Synthesis of a heterotrifunctional linker for the site-specific preparation of antibody-drug conjugates with two distinct warheads

Codelivery of multiple therapeutic agents with different anticancer mechanisms can overcome drug resistance as well as generate additive or synergistic anticancer effects that may enhance the antitumor efficacy. Antibody-drug conjugates (ADCs) can be used for highly specific delivery of multiple therapeutic agents with different anticancer mechanisms, though more research is required towards designing flexible platforms on which dual drug ADCs could be prepared. Herein, we describe the synthesis of a heterotrifunctional linker that could be used to construct flexible platforms for preparing dual-cytotoxic drug conjugates in a site-specific manner. As a proof of concept, we synthesized dual drug ADCs carrying monomethyl auristain E (MMAE, tubulin polymerization inhibitor) and pyrrolobenzodiazepine dimer (PBD, DNA minor groove alkylator). We then evaluated the dual drug ADCs for in vitro efficacy and confirmed the dual mechanism of action.     

Conformational analysis of site-specific DNA cross-links of cisplatin-distamycin conjugates

The requirement for novel platinum antitumor drugs led to the concept of synthesis of novel platinum drugs based on targeting cisplatin to various carrier molecules. We have shown [Loskotova, H., and Brabec, V. (1999) Eur. J. Biochem. 266, 392-402] that attachment of DNA minor-groove-binder distamycin to cisplatin changes several features of DNA-binding mode of the parent platinum drug. Major differences comprise different conformational changes in DNA and a considerably higher interstrand cross-linking efficiency. The studies of the present work have been directed to the analysis of oligodeoxyribonucleotide duplexes containing single, site-specific adducts of platinum-distamycin conjugates. These uniquely modified duplexes were analyzed by Maxam-Gilbert footprinting, phase-sensitive gel electrophoresis bending assay and chemical probes of DNA conformation. The results have indicated that the attachment of distamycin to cisplatin mainly affects the sites involved in the interstrand cross-links so that these adducts are preferentially formed between complementary guanine and cytosine residues. This interstrand cross-link bends the helix axis by approximately 35 degrees toward minor groove, unwinds DNA by approximately 95 degrees and distorts DNA symmetrically around the adduct. In addition, CD spectra of restriction fragments modified by the cisplatin-distamycin conjugates have demonstrated that distamycin moiety in the interstrand cross-links of these compounds interacts with DNA. This interaction facilitates the formation of these adducts. Hence, the structural impact of the specific interstrand cross-link detected in this study deserves attention when biological behavior of cisplatin derivatives targeted by oligopeptide DNA minor-groove-binders is evaluated.     

Synthesis of carotenoid-cysteine conjugates

Isozeaxanthin under acidic conditions forms an allylic cation which reacts readily with thiol nucleophiles. With N-acetylcysteine as a nucleophile the products obtained are carotenoid-cysteine conjugates in which the amino acid moiety is attached to the carotenoid via sulphur in position 4. The water solubility of the products can be increased by deprotection of the amino group. The antioxidant activity of the products were examined on human liver cells under conditions of hydrogen-peroxide induced oxidative stress.     

Synthesis of aminoglycoside-DNA conjugates

Development of aminoglycoside-nucleic acid conjugates is presented. Synthesis of a DNA dimer covalently linked to kanamycin and neomycin isothiocyanates has been carried out. The development of such conjugates will help couple the sequence specificity of nucleic acids to the electrostatic/shape complementarity of aminoglycoside antibiotics in binding nucleic acid targets.     

Synthesis of pyochelin-norfloxacin conjugates

Using synthetic functionalized analogues of pyochelin, a siderophore common to several pathogenic Pseudomonas and Burkholderia species, four fluoroquinolone-pyochelin conjugates were efficiently synthesized and evaluated for their biological activities.     

Synthesis of radiolabeled cytarabine conjugates

N4-Modified, novel Ara-C conjugate capable of radiolabeling with gamma ray-emitting (^99mTc) as well as positron emitting (¹⁸F) radionuclides, that is, N4-hydrazine derivative was synthesized. The radiolabeling of N4-(hydrazinonicotinyl)-1-β-arabinofuranosyl cytosine (HAra-C) with ^99mTc was performed with over 95% labeling yield. To label HAra-C with ¹⁸F, 4-fluoro(¹⁸F)-benzaldehyde was synthesized from 4-formyl-N,N,N-trimethylanilinium triflate in 30% radiochemical yield; it quantitatively formed hydrazone derivative with HAra-C within 45 min. The radiolabeled conjugates were analyzed by radio-UV-RP-HPLC. The cold precursors were characterized by ¹H, ¹³C NMR. Additionally, HAra-C was evaluated for cytotoxicity in lung adenocarcinoma (H441) cells and found to be comparable in cell killing efficiency to that of Ara-C. Uptake of ^99mTc-HAra-C in cultures of H441 cells and sensitive pancreatic cancer cells (MIAPaCa-2) was inhibited by nucleoside transporter inhibitor nitrobenzylthioinosine. The results suggest that ^99mTc-labeled HAra-C is a substrate for the membrane nucleoside transporters, and that it may be used in molecular imaging of nucleoside transporter expression for the verification of potential anticancer efficacy of nucleoside drugs, such as Ara-C and gemcitabine.     

