Synthesis of sulfonamide- and sulfonyl-phenylboronic acid-modified silica phases for boronate affinity chromatography at physiological pH

Two new types of boronate affinity solid phases were synthesized and characterized. The materials were prepared by silylation of porous silica gel with monochlorosilane derivatives containing synthetic sulfonyl- and sulfonamide-substituted phenylboronic acids. The new solid phases were evaluated for boronate affinity chromatography with aryl and alkyl cis-diol compounds and were found to be suitable for the retention of cis-diols under acidic conditions. Significant correlations between the retention factor (K) and the pH of the mobile phase demonstrate that the binding of cis-diols to the solid phases is best rationalized by chelation. Based on the lower pKa, caused by the electron-withdrawing effects of the sulfonyl and sulfonamide groups, these media display an enhanced affinity for cis-diols as compared with unsubstituted phenylboronic acid. Using isocratic elution, a mixture of various biologically relevant l-tyrosines, l-DOPA, and several catecholamines were resolved with a mobile phase composed of 0.05M phosphate buffer (pH 5.5). Mono-, di-, and triphosphates of adenosine were also separated at pH 6.0. Hence, the new boronate solid phase offers efficient affinity separation and purification of cis-diol-containing molecules under rather mild pH conditions.     

An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography

Conjugation of truncated recombinant staphylokinase (trSak) with polyethylene glycol (PEG) is an effective way to overcome its short plasma half-life and enhance its therapeutic potential. However, conventional amine directed PEGylation chemistry inevitably led to modification at its functionally important N terminus, which resulted in a significantly reduced bioactivity of trSak. In this study, a novel solid phase PEGylation process was developed to shield the N-terminal region of the protein from PEGylation. The process was achieved by oriented adsorption of an N-terminally His-tagged trSak (His-trSak) onto an immobilized metal-ion affinity chromatography (IMAC). His-trSak was efficiently separated and retained on IMAC media before reaction with succinimidyl carbonate mPEG (SC-mPEG, 5, 10 or 20 kDa). The IMAC derived mono-PEGylated His-trSak showed structural and stability properties similar to the liquid phase derived conjugate. However, isoelectric focusing electrophoresis analysis revealed that mono-PEGylated His-trSaks via solid phase PEGylation were more homogeneous than those from liquid phase PEGylation. Moreover, tryptic peptide mapping analysis suggested that a complete N-terminal blockage of IMAC bound His-trSak from PEGylation with 10 kDa- and 20 kDa-SC-mPEG. In contrast, only partial protection of the N-terminal region was obtained for 5 kDa-SC-mPEG. Bioactivities of 10 kDa- and 20 kDa-PEG-His-trSak conjugates without N-terminal PEGylation were significantly higher than those of randomly PEGylated products. This further demonstrated the advantage of our new on-column PEGylation strategy.     

4-Bromoacetamidoprocaine: An affinity ligand for brain muscarinic and nicotinic cholinergic receptors

This study describes the synthesis, receptor binding characteristics, and some behavioral effects of p-bromoacetamidoprocaine (BAP), a new affinity ligand for brain muscarinic and nicotinic cholinergic receptors. The reversible binding of [³H]QNB to rat brain membranes was inhibited in a concentration dependent and saturable manner by both procaine and BAP, with Ki values of 4×10^–6 and 3×10^–7 M, respectively, and complete inhibition at 1×10^–5 M. Both procaine and BAP, although at much concentrations, inhibited the binding of [³H]methylcarbamylcholine in a concentration dependent manner, with Ki values of 5×10^–5 and 1×10^–5 M, respectively, and complete inhibition for both at 1×10^–3 M. Plots of the % irreversible inhibition of [³H]QNB, [³H]nicotine, and [³H]MCC vs [BAP] yielded Ki values of 7×10^–8, 1×10^–4, and 6×10^–5 M, respectively. In behavioral studies BAP was able to antagonize the QNB-induced hyperactivity in mice; however, BAP did not appear to alter nicotine-induced seizure activity or other behavioral effects in mice. A plot of the time course of inhibition by BAP for [³H]QNB binding revealed that the inhibition was almost complete within 10 min exposure at 37°. The findings indicate that BAP is a useful affinity ligand for examining the biochemical and functional characteristics of brain cholinergic receptors, particularly the muscarinic which has an affinity near the nM concentration range.     

