Chemoenzymatic Synthesis and High-Throughput Screening of an Aminoglycoside−Polyamine Library:  Identification of High-Affinity Displacers and DNA-Binding Ligands

Abstract

Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and DNA-binding ligands. Regioselective lipase-catalyzed acylation, followed by chemical aminolysis, was used to generate vinyl carbonate and vinyl carbamate linkers, respectively, of the aminoglycosidic cores. These were further derivatized with polyamines, leading to library generation. A parallel batch-displacement assay was employed to identify the efficacy of the library candidates as potential displacers for protein purification. Using this approach, low-molecular-mass displacers with affinities higher than those previously observed have been identified. The aminoglycoside-polyamine library was also screened for DNA binding efficacy using an ethidium bromide displacement assay. These highly cationic molecules exhibited strong DNA-binding properties and may have potential for enhanced gene delivery.

Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite

“…A Jupiter 5 m C4 300A column (4.6 mm × 50 mm) was purchased from Phenomenex (Torrance, CA). Ribonuclease A from bovine pancreas (RNaseA), ribonuclease B from bovine pancreas (RNaseB), ␣-chymotrypsinogen A from bovine pancreas (␣-ChyA), cytochrome C from equine heart (CytC), lysozyme from chicken egg white (Lys), conalbumin from chicken egg white (Conal), hemoglobin from bovine blood (Hemo), myoglobin from equine heart (Myo), avidin from chicken egg white, subtilisin A from Bacillus, elastase from porcine pancreas, papain from papaya latex, bromelain from pineapple stem, alcohol dehydrogenase from equine liver, trypsinogen from bovine pancreas, catalase from bovine liver, aprotinin from bovine lung, aconitase from porcine heart, albumin from bovine serum, neomycin sulfate (displacer 1), paromomycin sulfate (2), bekanamycin sulfate (3), amikacin sulfate (4), spermine (5), bis(hexamethylene)triamine (7), spermidine (8), 1,4-bis(3-aminopropyl)piperazine (9), diethylenetriamine (10), 4,7,10-trioxa-1,13-tridecanediamine (11), N,Ndiethyl-1,3-propanediamine (12), N,N-diethyldiethylenetriamine (13), 2-(2-aminoethylamino)ethanol (14), spectinomycin dihydrochloride pentahydrate (15), l-arginine methyl ester dihydrochloride (16), l-lysine methyl ester dihydrochloride (17), N-hexylethylenediamine (18), piperazine (19), cyclohexylamine (20), acetic acid (21), malonic acid (22), succinic acid (23), adipic acid (24), isocitric acid lactone (25), trans-aconitic acid (26), 1,2,4-butanetricarboxylic acid (27), 1,2,3,4-butanetetracarboxylic acid (28), glycine (29), 3-guanidinopropionic acid (30), 5-aminovaleric acid (31), pantothenic acid (32), aspartic acid (33), l-␤-homoglutamic acid hydrochloride (34), guanidinosuccinic acid (35), l-2,3-diaminopropionic acid hydrochloride (36), lysine (37), arginine (38), meso-2,3,-diaminosuccinic acid (39), ethylenediaminetetrapropionic acid (40), glycerol (41), threitol (42), adonitol (43), dulcitol (44), malic acid (45), tartaric acid (46), mucic acid …”

“…Displacement chromatography has been successfully employed for the purification of proteins on multiple different stationary phases [2][3][4][5][6][7][8][9][10][11][12][13]. A wide variety of classes of displacers, such as polyelectrolytes [9], polysacchardies [14], low-molecular-mass dendrimers [11], amino acids [15], antibiotics [16] and aminoglycosidepolyamines [17] have been identified for protein displacement separations. Further, the application of low-molecular-weight displacers has attracted attention due to several distinct operational advantages [8].…”

“…Mass action selective displacers typically have an affinity for the resin that lies between that of the two solutes being separated and can be readily predicted using the steric mass action formalism [23,24]. While selective displacement chromatography has been successfully employed in ion exchange systems [5,9,11,[14][15][16][17][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38], to date it has not been applied to CHA or any other multi-modal resin system.…”

High Throughput Determination and QSER Modeling of Displacer DC‐50 Values for Ion Exchange Systems

“…Finally, the use of low MW displacers enables the operation of displacements in the selective displacement mode which results in elution of the weakly retained proteins in the induced salt gradient, displacement of the bioproduct of interest, and closely associated impurities, and the desorption of the more strongly retained impurities after breakthrough of the displacer front. A variety of low-molecular-mass displacers have been identified including protected amino acids (7), dendrimers (8), antibiotics (9), phloroglucinol based salts (10) and aminoglycoside-polyamines (11).…”

“…Although the HTS technique based on the "percentage of protein displaced" enabled quick screening of displacers, it was soon discovered that the "percentage of protein displaced" can measure displacer affinity based on only one fixed displacer concentration, which made high affinity displacers tend to lump together in the response factor used in those works. In an effort to better distinguish the relative efficacies of various high affinity displacers, a new response factor "the initial displacer concentration required for the elution of 50% bound protein" (DC-50) was developed and used successfully for a more accurate affinity analysis of a particular synthesis high affinity low molecular weight displacer library (11).…”