Purification System for Polybasic Amphiphiles (PAs)

低膜渗透性和广泛外排是细菌防御机制，其协同减少革兰氏阴性菌（GNB）中的化合物积累，导致内在耐药性，阻止抗菌剂实现其（预期）生物学效应。多元两亲物（PA）是一类新兴的“增效剂分子”，称为佐剂，通过增强外膜渗透性和减少外排来拯救抗菌药物。由于对人体的毒性和选择性问题，PA作为治疗剂的来源已被制药行业广泛忽视。然而，有多个基于PA的批准用于人的药物的例子，包括基于多粘菌素和氨基糖苷类的抗生素以及大环内酯阿奇霉素。此外，基于PA的抗微生物肽（凯萨林菌素）在人类感染部位释放作为第一道防线，表明PA的责任可以在人类中最小化/抑制。** 与非碱性两亲物相比，PA是具有挑战性的分子纯化。PA是高极性分子，（取决于pH值）以中性、混合或多离子化（聚阳离子）形式共存。目前，我的团队使用过时的低压反相快速色谱法通过反相纯化PA。该技术导致（i）低纯度水平和（ii）少量（通常为10个活跃的研究合作）的积累，从而将高达30%的研究时间用于纯化。为了解决这些挑战，帮助学生变得更有竞争力，并使用各种固相反相萃取技术对学生进行现代纯化技术的培训，需要PA的自动高压纯化系统，以便在半制备规模（5-50 mg）上快速获得高纯度（95%）的PA。** 多元两亲物的拟议纯化系统预计将作出重大贡献，包括在与细菌系统的生物化学、生物有机化学和微生物学相关的各种领域的潜在突破。这些研究所收集的信息将对制定化学战略产生直接、直接的重大影响，以解除细菌防御机制和应对GNB的抗菌素耐药性，并具有多种应用，包括食品和水安全、兽医健康、农业和安全。*****

Low membrane permeability and extensive efflux are bacterial defence mechanisms which synergistically reduce compound accumulation in Gram-negative bacteria (GNB) resulting in intrinsic resistance that prevent antibacterial agents from achieving their (intended) biological effect(s). Polybasic amphiphiles (PAs) are an emerging class of “potentiator molecules” termed adjuvants that rescue antibacterials from resistance in GNB by enhancing outer membrane permeability and reducing efflux. PAs have been widely neglected by the pharmaceutical industry as a source of therapeutics because of perceived toxicity and selectivity concerns in humans. However, there are multiple examples of PA-based approved drugs for human use including the polymyxin- and aminoglycoside-based classes of antibiotics as well as the macrolide azithromycin. In addition, PA-based antimicrobial peptides (cathelicidins) are released at the site of human infection as a first line of defense indicating that the liabilities of PAs can be minimized/suppressed in humans. ******In contrast to non-basic amphiphiles, PAs are challenging molecules to purify. PAs are highly polar molecules that (depending on pH) coexist in neutral, mixed or polyionized (polycationic) forms. Currently my group purifies PAs by reverse-phase using outdated low-pressure reverse phase flash chromatography. This technique results in (i) low purity levels and (ii) accumulation of small quantities (typically 10 active research collaborations) to spend up to 30 % of their research time on purification. To resolve these challenges, to help students to become more competitive and to train students in modern purification techniques using various solid phase reverse phase extraction techniques, an automated high-pressure purification system for PAs is required to enable rapid access to PAs with high purity ( 95%) on semi-preparative scale (5-50 mg). ******The proposed purification system for polybasic amphiphiles is expected to make significant contributions, including potential breakthroughs in a wide variety of areas associated with biological chemistry of bacterial systems, bioorganic chemistry and microbiology. The information gathered by these studies will have immediate, direct strong impacts on the development of chemical strategies to disarm bacterial defence mechanisms and counter antimicrobial resistance in GNB with multiple applications including food and water safety, veterinary health, agriculture and security. *****