Synthesis and immunogenicity of polysaccharide-protein conjugate composed of galactoglucoxylomannan of Cryptococcus laurentii

Galactoglucoxylomannan (GalGXMan) antigen of Cryptococcus laurentii was conjugated to protein carrier by a simple one step reaction. The conjugate was immunogenic in rabbits and reinjection elicited booster response with significant increase of serum IgG (H+L) level. Induction of this Ig-isotype was confirmed in experiments with Protein A. Effectiveness of immune serum to inhibit the growth of C. laurentii was demonstrated. The results indicate that the prepared conjugate could be considered as effective immunogen with potential for incorporation in the vaccine.     

Synthesis and characterization of a novel glycopolymer with protective activity toward human anti-alpha-Gal antibodies

An efficient and rapid synthesis of the derivative of the biocompatible polymer poly(styrene co-maleic acid) with Linear B disaccharide (Galili antigen) was achieved. The oligosaccharide portion was obtained by a transglycosylation reaction catalyzed by coffee bean alpha-D-galactosidase using p-nitrophenyl-alpha-D-galactopyranoside both as donor and as acceptor. The reaction was carried out in aqueous buffer without any organic cosolvent. The molar yield (30%) and the regioselectivity (82%) were significantly improved with respect to the data so far reported in the literature. The selective reduction of the p-nitrophenyl group afforded the p-aminophenyl derivative of Linear B disaccharide. Linkage of this derivative via an amidic bond to the poly(styrene co-maleic acid) was obtained by using N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. The products were chemically characterized by ionspray mass spectrometry, infrared, (13)C- and (1)H-nuclear magnetic resonance. The glycopolymer specifically reacts with human serum containing antibodies and with a mixture of partially purified human IgG and IgM anti-Linear B. It efficiently protects pig kidney PK15 cells from cytotoxic effects of human serum.     

Synthesis of a soluble catalase-dextran conjugate

Results of a systematic study of the conditions for preparation of soluble catalase-dextran conjugates, using the cyanogen bromide activation procedure, are reported. A protocol for the synthesis of such a conjugate with satisfactory retention of enzymatic activity and high efficiency of coupling is described.     

Synthesis and evaluation of a triplex-forming oligonucleotide-pyrrolobenzodiazepine conjugate

In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5' end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO-PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO-PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.     

13价肺炎球菌结合疫苗在老年人群中的免疫原性研究

老年人群对肺炎球菌疫苗的免疫应答不同于婴幼儿,在对老年人群肺炎球菌疫苗免疫原性的研究中,临床试验设计和判定标准均有所不同。现将美国13价肺炎球菌结合疫苗(13-valent conjugated vaccine,PCV13)在老年人群免疫原性研究的临床资料进行归纳和分析,为国内肺炎球菌疫苗在老年人群中的临床研究提供参考。     

Protective efficacy and immunogenicity of Vi-porin conjugate against Salmonella typhi.

A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyl dithio)-propionate (SPDP). After preparing the Vi-porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5-fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti-Vi antibody (IgG) levels (P < 0.01) than the mice immunized with Vi alone. Anti-porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate-immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.     

Synthesis,characterization and fluorescent properties of water-soluble glycopolymer bearing curcumin pendant residues

Curcumin is a potential natural anticancer drug with low oral bioavailability because of poor water solubility. The aqueous solubility of curcumin is enhanced by means of modification with the carbohydrate units. Polymerization of the curcumin-containing monomer with carbohydrate-containing monomer gives the water-soluble glycopolymer bearing curcumin pendant residues. The obtained copolymers (P1 and P2) having desirable water solubility were well-characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), UV–Vis absorption spectroscopy, and photoluminescence spectroscopy. The copolymer P2 with a molar ratio of 1:6 (curcumin/carbohydrate) calculated from the proton NMR results exhibits a similar anticancer activity compared to original curcumin, which may serve as a potential chemotherapeutic agent in the field of anticancer medicine.     

