Determining absolute configuration in flexible molecules: a case study.

Assigning absolute configuration of molecules continues to be a major problem. Determining absolute configuration in conformationally flexible systems is challenging, even for experts. Here, we present a case study in which we use a combination of molecular modeling, solution NMR, and X-ray crystallography to illustrate why it is difficult to use solution methods alone for configuration assignment. For the case examined, a comparison of calculated and experimental optical rotatory dispersion (ORD) data provides the most straightforward way to assign the absolute configuration.

Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin.

Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Peptidoglycan synthesis and degradation is a process in bacteria that involves multiple enzymes under strict temporal and spatial regulation. We used a set of small molecules that inhibit the transglycosylation step of peptidoglycan synthesis to discover genes that help to regulate this process. We identified a gene responsible for the susceptibility of Escherichia coli cells to killing by glycolipid derivatives of vancomycin, thus establishing a genetic basis for activity differences between these compounds and vancomycin.

Tandem action of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: novel glycopeptides.

The glycopeptides vancomycin and teicoplanin are clinically important antibiotics. The carbohydrate portions of these molecules affect biological activity, and there is great interest in developing efficient strategies to make carbohydrate derivatives. To this end, genes encoding four glycosyltransferases, GtfB, C, D, E, were subcloned from Amycolatopsis orientalis strains that produce chloroeremomycin (GtfB, C) or vancomycin (GtfD, E) into Escherichia coli. After expression and purification, each glycosyltransferase (Gtf) was characterized for activity either with the aglycones (GtfB, E) or the glucosylated derivatives (GtfC, D) of vancomycin and teicoplanin. GtfB efficiently glucosylates vancomycin aglycone using UDP-glucose as the glycosyl donor to form desvancosaminyl-vancomycin (vancomycin pseudoaglycone), with k(cat) of 17 min(-1), but has very low glucosylation activity, < or = 0.3 min(-1), for an alternate substrate, teicoplanin aglycone. In contrast, GtfE is much more efficient at glucosylating both its natural substrate, vancomycin aglycone (k(cat) = 60 min(-1)), and an unnatural substrate, teicoplanin aglycone (k(cat) = 20 min(-1)). To test the addition of the 4-epi-vancosamine moiety by GtfC and GtfD, synthesis of UDP-beta-L-4-epi-vancosamine was undertaken. This NDP-sugar served as a substrate for both GtfC and GtfD in the presence of vancomycin pseudoaglycone (GtfC and GtfD) or the glucosylated teicoplanin scaffold, 7 (GtfD). The GtfC product was the 4-epi-vancosaminyl form of vancomycin. Remarkably, GtfD was able to utilize both an unnatural acceptor, 7, and an unnatural nucleotide sugar donor, UDP-4-epi-vancosamine, to synthesize a novel hybrid teicoplanin/vancomycin glycopeptide. These results establish the enzymatic activity of these four Gtfs, begin to probe substrate specificity, and illustrate how they can be utilized to make variant sugar forms of both the vancomycin and the teicoplanin class of glycopeptide antibiotics.

Nonstatistical binding of a protein to clustered carbohydrates.

Carbohydrate-derivatized self-assembled monolayers (SAMs) are used as a model system to address issues involving cell-surface carbohydrate-protein interactions. Here we examine the influence of carbohydrate surface density on protein-binding avidity. We show that the binding selectivity of Bauhinia purpurea lectin switches from one carbohydrate ligand to another as the surface density of the carbohydrate ligands increases from values of chi(sugar) approximately 0.1-1.0. Polyvalent binding is possible at all surface densities investigated; hence, the switch in selectivity is not due simply to the achievement of a critical density that permits polyvalent contacts. Instead, secondary interactions at high surface densities promote a switch in carbohydrate-binding selectivity. These findings may have implications for how changes in the composition and the density of cell-surface carbohydrates influence biological recognition processes and regulatory pathways.

Vancomycin derivatives that inhibit peptidoglycan biosynthesis without binding D-Ala-D-Ala.

Vancomycin is an important drug for the treatment of Gram-positive bacterial infections. Resistance to vancomycin has begun to appear, posing a serious public health threat. Vancomycin analogs containing modified carbohydrates are very active against resistant microorganisms. Results presented here show that these carbohydrate derivatives operate by a different mechanism than vancomycin; moreover, peptide binding is not required for activity. It is proposed that carbohydrate-modified vancomycin compounds are effective against resistant bacteria because they interact directly with bacterial proteins involved in the transglycosylation step of cell wall biosynthesis. These results suggest new strategies for designing glycopeptide antibiotics that overcome bacterial resistance.

Design of compounds that increase the absorption of polar molecules.

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

Polyvalent binding to carbohydrates immobilized on an insoluble resin.

