Template-directed assembly of receptor signaling complexes

Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, CheA bound to and was activated approximately 180-fold by vesicle-bound CF. The extent of CheA activation was found to be independent of the level of covalent modification on the CF. Instead, the stability of the complex increased significantly as the level of covalent modification increased. Surface-assembled CF was also found to serve as a substrate for receptor methylation in a reaction catalyzed by the receptor methyltransferase, CheR. Since neither CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it is concluded that surface templating generates the organization among CF subunits required for biochemical activity.     

Stepwise assembly of platinum-folic acid conjugates

Folates and folate-like molecules represent a class of vectors for drug targeting and delivery. The folate receptor (FR) is overexpressed in many human cancers, therefore folate-cytotoxic drug conjugates can deliver therapeutic agents specifically to FR positive tumours. We have linked, directly through one of the carboxylate functionalities of the folic acid (α or γ), two different chelating arms for a cytotoxic platinum moiety, namely a diamine and a dicarboxylate. Five different derivatives were synthesised by multi-step procedures, purified and characterised; unfortunately, they resulted barely soluble in water, thereby precluding any biochemical and biological tests.     

Template-directed supramolecular assembly of a new type of nanoporous peptide-based material.

Dipeptides with two hydrophobic residues are known to often form crystals with hydrophobic or hydrophilic channels. One of the exceptions is Leu-Ile, which has been previously shown to crystallize as a nonporous 0.75 hydrate in the hexagonal space group P6(1) with Z' = 4. We have now found that in the presence of D-Leu, a second pseudopolymorph of the dipeptide is formed. The crystal structure has hydrophilic, water-filled channels with an irregular cross section of approximately 5.5 x 3.5 A and constitutes the first example of a crystal packing arrangement where the direction of the channels is perpendicular to the direction of hydrophobic stacking of side chains rather than parallel as in all other porous peptide-based materials.     

Solid phase assembly of defined protein conjugates

We have developed a solid-phase procedure for protein-protein conjugation that gives greater control over product size and composition than previous methods. Conjugates are assembled by sequential addition of activated proteins to the support under conditions suitable for maintaining the activity of the proteins. The total number of conjugate units to be prepared is fixed in the first step by the quantity of the first protein absorbed by the support. In each following step, the added protein links only to previously bound protein. The final conjugate is released to solution by cleaving the linker holding the first protein to the support. This stepwise assembly provides uniformly sized conjugates of the desired size and composition with placement of components at the desired positions within the structure. Using this approach, we have prepared a series of conjugates containing R-phycoerythrin as the central protein, with varying quantities of alkaline phosphatase and IgG with expected molecular masses ranging from 1.6 to 11.5 MDa. Size-exclusion chromatography and atomic force microscopy demonstrate homogeneity and control of the conjugate size. In an immunoassay for human thyroid stimulating hormone, the conjugates show signals consistent with their compositions.     

Synthesis and characterization of digoxin-phospholipid conjugates

The preparation of immunoreactive derivatives of digoxin for analytical applications is most often carried out by periodate cleavage of the terminal sugar ring (digitoxose) followed by reaction with an enzyme, protein, carrier, or related biological molecules. Here we report an improved and more efficient synthesis which was developed to provide digoxin-phospholipid conjugates useful for liposome immunoassay. The approach used involved the linking of the cleaved digitoxose through a carboxymethyl oxime functionality, which provides much improved yields of readily purified products. The synthetic modification should be applicable to the preparation of analogous phospholipid conjugates involving linkage through a sugar ring (digitoxin, ouabain, and related cardiac glycosides) or to those involving steroids (i.e., 3-digoxigenone) which can be modified to form oxime derivatives remote from key functionalities important for immunorecognition by specific antibody. The characterization of the digoxin-phospholipid conjugates with high-resolution NMR and fast atom bombardment mass spectrophotometry will also be discussed.     

Synthesis and characterization of insulin-transferrin conjugates

Receptor-mediated endocytosis can be exploited for improving the transcellular delivery of therapeutic proteins. Insulin conjugated to transferrin by forming disulfide bonds has been shown to improve insulin oral bioavailability in diabetic rats. We are developing a combination strategy involving complexation hydrogels as delivery vehicles for insulin-transferrin conjugates. The complexation hydrogels developed in our laboratory have been shown to be promising carriers for oral delivery of proteins and peptides. Integrating the strategies based on the complexation hydrogels and insulin-transferrin conjugates may prove to be a novel approach for oral delivery of insulin and other therapeutic proteins. In this work, electrospray ionization mass spectrometry (ESI-MS) was used to study the modification of insulin during its reaction with transferrin. The stability of the conjugated insulin to enzymatic degradation was also studied. ESI-MS studies confirmed the site-specific modifications of insulin. The transferrin conjugation of insulin was also shown to increase the stability of insulin to enzymatic degradation.     