Synthesis and biological evaluation of a folate-targeted rhaponticin conjugate

To improve the therapeutic effect of rhaponticin (RHA), a folate receptor (FR) targeted RHA conjugate was synthesized by utilizing a hydrophilic peptide spacer linked to folic acid (FA) via a releasable disulfide linker. This water-soluble conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Treatment of FRHA with a reducing agent indicated that the amino-reactive derivative of RHA would be released spontaneously following disulfide bond reduction within the endosomes. FRHA also proved to be active predominantly specific against FR-positive syngeneic and xenograft models in vivo, and possible curative activity resulted with minimal to moderate toxicity. The FRHA conjugate greatly enhanced the therapeutic effects and reduced the toxicity of RHA. In conclusion, FRHA represents a folate-targeted chemotherapeutic that can produce potent activity against established sc tumors. Hence, this report has a great significance in pharmacology and clinical medicine as well as methodology.     

Synthesis and evaluation of a DHA and 10-hydroxycamptothecin conjugate

We have synthesized a conjugate of cis-4,7,10,13,16,19-docosahexenoic acid (DHA) and 10-hydroxycamptothecin (HCPT), DHA-HCPT. The antitumor activity of DHA-HCPT was evaluated in vitro against L1210 leukemia cells and in experimental animal tumor models including L1210 leukemia, Lewis lung carcinoma, and colon 38 adenocarcinoma. DHA-HCPT showed a greatly improved antitumor efficacy compared to HCPT.     

Synthesis and solution characterization of a porphyrin-CCK8 conjugate.

In this paper we report the synthesis and a detailed NMR solution characterization of a new CCK8 analogue and its indium(III) complex, PK-CCK8 and In-PK-CCK8. The new compounds contain a porphyrin moiety covalently bound through an amide bond to the side chain of a Lys residue introduced at the N-terminus of CCK8. A molecular dynamics simulation, based on the NMR structure of the complex between CCK8 and the N-terminal extracellular arm of the CCK(A) receptor, is also reported. Both the NMR study and the molecular dynamics simulation indicate that the porphyrin-peptide conjugate might be able to bind to the CCK(A) receptor model. The results of the molecular dynamics calculations show that the conformational features of the CCK8/CCK(A) receptor model complex and of the PK-CCK8/CCK(A) receptor-model complex are similar. This evidence supports the view that the introduction of the porphyrin-Lys moiety does not influence the mode of ligand binding to the CCK(A) receptor model. The NMR structure of PK-CCK8 in DMSO consists of a well defined pseudo-helical N-terminal region, while the C-terminal region is flexible. Moreover, the absence of NOE contacts between the porphyrin and the peptide indicates that the macrocyclic ring is directed away from the peptide region involved in the binding with the receptor.     

Synthesis and biological evaluation of a carbocyclic azanoraristeromycin siderophore conjugate

Synthesis and biological evaluation of a carbocyclic azanoraristeromycin siderophore conjugate 22 is reported. Coupling of previously prepared L-alanyl-4'-azanoraristeromycin 19 with protected tripeptide trihydroxamate 20, followed by hydrogenolytic removal of all protecting groups, provided the first carbocylic azanoraristeromycin siderophore conjugate (22, 8 with iron). Compounds 19 and 22 showed inhibitory activity against tumor cells, and conjugate 22, in particular, displayed significant activity against those viruses (i.e. reo, parainfluenza, vaccinia, cytomegalo) that are known to be inhibited by S-adenosylhomocysteine hydrolase inhibitors.     

Synthesis and activity of a folate targeted monodisperse PEG camptothecin conjugate

A folate targeted camptothecin small molecule drug conjugate (SMDC) was synthesized using a monodisperse PEG spacer linked to folate via a releasable disulfide carbonate linker. Cell cytotoxicity in human KB cells exhibited an IC₅₀ of 6 nM. Importantly, activity of the prodrug was blocked by excess folate, demonstrating receptor-mediated celluar uptake of the PEG conjugate.     

