Dextran and 5-aminosalicylic acid (5-ASA) conjugates: synthesis, characterisation and enzymic hydrolysis

Mesalamine (5-aminosalicylic acid) is the drug of choice for the treatment of Crohn's disease. A scheme for the synthesis of 5-aminosalicylic acid (5-ASA) conjugates of dextrans was developed with a focus on Crohn's disease applications. Dextrans were oxidised using sodium periodate (NaIO(4)), where the aldehyde groups formed were coupled with the alpha-amino (-NH(2)) group of 5-ASA. The resulting imine bonds were unstable in water and were consequently reduced to secondary amine groups. The effects of different aspects of the conjugation reaction were studied. These included the following: the molecular weight of the dextrans, the molar proportion of NaIO(4) to the dextrans (for periodate oxidation), the pH of the conjugation solutions, the ratio 5-ASA to oxidised polysaccharide and the relationship between the degree of conjugation and the amount of enzyme hydrolysis. Conjugates incubated in HCl were stable in 0.5 and 1.0M HCl, but they underwent degradation in 2.0 and 4.0M HCl. Dextrans (MW 20,000) with various degrees of oxidation (12%, 26%, 46%, 65%, 90% and 93%) were also prepared. Each oxidised dextran sample was conjugated with 5-ASA, and the product was quantified by high-performance liquid chromatography (HPLC). Dextrans with a maximum degree of oxidation (93%) unsurprisingly gave maximum conjugation of 5-ASA (49.1mg per 100mg of product) but were resistant to dextranase hydrolysis. Less oxidised dextrans (12%) conjugated proportionally less 5-ASA (15.1mg per 100mg of product) but were successfully hydrolysed by dextranase, suggesting their potential applications for the treatment of Crohn's disease in the distal ileum and proximal colon.     

New regioselective derivatives of sucrose with amino acid and acrylic groups

We report here a range of new sucrose derivatives obtained from '3-ketosucrose' in aqueous medium with few reaction steps. As an intermediate, 3-amino-3-deoxy-alpha-D-allopyranosyl beta-D-fructofuranoside (1) was obtained via the classical route of reductive amination with much improved yield and high stereoselectivity. Building blocks for polymerization were synthesized by introduction of acrylic-type side chains, for example, with methacrylic anhydride. Corresponding polymers were synthesized. Aminoacyl and peptide conjugates were obtained through conventional peptide synthesis with activated and protected amino acids. Deprotection yielded new glycoderivatives having an unconventional substitution pattern, namely 3-(aminoacylamino) allosaccharides. Both mono- and di-peptide conjugates of allosucrose have been synthesized.     

A novel 5-fluorouracil prodrug using hydroxyethyl starch as a macromolecular carrier for sustained release

The objective of this study was to develop a sustained-release drug delivery system for 5-fluorouracil (5-FU) to improve its short half-life. 5-Fluorouracil-1-acetic acid (FUAC) was prepared and then conjugated to hydroxyethyl starch (HES) through ester bonds. The conjugates were relatively stable in acidic buffer solution at pH 5.8 and slowly released FUAC but became more sensitive to hydrolysis with an increase in the pH and temperature. The conjugates were degraded to FUAC both in human and rat plasma with half-time life of 20.4 h and 24.6 h, respectively. Both 5-FU and FUAC were released in a rat liver homogenate following a 12 h incubation of the conjugates. The pharmacokinetic behavior was evaluated in rats after intravenous injection of 5-FU, FUAC and the conjugates. The drug release data in vitro and in vivo indicated that HES is a promising carrier for the sustained-release of antitumor drugs.     

Artificial enzymes, “Chemzymes”: current state and perspectives

Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successfully perform Michaelis–Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called “Chemzymes”, based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well as chemzymes that catalyze conjugate additions, cycloadditions, and self-replicating processes. The focus will be mainly on cyclodextrin-based chemzymes since they have shown to be good candidate structures to base an enzyme model skeleton on. In addition hereto, other molecules that encompass binding properties will also be presented.     

Synthesis and structural characterization of bioactive peptide conjugates using thioether linkage approaches.

Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.     

An automated assay for fecal estrogen conjugates in the determination of sex in avian species

Recently captive propagation of psitticines has become necessary in order to preserve and breed endangered species. As habitat destruction increases and importation restrictions become more stringent, the need for a reliable and noninvasive technique for sex assignment is essential to improve the productivity of breeding and raising birds in captivity. One aspect of breeding is the proper assignment of breeding pairs; however, sex identification can be challenging due to the lack of sexual dimorphism in approximately 30% of all avian species. Historically, visual, behavioral, surgical, cytogenetic, and endocrine methods have been utilized in sex identification of avian species; however, the practicality, safety, and reliability of these techniques have been questioned. Therefore, there remains a need for an accurate, simple, and non-invasive method to identify the sex of monomorphic birds so that the formation of breeding pairs and the success of breeding programs is facilitated. The data in this study support the concept that an automated fluorescence-immunoassay which measures fecal estrogen conjugates can have an 87% success rate in determining the sex of psitticines, as demonstrated with cockatiels, while offering the advantages of non-invasiveness, simplicity, and speed.     