A novel radioiodinated high affinity ligand for the D2-dopamine receptor: Characterization of its binding in bovine anterior pituitary membranes

A novel high affinity dopaminergic ligand, N-(p-aminophenethyl)spiroperidol, has been synthesized and radioiodinated to a specific radioactivity of 2175 $\text{Ci}\text{mmol}$. Binding of this ligand to bovine anterior pituitary membranes is: (i) rapid (40–60 min to equilibrium at 25°C) and reversible t_{$\text{1}\text{2}$} = 1 h at 25°C); (ii) saturable and of high affinity (K_D ~ 20 pM) and (iii) displays a typical D₂-dopaminergic specificity. The ligand, which identifies the same number of receptor sites as other tritiated antagonist ligands, can be used in different tissues and preparations to delineate the characteristics of the D₂ receptor. Thus, this high affinity, high specific radioactivity ligand (N-(p-amino-m-[¹²⁵I]iodophenethyl)spiroperidol) represents a tool which until now had not been available for the characterization of the D₂-dopamine receptor.     

固化金属离子亲和层析富集磷酸化多肽方法的选择及优化

在磷酸化蛋白质组学研究中,根据是否需要对待富集样品进行甲酯化处理,可将固化金属离子亲和层析（IMAC）方法分为两类,即需要甲酯化处理的IMAC方法（ME—IMAC）和不需要甲酯化处理的IMAC方法（Non—ME—IMAC）。要实现对磷酸化多肽的有效富集和鉴定,就必须对富集方法进行选择和优化。利用基质辅助激光解析离子化串联飞行时间质谱（MALDI—TOF—MS）对两种方法富集的磷酸化多肽进行了比较研究。结果表明,ME—IMAC方法容易发生样品丢失,质谱结果的分析也比较复杂,而Non—ME—IMAC方法则不仅操作简单而且富集效果理想。另外,优化了Non—ME—IMAC方法的实验条件,指出最佳的结合溶液是8％ACN／0．3％TFA,最佳的洗脱溶液是0．1mol／LEDTA（pH值8．0）,从而建立了一套完整而简单有效的磷酸化多肽富集方法。     

Use of commercial anion-exchange resins as solid support for peptide synthesis and affinity chromatography

This report demonstrates that due to the presence of residual reactive sites in their matrices, classical diethylaminoethyl-attaching commercial anion-exchanger resins such as DEAE-MacroPrep and DEAE-Sephadex A50 supports can be used for peptide synthesis. Moreover, due to the high stability of the peptide-resin bond in the final cleavage treatments, desired peptidyl-resins free of side-chain protecting groups, which enables them to be further used as solid support for affinity chromatography, can be obtained. To demonstrate this potentiality, a fragment corresponding to the antigenic and immunodominant epitope of sporozoites of the Plasmodium falciparum malaria parasite was synthesized in these traditional resins and antibody molecules generated against the peptide sequence were successfully retained in these peptidyl supports. Due to the maintenance of their original anion-exchange capacities, the present findings open the unique possibility of applying, simultaneously, dual anion-exchange and affinity procedures for purification of a variety of macromolecules.     

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.     

Affinity chromatography of GroEL chaperonin based on denatured proteins: Role of electrostatic interactions in regulation of GroEL affinity for protein substrates

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.     

