Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd ‫؍‬ 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10 5 -10 7 yeast surfacedisplayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wildtype complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

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Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this, <450 variants were screened, which allowed accurate assessment of enzyme performance using a low-throughput ultra performance liquid chromatography assay. During the course of this work, crystal structures of Vfat wild type and an improved variant (Vfat variant r414) were solved and they are reported here for the first time. This work also provides insight into the critical residues for substrate specificity for the transamination of (R)-ethyl 5-methyl 3-oxooctanoate and structurally related β-ketoesters.

Amphiphilic gadolinium complexes were investigated as potential magnetic resonance imaging (MRI) contrast agents. A series of complexes was synthesized in order to study the effect of hydrophilic phosphodiester groups on albumin binding, relaxivity, and blood half-life in rats. Thus, compound 11a, a diethylenetriaminepentaacetato aquo gadolinium(III) (Gd-DTPA) derivative with an octyl substituent, was synthesized and compared to 5b, the analogous octyl derivative containing a phosphodiester linkage between the gadolinium chelate and the alkyl chain. Likewise, 11b, a naphthyl Gd-DTPA derivative, was compared to the naphthyl phosphodiester derivative 5c. A direct comparison is not available for 5a, a 4,4-diphenylcyclohexyl phosphodiester Gd-DTPA derivative; however, its pharmacokinetic properties mirror those of the other phosphodiester derivatives. Although the introduction of the phosphodiester moiety decreased log P by approximately 1.7 units, albumin binding data obtained in 4.5% human serum albumin (HSA) indicated that derivatives containing the phosphodiester group exhibited somewhat higher albumin affinity than their alkyl analogues (54 +/- 5 and 44 +/- 4% for 5b and 11a, respectively; 40 +/- 4 and 30 +/- 3% for 5c and 11b, respectively). Both classes of agents were characterized by enhanced relaxivity in the presence of 4.5% HSA (r1 = 16-42 mM(-1) s(-1) at 20 MHz and 37 degrees C) as compared with the relaxivity values measured in phosphate-buffered saline (PBS) alone (r1 = 4.6-6.6 mM(-1) s(-1) at 20 MHz and 37 degrees C). Pharmacokinetic data indicated that compound 5b had a half-life of 14.3 +/- 1.8 min in the rat as compared with a half-life of 6.20 +/- 0.04 min for the non-phosphodiester analogue 11a. Similarly, the half-life obtained for the phosphodiester 5c was 14.3 +/- 1.7 min as compared with a half-life of 6.80 +/- 0.03 min for 11b. The percent biliary excretion was significantly lower for the phosphodiester compounds than for non-phosphodiester analogues (17.7 +/- 4.0 and 66.9 +/- 3.4% for 5b and 11a, respectively; 17.0 +/- 1.6 and 64.3 +/- 9.0% for 5c and 11b, respectively). The percent biliary excretion (15.8 +/- 4.4%) and plasma half-life in the rat (23.1 +/- 2.9 min) for 5a are consistent with the extended plasma half-life of the other phosphodiester derivatives. Taken together, the enhanced relaxivity and extended blood half-life of the phosphodiester derivatives support the concept of using endogenous albumin binding to achieve blood pool-like properties for small-molecule magnetic resonance imaging (MRI) contrast agents.

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The amphiphilic gadolinium complex MS-325 ((trisodium-{(2-(R)-[(4,4-diphenylcyclohexyl) phosphonooxymethyl] diethylenetriaminepentaacetato) (aquo)gadolinium(III)}) is a contrast agent for magnetic resonance angiography (MRA). MS-325 comprises a GdDTPA core with an appended phosphodiester moiety linked to a diphenylcyclohexyl group to facilitate noncovalent binding to serum albumin and extension of the plasma half-life in vivo. The chiral DTPA ligand (R) was derived from L-serine, and upon complexation with gadolinium, forms two interconvertible diastereomers, denoted herein as isomers A and B. X-ray crystallography of the tris(ethylenediamine)cobalt(III) salt derivative of isomer A revealed a structure in the polar acentric space group P32. The structure consisted of three independent molecules of the gadolinium complex in the asymmetric unit along with three Delta-[Co(en)3]3+ cations, and it represents an unusual example of spontaneous Pasteur resolution of the cobalt cation. The geometry of the coordination core was best described as a distorted trigonal prism, and the final R factor was 5.6%. The configuration of the chiral central nitrogen of the DTPA core was S. The Gd-water (2.47-2.48 A), the Gd-acetate oxygens (2.34-2.42 A), and the Gd-N bond distances (central N, 2.59-2.63 A; terminal N, 2.74-2.80 A) were similar to other reported GdDTPA structures. The structurally characterized single crystal was one of two interconvertable diastereomers (isomers A and B) that equilibrated to a ratio of 1.81 to 1 at pH 7.4 and were separable at elevated pH by ion-exchange chromatography. The rate of isomerization was highly pH dependent: k1 = (1.45 +/- 0.08) x 102[H+] + (4.16 +/- 0.30) x 105[H+]2; k-1 = (2.57 +/- 0.17) x 102[H+] + (7.54 +/- 0.60) x 105[H+]2.