Synthesis and bioactivity of new Finasteride conjugate

Finasteride is a synthetic 4-azasteroid compound that acts by inhibiting type II 5α-reductase, the enzyme that converts the androgen testosterone to 5α-dihydrotestosterone. It was approved by the US FDA for the treatment of benign prostatic hyperplasia and male pattern baldness. Here the acylation product of Finasteride C-18 amide N-polimod was synthesized by employing acylation reaction with polimod amide as a pivotal intermediate. The structure of the key intermediate and target molecule was confirmed by infrared spectrum, ¹H NMR and ¹³C NMR spectra and mass spectrum, and the inhibition of the steroid 5α-reductase and the rats’ benign prostatic hyperplasia by the new Finasteride conjugate and Finasteride was also determined. The inhibition of the Finasteride conjugate on 5α-reductase was stronger than that of Finasteride. Prostate hyperplasia of rats was reduced by Finasteride conjugate treatment similar to the Finasteride treatment. However, the Finasteride conjugate treated animals showed better viable condition than the Finasteride treated ones, suggesting the new compound may have improved toxicity profile than Finasteride.     

Synthesis and evaluation of a novel lipid-peptide conjugate for functionalized liposome

A novel lipid analog based on amino acids for liposome modification was developed. It consisted of three different kinds of amino acid derivatives and two fatty acids, and can react directly with the peptide synthesized first on resin by Fmoc solid-phase synthesis. In this study, lipid analog conjugated with HIV-TAT peptide (domain of human immunodeficiency virus TAT protein) was synthesized and successfully incorporated into liposome. The liposome containing the lipopeptide bearing HIV-TAT exhibited efficient cellular uptake.     

Synthesis and characterization of a hemoglobin-ribavirin conjugate for targeted drug delivery

A novel conjugate of human hemoglobin (Hb) and the nucleoside analogue ribavirin (RBV) was synthesized to demonstrate the utility of Hb as a biocompatible drug carrier for improved drug delivery in the treatment of liver disease. RBV is used in combination with interferon for the treatment of hepatitis C, but its side effects can result in dose limitation or discontinuation of treatment. Targeted delivery of RBV may help to prevent or minimize its toxicity. The hemoglobin-ribavirin conjugate (Hb-RBV) was designed to release bioactive drug upon endocytosis by cells and tissues involved in extracellular Hb catabolism and clearance. Ribavirin-5'-monophosphate (RBV-P) was prepared from RBV and activated as the 5'-monophosphorimidazolide (RBV-P-Im) for reaction with carbonmonoxyhemoglobin to yield Hb-RBV consisting of multiple RBV drugs covalently attached as physiologically labile phosphoramidates via their 5'-hydroxyl groups. A molar drug ratio of six to eight RBV molecules per Hb tetramer was obtained with near complete haptoglobin (Hp) binding of the drug modified Hb maintained. The conjugate complex (Hp-Hb-RBV) was selectively taken up in vitro by cells that express the hemoglobin-haptoglobin receptor, CD163. Recovered ribavirin enzymatically cleaved from Hb-RBV showed equipotent antiproliferative activity compared to control unconjugated RBV against human HepG2 and mouse AML12 liver cell lines. Based upon the reported high level of Hb uptake in the liver, Hb-RBV may be useful in the treatment of certain liver diseases, as well as inflammatory disorders associated with CD163-positive macrophages.     