A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG‐binding domain of Protein G

This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab‐binding domain of Streptococcal Protein G (SpG‐domain III). The ligand (A2C7I1) was synthesized by the four‐component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer‐aided analysis suggests a putative binding site on the C_H1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization–mass spectrometry and ¹H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml^?1 and a dissociation constant of 5.34 × 10^?5 M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA‐specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N‐terminal sequence was found in two selected peptides: F4‐2 (KPRSMLH) and F5‐4 (KPRSVSG). The peptide F5‐4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5‐4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage‐derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Nanoprobe-based immobilized metal affinity chromatography for sensitive and complementary enrichment of multiply phosphorylated peptides

Magnetic nanoparticles (MNP, <100 nm) have rapidly evolved as sensitive affinity probes for phosphopeptide enrichment. By taking advantage of the easy magnetic separation and flexible surface modification of the MNP, we developed a surface‐blocked, nanoprobe‐based immobilized metal ion affinity chromatography (NB‐IMAC) method for the enhanced purification of multiply phosphorylated peptides. The NB‐IMAC method allowed rapid and specific one‐step enrichment by blocking the surface of titanium (IV) ion‐charged nitrilotriacetic acid‐conjugated MNP (Ti⁴⁺‐NTA‐PEG@MNP) with low molecular weight polyethylene glycol. The MNP demonstrated highly sensitive and unbiased extraction of both mono‐ and multiply phosphorylated peptides from diluted β‐casein (2×10^?10 M). Without chemical derivation or fractionation, 1283 phosphopeptides were identified from 400 μg of Raji B cells with 80% purification specificity. We also showed the first systematic comparison on the particle size effect between nano‐sclae IMAC and micro‐scale IMAC. Inductively coupled plasma‐mass spectrometry (ICP‐MS) analysis revealed that MNP had a 4.6‐fold higher capacity for metal ions per unit weight than did the magnetic micro‐sized particle (MMP, 2–10 μm), resulting in the identification of more phosphopeptides as well as a higher percentage of multiply phosphorylated peptides (31%) at the proteome scale. Furthermore, NB‐IMAC complements chromatography‐based IMAC and TiO₂ methods because <13% of mono‐ and 12% of multiply phosphorylated peptide identifications overlapped among the 2700 phosphopeptides identified by the three methods. Notably, the number of multiply phosphorylated peptides was enriched twofold and threefold by NB‐IMAC relative to micro‐scale IMAC and TiO₂, respectively. NB‐IMAC is an innovative material for increasing the identification coverage in phosphoproteomics.     

Single step immobilized metal ion affinity precipitation/chromatography based procedures for purification of concanavalin A and Cajanus cajan mannose-specific lectin

Concanavalin A and a mannose-specific lectin could be precipitated specifically from extracts of jack bean and Cajanus cajan seeds, respectively, using metal charged EGTA. Single step purification of the lectins was also possible using iminodiacetic acid-Sepharose charged with metal ions. Nondenaturing electrophoresis in polyacrylamide gel and that performed in presence of SDS ascertained homogeneity of the isolated lectins. The migration behavior of the purified lectins was comparable with those of the lectins purified using alternative procedures.     

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.     

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine

Fusion of peptide‐based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram‐range amounts of proteins. IMAC‐Ni(II) columns have become the natural partners of 6xHis‐tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His‐tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur‐containing molecules. In this work, we evaluated two different cysteine‐ and histidine‐containing six amino acid tags linked to the N‐terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine‐containing tagged GFPs were able to bind to IMAC‐Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC‐Ni(II) system reaches less than 20% recovery of the cysteine‐containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC‐Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.     

Design,synthesis and application of benzyl‐sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

The human anti‐human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity‐ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4‐aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS‐Trz‐4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS‐Trz‐4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two‐step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S‐Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS‐Trz‐4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and enzyme‐linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Cross-flow ultrafiltration of proteins through asymmetric polysulfonic membranes: I. Retention curves and pore size distributions

Flux and retention of 0.1%w/w aqueous solutions of several proteins [lysozyme, pepsin, bovine serum albumin (BSA), lipase, and gamma-globulin] with molecular weights of 14.6, 36, 67, 801 and 150 kDa are studied when they are tangentially filtered, with transmembrane pressure differences until 1 MPa and circulation velocities in the re-tentate loop from 0.04 to 1.98 m/s (laminar regime), through two asymmetric polysulfone commercial membranes (E-100 with a nominal pore size of 0.01 mum and E-500 with a nominal pore size of 0.04 mum). Results are analyzed with the film theory for the concentration-polarization phenomenon, obtaining the mass transfer coefficient along with the apparent and true retention coefficients for the cell used, as a function of the feed circulation velocity and the molecular weight of the solute. The standard retention curves lead to pore size distributions differing from the nominal ones. These differences can be attributed to the modifications of the membranes when they are in operational conditions, probably due to protein adsorption. (c) 1995 John Wiley & Sons, Inc.     