Endocrine monitoring of the ovarian cycle and pregnancy in the saddle-back tamarin (Saguinus fuscicollis) by measurement of steroid conjugates in urine

Direct measurements of urinary immunoreactive estrone conjugates (E1C) and pregnanediol glucuronide (PdG), were applied to monitoring the ovarian cycle (n = 9) and pregnancy (3 full term pregnancies, 2 mid-term abortions) in Saguinus fuscicollis. During the ovarian cycle, urinary E1C concentrations revealed a high degree of day-to-day variability and appeared to be uninformative in reflecting cyclic ovarian function. In contrast, PdG was a reliable indicator of ovarian cyclicity with excretion patterns corresponding well with plasma progesterone profiles. Luteal phase PdG concentrations were on average 4–7–fold higher than corresponding follicular phase values. On the basis of changes in circulating progesterone, a mean cycle length of 25.7 ±1.0 days with an average follicular phase of 7.1 ± 0.6 days and a mean luteal phase of 18.6 ± 0.7 days, was found (n = 14 cycles). Following conception, both urinary steroid conjugate concentrations increased and elevated levels were maintained beyond the normal luteal phase length, allowing pregnancy to be determined at around day 25–30. During mid- to late pregnancy, PdG levels declined while E1C concentrations continued to be elevated until approximately 6 weeks before parturition when a decrease occurred. Both hormones showed a clear and rapid fall to follicular phase values following termination of pregnancy at either parturition or mid-term abortion. Post partum ovulations (n = 5) occurred on average 17–18 days following birth with four ovulations leading to conceptions. The results demonstrate the potential of urinary steroid conjugate analysis as a practical and reliable method for non-invasive monitoring of reproductive status in the female saddle-back tamarin. © 1995 Wiley-Liss, Inc.     

Intracellular drug delivery: Potential usefulness of engineered Shiga toxin subunit B for targeted cancer therapy

A treasure trove of intracellular cancer drug targets remains hidden behind cell membranes. However, engineered pathogen-derived toxins such as Shiga toxins can deliver small or macromolecular drugs to specific intracellular organelles. After binding to ganglioglobotriaosylceramide (Gb3, CD77), the non-toxic subunit B (StxB) of the Shiga-holotoxin is endocytosed and delivers its payload by a unique retrograde trafficking pathway via the endoplasmic reticulum to the cytosol. This review provides an overview of biomedical applications of StxB-based drug delivery systems in targeted cancer diagnosis and therapy. Biotechnological production of the Stx-material is discussed from the perspective of developing efficacious and safe therapeutics.     

Palmilycorine and lycoriside: acyloxy and acylglucosyloxy alkaloids from crinum asiaticum

Two new types of alkaloidal conjugates, a C₁₆-acyloxy derivative, named palmilycorine, and an acylglucosyloxy derivative, named lycoriside, were isolated from the fruits of Crinum asiaticum. The presence of these compounds was also detected in the fleshy scale leaves and in roots of this species. The structures of the two compounds were established as 1-O-palmitoyllycorine (1) and lycorine-1-O-(6′-O-palmitoyl-β-D-glucopyranoside)(2), respectively, on the basis of chemical transformation and comprehensive spectral evidence. The biological effects of the alkaloids were evaluated.     

Design and synthesis of structurally defined heparan sulfate (HS)-FK506 conjugates as an exogenous approach to investigate biological functions of nucleus HS

Although heparan sulfate (HS) is widely implicated in numerous physiological and pathological processes, the biological function of nucleus HS remains underexplored, largely due to its complex structure and high hydrophilic property. To supplement these efforts, ideal vehicles are drawing attention as they combine attractive features including lipid solubility for penetrating cell membrane, high affinity binding to its target receptor, metabolic stability, and no cellular actions resulting in toxicity. Herein, we develop a convenient and promising strategy to prepare HS-FK506 conjugates for membrane transport and entry into nucleus, where click chemistry takes easily place between the exocyclic allyl group of a clinic drug FK506 and thiol as a handle incorporated into HS analogues. HS derivatives for constructing the conjugates were synthesized using a cutting-edge chemoenzymatic method. Meantime, [³⁵S] labeled 3′-phosphoadenosine 5′-phosphosulfate (PAP³⁵S) and [¹⁴C] glucuronic acid (Glc A) were adopted to label HS-FK506 conjugates, respectively, to evaluate their efficiency of nucleus entry, as a result, ¹⁴C Glc A was sensitive, effective and reliable whereas PAP³⁵S gave rise to a mixture of labeled compounds, hampering the understanding of structure-function relationship of nucleus HS. Compared with the corresponding HS, the amount of HS-FK506 conjugates to translocate into nucleus from radioactive assay experiments sharply increased, e.g. tridecasaccharide-FK506 1d increased by approximate 10 folds, offering a simple and robust platform for enabling hydrophilic compounds including carbohydrates to translocate into nucleus and shedding light on their biological functions.