, catalase, superoxide dismutase, glutathione and hydroxyproline were investigated in wound tissues. Histopathological examination was also performed. The hydroxyproline content of the granulation tissue and the glutathione levels were both significantly higher in the treatment group than in the control group (p < 0.05 for both); while the malondialdehyde levels were significantly lower in the treatment group (p < 0.05). These results were supported with histological results. The ethanol extract of C. coggygria Scop could be considered as an effective agent in wound healing in accordance with its traditional use.     

对甲氧基苯磺酰氯柱前衍生测定尿中游离羟脯氨酸与脯氨酸

以对甲氧基苯磺酰氯为衍生试剂,建立了分离测定人尿中游离羟脯氨酸和脯氨酸的高效液相色谱方法。考察了衍生温度、衍生缓冲液pH值、衍生时间、衍生剂用量对衍生反应的影响以及流动相组成、流动相缓冲液浓度、pH值和柱温对分离的影响。在优化条件下,羟脯氨酸和脯氨酸分别在5～100μmol/L和5～250μmol/L范围内呈良好线性,相关系数分别为0.999 4、0.999 5,检出限(S/N=3)分别为0.50 nmol/L和0.20 nmol/L,回收率(n=5)分别为95%～99%和96%～102%,相对标准偏差分别为2.1%～4.3%和2.0%～4.8%。该方法可用于人尿中游离羟脯氨酸和脯氨酸的定性定量分析。     

反相高效液相色谱法测定人肌腱中的胶原蛋白

建立了测定人肌腱中胶原蛋白含量的高效液相色谱法。动物肌腱中的胶原蛋白经酸水解后生成包括羟脯氨酸在内的氨基酸混合物,用2,4-二硝基氯苯对水解产物衍生化,然后以0.01 mol/L乙酸钠-乙酸缓冲液(pH 6.0)-乙腈(体积比为80∶20)为流动相,经反相C18柱分离,紫外检测器于360 nm波长处检测来测定羟脯氨酸的含量。羟脯氨酸是胶原蛋白的特异性氨基酸且含量稳定,因而可通过样品中羟脯氨酸的含量来计算胶原蛋白的含量。该方法对羟脯氨酸的检出限为3 μg/L,羟脯氨酸为3 μg/L~100 mg/L时与峰面积的线性关系良好;样品测定的相对标准偏差为1.95%,加标回收率为98.4%~110.8%。对60份人肌腱样品中胶原蛋白的含量进行了测定。所建立的方法灵敏、准确、干扰少,适用于肌腱中胶原蛋白含量的测定。     

Acidified Nitrite Accelerates Wound Healing in Type 2 Diabetic Male Rats: A Histological and Stereological Evaluation

Impaired skin nitric oxide production contributes to delayed wound healing in type 2 diabetes (T2D). This study aims to determine improved wound healing mechanisms by acidified nitrite (AN) in rats with T2D. Wistar rats were assigned to four subgroups: Untreated control, AN-treated control, untreated diabetes, and AN-treated diabetes. AN was applied daily from day 3 to day 28 after wounding. On days 3, 7, 14, 21, and 28, the wound levels of vascular endothelial growth factor (VEGF) were measured, and histological and stereological evaluations were performed. AN in diabetic rats increased the numerical density of basal cells (1070 ± 15.2 vs. 936.6 ± 37.5/mm³) and epidermal thickness (58.5 ± 3.5 vs. 44.3 ± 3.4 μm) (all p < 0.05); The dermis total volume and numerical density of fibroblasts at days 14, 21, and 28 were also higher (all p < 0.05). The VEGF levels were increased in the treated diabetic wounds at days 7 and 14, as was the total volume of fibrous tissue and hydroxyproline content at days 14 and 21 (all p < 0.05). AN improved diabetic wound healing by accelerating the dermis reconstruction, neovascularization, and collagen deposition.     

Proline‐based polymeric monoliths: Synthesis,characterization, and applications as organocatalysts in aldol reaction

Proline‐based polymer monoliths were synthesized via green protocol using lipase‐catalyzed esterification of methacrylic acid and 4‐hydroxyproline. Prolinyl methacrylate thus prepared was polymerized in situ as crosslinked monolith. The monolith was characterized by various techniques such as Fourier transform infrared, ¹H‐NMR, ¹³C‐NMR, scanning electron microscopy (SEM), X‐ray diffraction (XRD), and nitrogen analysis and used as catalyst in aldol reactions. The swelling behavior of the monolith was also studied as function of various external parameters like pH and temperature. The monoliths synthesized with 1% crosslinker was selected as candidate monolith for use as catalyst in aldol reaction, which was studied as a function of time, temperature, substrate structure, and amount of water:EtOH. The catalysts exhibited high efficiency in the cross aldol reaction, especially with the aromatic substrates having electron withdrawing substituent, and also good activity retention was observed when recycleability was studied up to five cycles. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1007–1015, 2010     

Improving detection sensitivity of amino acids in thyroid tissues by using phthalic acid as a mobile phase additive in hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry

In this work, 0.08 mmol L⁻¹ of phthalic acid was introduced as a mobile phase additive to quantify free amino acids (AAs) by hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The addition of phthalic acid significantly increased the signal intensity of protonated AA ions, resulting from the decrease of the relative abundance of AA sodium adducts. Meanwhile, the chromatographic peak shapes of AAs were optimized. As a consequence, there was a noticeable increase in the sensitivity of detection for AAs. The limits of detection (LOD) and quantification (LOQ) of the AAs ranged from 0.0500 to 20.0 ng mL⁻¹ and from 0.100 to 50.0 ng mL⁻¹, respectively, which were 4–50 times lower compared to the values measured without the addition of phthalic acid. The enhanced detection and separation of AAs were obtained by merely adding phthalic acid to the mobile phase without changing other conditions. Eventually, this simple method was validated and successfully applied to the analysis of twenty-four kinds of free AAs in human thyroid carcinoma and para-carcinoma tissues, demonstrating a significant increase of most AAs in thyroid carcinoma tissues (p < 0.05).     

从(4R)-羟基-(S)-脯氨酸合成4-取代脯氨酸

叙述了以(4R) 羟基 (S) 脯氨酸为原料进行的几种新型手性催化剂的简便合成方法. (4S) 苯氧基 (S) 脯氨酸 1a由N 乙氧羰基 (4R) 羟基 (S) 脯氨酸甲酯经Mitsunobu反应, C 4构型发生翻转, 水解后得到; (4R) 芳甲氧基 (S) 脯氨酸由N 叔丁氧羰基 (4R) 羟基 (S) 脯氨酸用ArCH2X醚化后脱去N 保护基得到, C 4构型不受影响.