Per-capita productivity in a social wasp: no evidence for a negative effect of colony size

Optimal colony size in eusocial insects likely reflects a balance between ecological factors and factors intrinsic to the social group. In a seminal paper Michener (1964) showed for some species of social Hymenoptera that colony production of immature stages (productivity), when transformed to a per-female basis, was inversely related to colony size. He concluded that social patterns exist in the social insects that cause smaller groups to be more efficient than larger groups. This result has come to be known as “Michener’s paradox” because it suggests that selection on efficiency would oppose the evolution of the large and complex societies that are common in the social insects. Michener suggested that large colony size has other advantages, such as improved defense and homeostasis, that are favored by selection. For his analysis of swarm-founding wasps, Michener combined data from colonies of different species and different developmental stages in order to obtain adequate sample sizes; therefore, his study did not make a strong case that efficiency decreases with increasing colony size (across colonies) in these wasps. We tested Michener’s hypothesis on the Neotropical swarm-founding wasp Parachartergus fraternus, while controlling for stage of colony development. We found that small colonies were more variable in percapita productivity relative to larger colonies, but found no evidence for a negative relationship between efficiency and size across colonies. Received 1 February 2006; revised 5 May 2006; accepted 11 May 2006.     

Body size and sexual size dimorphism in primates: influence of climate and net primary productivity

Understanding the evolution of body size and sexual size dimorphism has been a longstanding goal in evolutionary biology. Previous work has shown that environmental stress can constrain male‐biased sexual size dimorphism at the population level, but we know little about how this might translate to geographical patterns of body size and sexual size dimorphism at the species level. Environmental constraints due to a highly seasonal, resource‐poor and/or variable environment have often been cited to explain the unusual lack of sexual size dimorphism among Madagascar's diverse and numerous primate taxa; however, empirical tests of this hypothesis are lacking. Using a phylogenetic approach and a geographical information system platform, we explored the role of seasonality, interannual variability and annual measures of temperature and rainfall, and net primary productivity on patterns of body size and sexual size dimorphism across 130 species of primates. Phylogenetically controlled comparisons showed no support for a role of environmental constraints in moderating sexual size dimorphism at the interspecific level, despite significant associations of environmental variables with body mass. Results suggest that the focus of discussions that have dominated in the last two decades regarding the role of environmental constraints in driving patterns of monomorphism of Madagascar's lemurs should be reconsidered; however, the conundrum remains.     

A novel and conserved pocket of human kappa-Fab fragments: design, synthesis, and verification of directed affinity ligands

Antibodies of type IgG may be divided into two classes, called lambda or kappa, depending on the type of light chain. We have identified a conserved pocket between the two domains CH1 and CL of human IgG kappa-Fab, which is not present in the lambda type. This pocket was used as a target docking site with the purpose of exploring the possibilities of designing affinity ligands that could function as such even after immobilization to gel. The idea of the design arose mainly from the results of the saturated transfer difference (STD-NMR) screening of 46 compounds identified by means of virtual docking of 60 K diverse compounds from the Available Chemicals Directory (ACD). Surface plasmon resonance (SPR) was used as an alternative method to monitor binding in solution. A total of 24 compounds belonging to a directed library were designed, synthesized, and screened in solution. They consist essentially of an amino acid condensed to a N,N'-methylated phenyl urea. STD-NMR results suggest that a small hydrophobic side chain in the condensed amino acid promotes binding, whereas a hydroxyl-group-containing side chain implies absence of STD-NMR signals. Three compounds of the directed library were immobilized and evaluated as chromatographic probes. In one case, using D-Pro as the condensed amino acid, columns packed with ligand-coupled Sepharose (Amersham Biosciences) retained two different monoclonal samples of kappa-Fab fragments with different variable regions, whereas a sample of monoclonal lambda-Fab fragments was not retained under similar chromatographic conditions.