Numerous studies have established that polyvalency is a critical feature of cell surface carbohydrate recognition. Nevertheless, carbohydrate-protein interactions are typically evaluated by using assays that focus on the behavior of monovalent carbohydrate ligands in solution. It has generally been assumed that the relative affinities of monovalent carbohydrate ligands in solution correlate with their polyvalent avidities. In this paper we show that carbohydrate ligands synthesized directly on TentaGel beads interact with carbohydrate-binding proteins in a polyvalent manner. The carbohydrate-derivatized beads can, therefore, be used as model systems for cell surfaces to evaluate polyvalent carbohydrate-protein interactions. By using a combinatorial approach to synthesize solid-phase libraries of polyvalent carbohydrates, one can rapidly address key issues in the area of cell surface carbohydrate recognition. For example, studies reported herein demonstrate that there is an unanticipated degree of specificity in recognition processes involving polyvalent carbohydrates. However, the correlation between polyvalent avidities and solution affinities is poor. Apparently, the presentation of carbohydrates on the polymer surface has a profound influence on the interaction of the ligand with the protein receptor. These findings have implications for how carbohydrates function as recognition signals in nature, as well as for how polyvalent carbohydrate-protein interactions should be studied.

Combinatorial approaches to carbohydrates.

Carbohydrates comprise an extremely important class of biological molecules but little is known about how they mediate their effects. This gap in understanding is largely due to the fact that obtaining pure carbohydrates in amounts large enough for biochemical studies is extremely difficult. The advent of combinatorial strategies to make carbohydrates promises to revolutionize the field of carbohydrate chemistry and biochemistry.

Parallel synthesis and screening of a solid phase carbohydrate library.

A solid phase carbohydrate library was synthesized and screened against Bauhinia purpurea lectin. The library, which contains approximately 1300 di- and trisaccharides, was synthesized with chemical encoding on TentaGel resin so that each bead contained a single carbohydrate. Two ligands that bind more tightly to the lectin than Gal-beta-1,3-GalNAc (the known ligand) have been identified. The strategy outlined can be used to identify carbohydrate-based ligands for any receptor; however, because the derivatized beads mimic the polyvalent presentation of cell surface carbohydrates, the screen may prove especially valuable for discovering new compounds that bind to proteins participating in cell adhesion.

Cationic facial amphiphiles: a promising class of transfection agents.

A promising class of compounds for DNA transfection have been designed by conjugating various polyamines to bile-acid-based amphiphiles. Formulations containing these compounds were tested for their ability to facilitate the uptake of a beta-galactosidase reporter plasmid into COS-7 cells. Dioleoyl phosphatidyl ethanolamine (DOPE) formulations of some of the compounds were several times better than Lipofectin at promoting DNA uptake. The most active compounds contained the most hydrophilic bile acid components. The activity is clearly not related to affinity for DNA: the hydrophobic bile acid conjugates were found to form stable complexes with DNA at lower charge ratios than the hydrophilic conjugates. We suggest that the high activity of the best compounds is related to their facial amphiphilicity, which may confer an ability to destabilize membranes. The success of these unusual cationic transfection agents may inspire the design of even more effective gene delivery agents.

Strategies for the design of minor groove binders: a re-evaluation based on the emergence of site-selective carbohydrate binders.

Studies on minor groove binders provide new insights into DNA structure and recognition, information that may in the future serve as the basis for the design of synthetic binders targeted to particular minor groove sites. Carbohydrate-based minor groove binders are emerging as a particularly interesting and important class of ligands for DNA.

Use of triethylene glycol to mimic oligosaccharides: design and synthesis of a ligand based on chromomycin A3.

Chromomycin A3 (CRA3) is an antitumor antibiotic that binds to DNA. It contains an acac-like metal binding site and forms a 2:1 complex with Mg2+. Interestingly, acac ligands similar to CRA3 form 1:1 complexes with Mg2+. We have previously shown that the unusual stability of the 2:1 CRA3-Mg2+ complex is related to a favorable intermolecular interaction between the CDE trisaccharide of one CRA3 molecule and the chromophore of the other. We have used this knowledge to design and synthesize a very simple molecule in which a triethylene glycol chain mimics the CDE trisaccharide of CRA3. This minimalist ligand behaves like CRA3 with respect to dimer formation. This result sheds light on how the CRA3 sugars function to stabilize the dimer. At the same time, the work provides a starting point for investigating the relationship between dimer formation and DNA binding. Starting from these relatively simple metal complexes, it should be possible to develop a better understanding of the structural requirements for DNA binding by CRA3 and related molecules.

Isomorphous binding of mercury-substituted thiosaccharides to pertussis toxin crystals yields crystallographic phases.