A mutational analysis of the femtomolar-affinity anti-fluorescein antibody 4M5.3, compared to its wild-type progenitor, 4-4-20, indicates both context-dependent and -independent mutations are responsible for the 1800-fold affinity improvement. 4M5.3 was engineered from 4-4-20 by directed evolution and contains 14 mutations. The seven mutations identified as present in each of 10 final round affinity maturation clones were studied here. Affinities of the 4-4-20 single mutant addition and 4M5.3 single site reversion mutants were compared. These experiments identified four mutations, of these seven, that were context-dependent in their contribution to higher affinity. A simplified mutant containing only these seven mutations was created to analyze complete double mutant cycles of selected sets of mutations. Specific mutational sets studied included the ligand contact mutations, the heavy chain CDR3 mutations, the heavy chain CDR3 mutations plus the neighboring residue at site H108, and the early and late acquired mutations on the directed evolution pathway. The heavy chain CDR3 mutational set and the ligand-contacting mutations were shown to provide -1.4 and -2.0 kcal/mol, respectively, of the total -3.5 kcal/mol change in free energy of binding of the seven-site consensus mutant. The mutations acquired late in the directed evolution rounds provided much of the change in free energy without the earlier acquired mutations (-3.1 kcal/mol of the total -3.5 kcal/mol). Prior structural data and electrostatic calculations presented several hypotheses for the higher affinity contributions, some of which are supported by these mutational data.

955wileyonlinelibrary.com www.particle-journal.com www.MaterialsViews.com COMMUNICATION recent studies confi rm that cellular Ca 2+ overload can lead to cytotoxicity and trigger either apoptotic or necrotic cell death. [ 17 ] Moreover, chitosan with a molar mass greater than 10 KDa is soluble only under low pH conditions (mostly pH < 6), which further restricts the application of this hydrogel system. [ 18,19 ] To develop a cation-free hydrogel system that is capable of encapsulation and has a specifi c morphology is of particular biological and medical interest for perfecting drug delivery methods and cellular encapsulation. In this study, OC was used as a crosslinker to form a pectin hydrogel for the fi rst time. Briefl y, pectin solutions were extruded into the OC solution where hydrogel capsules successfully formed ( Scheme 1 ). Ionic interaction is the major force that contributes to the pectin-OC hydrogel formation, which is a single-step process. Pectin and chitosan have complementary charges and could form polyelectrolytes when at the proper pH. The gel formation process highly depends on charge density and the degree of ionization of both pectin and OC. Furthermore, the hydrogel morphology strongly depends on concentrations of both pectin and OC. [ 19 ] Interestingly, the hydrogel that was produced shows a redblood-cell-like (biconcave) morphology. When the pectin-chitosan hydrogels were formed, their erythrocyte morphology was immediately visible ( Figure 1 E). The morphology is a critical factor that determines the application of the hydrogel system. The erythrocyte shape may offer advantages over spheres, especially when the hydrogel systems are expected to be used for encapsulation and delivery vesicles. For example, the surface area-to-volume ratio is important to biological applications because the rate of diffusion through the capsule membrane needs to be adequate to support the volume within the capsule. [ 20 ] As the size of a sphere increases, the overall surface area to volume ratio decreases, and eventually, the transport rate will no longer support the encapsulated volume. When it comes to the erythrocyte morphology, the cylinder model could be used to simplify the analysis. Unsurprisingly, when comparing spheres and cylinders of the same diameters, the cylindrical shape has a greater surface area-to-volume ratio as long as the height of the cylinder is less than the diameter. In general, transport through a capsule membrane takes place by diffusion. This type of diffusion will take place through the shortest distance from the core of the particle to the exterior. [ 20,21 ] For spheres, this creates a possible dead zone in the center of the capsule, where molecules may be unable to reach. This problem is not present in a cylinder, with a height that is much less than the diameter, due to the shorter distance from the center to the top or bottom surface of the cylinder as compared with a sphere. [ 21 ] Overall, the biconcave disc shape exhibits important features in biological systems. For ex...