Synthesis and characterization of positively charged porphyrin-peptide conjugates

The total syntheses of 14 porphyrin conjugates containing one to four positively charged amino acids and two distinct linkers are described. All conjugates were fully characterized using spectroscopic methods, and the X-ray structure of a porphyrin isothiocyanate precursor was obtained. In vitro studies using HEp2 cells show that these conjugates have low cytotoxicity (IC50 > 250 microM) and that the extent of their cellular uptake depends significantly on the number, nature, and sequence of amino acids in the peptide, and on the presence of a centrally chelated metal ion. Metal-free conjugates bearing three consecutive arginine residues accumulated the most within cells. On the other hand, the preferential sites of subcellular localization were found to be independent from the number, nature, and sequence of amino acids in the conjugate, the linker, and coordinated metal ion; it is suggested, based on theoretical calculations, that the peptides in these conjugates fold over the porphyrin macrocycle in order to maximize intramolecular hydrophobic interactions.     

Synthesis and characterization of membrane-active GALA-OKT9 conjugates

Cellular processing of immunotoxins is inefficient, limiting the overall effectiveness of current immunotoxin therapies. Specifically, translocation of ribosome-inactivating toxins across intracellular membranes is agonizingly slow. In one strategy to improve immunotoxin efficacy, membrane-active peptides are attached to immunotoxins to facilitate transfer of the toxic moiety across a cellular membrane to the cytosol. pH-sensitive peptides are of particular interest, as the membrane activity can be localized to the endosomal/lysosomal pathway, reducing nonspecific interactions at the cell surface. In this study, GALA, a pH-sensitive peptide that forms multimeric pores in membranes, was chemically attached to OKT9, an anti-transferrin receptor mAb. Conjugates were tested by measuring release of encapsulated dyes from liposomes to determine the extent to which the membrane-lytic properties of GALA were retained. The most significant feature affecting the lytic properties of GALA-OKT9 conjugates was the number of attached GALA per OKT9. Conjugates with a single GALA per OKT9 caused almost no leakage while conjugates with two or three GALA per OKT9 caused significant leakage in a concentration-dependent manner. Invariably, GALA-OKT9 conjugates were significantly less active than unconjugated GALA, attributable to a decrease both in partitioning and in surface aggregation. No improvement in membrane-lytic activity was achieved by using a longer, more flexible poly(ethylene glycol) cross-linker. Attachment of GALA via C- versus N-terminal linkage had no effect on membrane-lytic properties. Size-selective release of high molecular weight dextrans was almost identical for conjugated and unconjugated GALA, suggesting that GALA forms the same pore structure regardless of conjugation state.     

Synthesis and characterization of fluorescent Lucifer yellow-lipid conjugates

The syntheses of fluorescent lipid probes composed of Lucifer yellow dyes linked to either cholesterol or phospholipids are described. The spectral properties of these probes are characterized, and the probes are evaluated for use with model membranes and with live animal and plant cells. Of the probes synthesized, the cholesterol derivative is the easiest to prepare and appears to be the most useful because it readily labels the plasma membrane of live cells and maintains a high ratio of cell surface-to-cytoplasmic fluorescence.     

Template-directed reactions of aminonucleosides

Summary We have studied the reactions between adenosine 5-phosphorimidazolide and 9-(2-amino-2-deoxyxylofuranosyl) adenine (I) or 3-methylamino-3-deoxyadenosine (II), both with and without a poly (U) template. We find that both amino compounds react much more rapidly than does adenosine, in the absence of a template. The rate of reaction is greatly enhanced by a poly (U) template in the case of I, but the enhancement is slight in the case of II.Abbreviations A adenosine - xylo A_NH2 9-(2-amino-2-deoxy--D-xylofuranosyl) adenine - A_NHMe 3-methylamino-3-deoxyadenosine - ImpA adenosine 5-phosphorimidazolide - A³ pA adenylyl-[35]-adenosine - A² pA adenylyl-[25]-adenosine - U_NPA adenylyl-[52]-2-amino-2-deoxyuridine - xylo A_NPA 9-[adenylyl-(52)-2-amino-2-deoxy--D-xylofuranosyl]adenine - A_(NMe)pA adenylyl-[53]-3-methylamino-3-deoxyadenosine - pA adenosine 5phosphate - AppA P₁, P₂-diadenosine 5pyrophosphate - (pA)_n n = 2, 3 [2-5]-linked oligomers of pA - A² pA² pA [2-5]-linked trinucleoside diphosphate of A - poly (U) polyuridylic acid     

Transglutaminase-mediated macromolecular assembly: production of conjugates for food and pharmaceutical applications

Various strategies have been explored in the last 20 years to modify the functional properties of proteins and, among these, protein/polymer conjugation resulted one of the most successful approaches. Thus, the surface modification of polypeptides of potential industrial interest by covalent attachment of different macromolecules is nowadays regarded as an extremely valuable technique to manipulate protein activities. Protein derivatives with a number of either natural or synthetic polymers, like different polysaccharides or polyethylene glycol, have been obtained by both chemical and enzymatic treatments, and in this context, the crosslinking enzyme transglutaminase is attracting an increasing attention as a simple and safe means for protein processing in vitro. In this short review, we summarized the most significant experimental findings demonstrating that a microbial form of the enzyme is an effective tool to obtain several biopolymer-based conjugates potentially useful for both food and pharmaceutical applications.     

Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels

Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3′ or 5′ end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3′ end and an electrochemical label at 5′ end.     

Preparation and characterization of paramagnetic polychelates and their protein conjugates

The gadolinium complexes of poly-L-lysine-poly(diethylenetriamine-N,N,N',N",N"-pentaacetic acid) (Gd-PL-DTPA) and poly-L-lysine-poly(1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetr aacetic acid) (Gd-PL-DOTA) and their conjugates with human serum albumin (HSA) have been prepared and characterized. Poly-L-lysine (PL, degree of polymerization approximately 100) was N-acylated with a mixed anhydride of the chelating ligand (DTPA or DOTA). Sixty to ninety chelating groups per molecule of PL could be attached in this way. Following purification of the polychelate by size-exclusion chromatography, the gadolinium complexes were prepared by standard methods and conjugated to HSA with heterobifunctional cross-linking reagents. The molar relaxities of these macromolecular species were 2-3-fold higher than those of the corresponding monomeric metal complexes [( Gd(DTPA)] and [Gd(DOTA)]). The conjugation conditions were optimized to produce conjugates containing 60-90 metal centers per molecule of HSA (ca. one polychelate per protein).     

Template-directed synthesis on short oligoribocytidylates

Summary The oligonucleotides C(pC)_n, n=4, 5, 6, 7, are efficient templates for the oligomerization of guanosine-5-phospho-2-methylimidazole (2-MeImpG). They yield oligomeric products that are substantially less regiospecific than those obtained on polycytidylate [poly(C)]. The overall distributions of products obtained on oligo(C)s are generally similar to those of products obtained on oligodeoxycytidylates [oligo(dC)s], but there are substantial differences in the ratios of isomers.The 3–5-linked dinucleoside monophosphate GpG efficiently initiates oligomer formation with 2-MeImpG on oligo(C) templates. The pattern of products obtained by chain extension parallels closely that of products obtained directly from 2-MeImpG, except that the former products lack the 5-terminal phosphate group.     

Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids

Synthesis of reference standards is needed to determine the presence and function of steroid glucuronides in the brain or other tissues, because commercial sources of steroid glucuronide standards are limited or unavailable. In the present study porcine, rat, and bovine liver microsomes were tested to evaluate their ability to glucuronidate eight neurosteroids and neuroactive steroids of various types: dehydroepiandrosterone, pregnenolone, isopregnanolone, 5alpha-tetrahydrodeoxycorticosterone, corticosterone, cortisol, beta-estradiol, and testosterone. In general, the glucuronidation efficiency of rat liver was rather poor compared with that of bovine and porcine liver microsomes. Since porcine liver apparently has a relatively large amount of dehydrogenase, its microsomes also produced dehydrogenated steroids and their glucuronides, as well as various regioisomers in which the site of glucuronidation varied. In contrast, bovine liver microsomes produced mainly a single major glucuronidation product and few dehydrogenation products and gave the best overall yield for two-third of the steroids tested. The enzymatic synthesis of five glucuronides of four steroids was carried out and the conditions, purification, and analytical methods for the glucuronidation products were optimized. The steroid glucuronides synthesized were characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (yield 10-78%) and good purity (>85-90%), which is sufficient for LC-MS/MS method development and analyses of steroid glucuronides in biological matrices such as brain, urine, or plasma.     

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

Antibody-drug conjugates (ADCs) are a growing class of biotherapeutics in which a potent small molecule is linked to an antibody. ADCs are highly complex and structurally heterogeneous, typically containing numerous product-related species. One of the most impactful steps in ADC development is the identification of critical quality attributes to determine product characteristics that may affect safety and efficacy. However, due to the additional complexity of ADCs relative to the parent antibodies, establishing a solid understanding of the major quality attributes and determining their criticality are a major undertaking in ADC development. Here, we review the development challenges, especially for reliable detection of quality attributes, citing literature and new data from our laboratories, highlight recent improvements in major analytical techniques for ADC characterization and control, and discuss newer techniques, such as two-dimensional liquid chromatography, that have potential to be included in analytical control strategies.     

Synthesis and characterization of covalently linked single-stranded DNA oligonucleotide-dendron conjugates

A solution-phase synthesis and characterization of covalent DNA-dendron conjugates is presented. Thiol-terminated 12-base oligonucleotides were added to second- and third-generation triazine-based dendrons via thiol/disulfide exchange chemistry. Single-stranded DNA oligonucleotides were successfully attached to dendrons at the core, the periphery, and both. Proof of structure for these architectures is derived primarily from mass spectrometry and polyacrylamide gel electrophoresis and complemented by labeling analysis using Ellman's reagent and degradation analysis using a reducing agent.     

Template-directed synthesis of acyclic oligonucleotide analogues

Bis-phosphoimidazolides of an analogue of adenosine (in which ribose is replaced by an acyclic chain) and of two related analogues of guanosine undergo oligomerization in the presence of complementary polynucleotide templates. Data on the template- and nontemplate-directed reactions are presented, and the possible relevance to origins of life is discussed.