An isomorphous derivative of pertussis toxin crystals was prepared using a 2-alpha-mercuric analog of N-acetyl neuraminic acid in a method analogous to the use of inhibitors labelled with heavy atoms to solve crystal structures of enzymes. This derivative exploits the specific binding between pertussis toxin and terminal sialic acid residues on receptor glycoproteins. Difference Patterson maps yielded heavy-atom sites which refined with good statistics, indicating that the protein probably does not undergo a conformational change on receptor binding. Mercuric analogs of other monosaccharides should be easily obtainable using the same synthetic strategy, suggesting a general method for derivatizing crystals of carbohydrate-binding proteins.

The sugars in chromomycin A3 stabilize the Mg(2+)-dimer complex.

Chromomycin A3 (CRA3) is a glycosylated antitumor antibiotic that binds as a dimer to the minor groove of DNA, with a Mg2+ cation (or another divalent cation with a radius less than 0.85 A) forming the center of the dimer. It has been shown that the chromose sugars are necessary for DNA binding [Kaziro & Kamiyama (1967) J. Biochem. (Tokyo) 62, 424-429; Kamiyama (1968) J. Biochem. (Tokyo) 63, 566-572], although the reason for this has not been addressed. We have investigated the role that the chromose sugars play in metal complexation in solution (methanol) by comparing the optical behavior of CRA3 and its aglycon, CRN, in the presence of various divalent metals (Mg2+, Ni2+, and Ca2+). The results show that CRA3 forms a dimeric complex [i.e., (CRA3)2M, where M is a metal ion] in the presence of 1 mol equiv of either Ni2+ or Mg2+ but a 1:1 complex in the presence of the much larger Ca2+. In contrast, CRN forms a 1:1 complex (CRN.M)+ with all three metals under identical conditions (1:1 mole ratio of drug to metal). Thus, for the smaller metal ions the sugars stabilize the 2:1 CRA3-metal complex in solution. NMR data on the 2:1 CRA3-Mg2+ complex show that the trisaccharide of one CRA3 molecule lies in close proximity to the chromophore of the other CRA3 molecule. This interaction, which is also present in the Mg(2+)-CRA3-DNA complex [Gao & Patel (1989) Biochemistry 28, 751-762], appears to be related to the stability of the dimer in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Cleavage behavior of calicheamicin gamma 1 and calicheamicin T.

Calicheamicin gamma 1 is a potent antitumor antibiotic that cleaves DNA with a high degree of specificity; there is much interest in the recognition process. We have investigated the DNA-cleaving properties of calicheamicin T, a truncated derivative of calicheamicin. We show that calicheamicin T cleaves DNA in a double-stranded fashion, indicating that the first two sugars are sufficient to facilitate binding of the aglycone in the minor groove. However, calicheamicin T cleaves DNA nonselectivity. This result suggests that cyclization kinetics do not determine the cleavage specificity of the intact drug. Instead, cleavage specificity probably reflects binding specificity. Because of the recognition sites reported in the original cleavage paper, calicheamicin has been assumed to recognize oligopyrimidine DNA sequences containing G-C base pairs. We show here that calicheamicin also cuts efficiently at A.T tracts, sometimes in preference to G.C-rich homopyrimidine tracts. Crystallographic data and experiments with chemical probes indicate that DNA sequences including G.C base pairs have significantly different local conformations from DNA sequences containing several (four or more) sequential A.T base pairs. This difference makes it unlikely that calicheamicin simply senses inherent groove conformation and suggests that there is some degree of "induced fit." The ability to recognize both A.T- and G.C-rich oligopyrimidine sequences with a high degree of specificity makes calicheamicin an unusual minor-groove binder.

Sodium permeability and myocardial resistance to cell swelling during metabolic blockade.

The role of cell membrane permeability to sodium in cell volume regulation during inhibition of the sodium-potassium exchange pump with ouabain and during total metabolic blockade was evaluated in sections of guinea pig renal cortex, ventricle, and atrium incubated in Krebs-Henseleit solution. In all tissues, 2 and 3 h of ouabain and metabolic blockade resulted in similar marked losses of potassium and parallel continuous reductions in resting membrane potentials. Only metabolic blockade of renal cortex increased cell water, chloride, and total monovalent cations (potassium plus sodium) significantly. Compared to ouabain, metabolic blockade markedly increased the rate of cellular washout of 24Na+ from renal cortex (t 1/2 reduced by 47%), which was significantly greater than reductions in t 1/2 from ventricle (16%) and atrium (15%). Thus, inhibition of sodium-potassium exchange pump activity was not sufficient to produce cell swelling unless associated with marked increases in cell membrane permeability to sodium, in which case sodium influx exceeded potassium loss and substantial increases in monovalent cations, chloride, and water occurred.