肺炎链球菌18C型糖蛋白结合物的制备及其免疫原性

制备肺炎链球菌18C型荚膜多糖－破伤风类毒素结合物（CPS－TT）,测定结合物的理化性质,抗原特异性及其在动物中的免疫原性。结果显示,结合物能与相应的多糖和破伤风抗血清形成明显的沉淀线,蛋白／多糖比率为1．86,结合物分子大小（Kd值）为0．058。注射小鼠后可诱导明显的抗体应答,而且随着注射针次的增加,抗体反应水平明显增高,显示加强效应。结果表明,制备的肺炎链球菌糖蛋白结合物抗原性良好,具有胸腺依赖性的特性,在小鼠中显示较好的免疫原性。     

Synthesis of a metallopeptide-PNA conjugate and its oxidative cross-linking to a DNA target

A nickel(II)-PNA bioconjugate was prepared by formation of a salicylaldimine complex with the amino terminus of a peptide-PNA hybrid with the sequence Arg-His-Gly-[TACCTAGCAT]PNA-Arg-CONH2. Hybridization to complementary oligodeoxynucleotides was demonstrated, and covalent adduct formation was observed upon addition of KHSO5 as oxidant. In the absence of PNA, the reactivity of the phenolic radical generated as an intermediate was found to be G > T > C, A; by inclusion of the PNA delivery agent, cross-links between the two oligomers could be observed with T and C bases in the vicinity of the nickel complex, although G was still the most reactive site. The metal complex could be removed by treatment with EDTA following which the Schiff base linkage was readily hydrolyzed. The final result in this case is a salicylaldehyde moiety appended at the target site in DNA.     

Synthesis and preliminary antitumor activity evaluation of a DHA and doxorubicin conjugate

A conjugate of DHA and doxorubicin (DHA-Dox) was synthesized, and its antitumor activity was evaluated in vitro against L1210 leukemia cells and in experimental animal tumor models including L1210 leukemia and B16 melanoma. DHA-Dox showed a greatly improved antitumor efficacy compared to free doxorubicin.     

Synthesis and characterization of a pyrrole-alginate conjugate and its application in a biosensor construction

N-(3-Aminopropyl)pyrrole was covalently coupled with alginate in an aqueous-phase reaction by means of carbodiimide-mediated activation chemistry to provide a pyrrole-alginate conjugate for subsequent use in biosensor applications. The pyrrole-alginate conjugate was quantified by UV spectroscopy at 230 nm, by an HPSEC-MALLS analytical method, as well as by FTIR and 13C NMR spectroscopies. The new pyrrole-alginate conjugate was used for the immobilization of polyphenol oxidase (PPO) onto an electrode surface by physical entrapment resulting from the gellification process and electrochemical polymerization of the pyrrole groups. The efficiency of this cross-linking approach (chemical and electrochemical) was investigated by comparing the amount of enzyme released from polypyrrole-alginate and regular alginate. In addition, biosensors were prepared by entrapment of the PPO in polypyrrole-alginate and regular alginate matrixes and their performance for the amperometric determination of catechol chosen as a model analyte was examined, yielding a sensitivity of 350 and 80 microA M(-1) cm(-2), respectively, for polypyrrole-alginate and alginate biosensors.     

Synthesis and characterization of a biotin-alginate conjugate and its application in a biosensor construction

Biotin was covalently coupled with alginate in an aqueous-phase reaction by means of carbodiimide-mediated activation chemistry to provide a biotin-alginate conjugate for subsequent use in biosensor applications. The synthetic procedure was optimized with respect to pH of the reaction medium (pH 6.0), the degree of uronic acid activation (20%), and the order of addition of the reagents. The biotin-alginate conjugate was characterized by titration with 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS), 4-hydroxyazobene-2'-carboxylic acid (HABA) and by an HPSEC-MALLS analytical method as well as by FTIR and 13C NMR spectroscopy. As a compromise between the need for a high percent of molar modification of the alginate, on one hand, and sufficient gelling capability, on the other hand, an optimal modification of 10-13% of biotin-alginate was used. The new biotin-alginate conjugate was used for the encapsulation of bioluminescent reporter cells into microspheres. A biosensor was prepared by conjugating these biotinylated alginate microspheres to the surface of a streptavidin-coated optical fiber, and the performance of the biosensor was demonstrated in the determination of the antibiotic, mitomycin C as a model toxin.