Synthesis of uniform protein-polymer conjugates

We have developed a novel technique to synthesize near-uniform protein-polymer conjugates by initiating atom transfer radical polymerization of monomethoxy poly(ethylene glycol)-methacrylate from 2-bromoisobutyramide derivatives of chymotrypsin (a protein-initiator). Polymerization initiated from the monosubstituted protein-initiator resulted in the conjugate containing a single, near-monodisperse polymer chain per protein molecule with polydispersity index 1.05. Increasing the number of conjugated 2-bromoisobutyramide initiators per molecule of protein increased the molecular weights and polydispersity indices of the final protein-polymer conjugates. The generic nature of this technique was demonstrated by initiating polymerization of nonionic, cationic, and anionic monomers from the protein-initiator. Protein-polymer conjugates synthesized by this novel technique retained 50-86% of the original enzyme activity. The technique described herein should be useful in synthesizing well-defined protein-polymer conjugates exhibiting a wide range of physical and chemical properties.     

Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting

Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in C_H2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.     

Synthesis of bioadhesive lectin-HPMA copolymer-cyclosporin conjugates

An amino group containing cyclosporin A (CsA) derivative has been synthesized and conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an aromatic azo bond, which can be specifically cleaved by azoreductase activity in colon to release the drug for the treatment of colon diseases. Lectins, peanut (Arachis hypogea) agglutinin (PNA) and wheat germ agglutinin (WGA), have been conjugated to HPMA copolymer-CsA derivative conjugates (PCsA), respectively, to give bioadhesive conjugates. The PNA and WGA are the targeting proteins that can bind to diseased colon tissue and healthy tissue, respectively. There were on average four P(CsA) copolymer chains attached on one WGA molecule with a drug content of 16.0 wt % and five P(CsA) copolymer chains attached on one PNA molecule with a drug content of 11.5 wt %. The incubation of a P(CsA) copolymer with the rat cecal contents resulted in the cleavage of the azo bond and release of the cyclosporin derivative. The biological evaluation of the conjugates is under way.     

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 applications of oligonucleotide-carbohydrate conjugates

Nowadays, oligonucleotide-carbohydrate conjugates are used in antisense biotechnology and in the study of glycosylated DNA functioning in vitro. The application of mono- and disaccharide phosphoramidites, solid-phase supports with immobilized carbohydrates, glycosylated nucleoside phosphoramidites, and postsynthetic conjugation of reactive sugar derivatives with oligonucleotides for preparation of oligonucleotide-carbohydrate conjugates have been systematically studied. The advantages and disadvantages of these approaches are considered. Possible strategies for synthesis of glycoclusters with different topologies conjugated to DNA are discussed. Applications of oligonucleotide-carbohydrate conjugates are highlighted. Studies of interactions of glycosylated oligonucleotides with proteins and effective cell-specific delivery of oligonucleotide-carbohydrate conjugates are discussed.     

Synthesis of immunoglobulin conjugates with dehydrogenases

Three methods of synthesis of immunoglobulin conjugates with malate, lactate and glucose-6-phosphate dehydrogenases, involving the sodium metaperiodate oxidation of immunoglobulin carbohydrate component, use of water-soluble carbodiimide and the one-step glutaraldehyde technique, were compared. The glutaraldehyde method was shown to give immunoglobulin-dehydrogenase conjugates with high catalytic and immunochemical activity, which may be useful for enzyme-immunoassay.     

Synthesis and electrochemistry of anthraquinone-oligodeoxynucleotide conjugates

Electroactive oligodeoxynucleotides (ODNs) with specific base sequences have a potential application as electrical sensors for DNA molecules. To this end, a phosphoramidite that bears a 9, 10-anthraquinone (AQ) group tethered to the 2'-O of the uridine via a hexylamino linker, 2'-O-[6-[2-oxo(9, 10-anthraquinon-2-yl)amino]hexyl]-5'-O-(4,4'-dimethoxytrityl)uridi ne 3'-[2-(cyanoethyl)bis(1-methylethyl)phosphoramidite] (3), has been synthesized and used to prepare three ODNs with tethered AQs using standard phosphoramidite chemistry. The synthetic methodology thus allows the synthesis of ODNs with electroactive tags attached to given locations in the base sequence. Cyclic voltammetric behavior of these AQ-ODN conjugates was examined in aqueous buffer solutions at a hanging mercury drop electrode. At slow sweep rates, nearly reversible two-electron waves characteristic of an adsorbed anthraquinone/hydroquinone redox couple was observed for all of the AQ-ODN conjugates. Approximate Langmuirian isotherms were found for the AQ-ODNs with molecular footprints, calculated from the saturation coverages, that scaled with molecular size. The cyclic voltammetric response of the duplexes formed from the AQ-ODNs and their complementary ODN was complicated by the competitive adsorption of the individual ODNs and possibly the duplex species as well.     

Direct site-specific cleavage of double-stranded DNA by conjugates of bleomycin A5 with triplex-forming oligonucleotide

Monofunctional conjugates of 15-mer triplex-forming oligonucleotide (TFO) with covalently attached bleomycin A5 residue at the 5′-end (Blm-p15) were synthesized. Bifunctional conjugates of TFO containing, in addition to Blm, the residues of intercalator 6-chloro-2-methoxy-9-aminoacridine (Acr) or N-(2-hydroxyethyl)phenazinium (Phn) were obtained for the first time. The Acr and Phn residues were attached to the 3′-phosphate group of TFO through L1 and L2 linkers, respectively, resulting in the compounds Blmp15pL1-Acr and Blm-p15pL2-Phn. The values of dissociation constants of the corresponding triplexes were evaluated using the gel retardation method. The Acr residue in Blm-p15pL1-Acr was shown to enhance the stability of the formed triplex by one order of magnitude. It was demonstrated that all synthesized conjugates are capable of specifically and nonspecifically damaging a target DNA, forming direct breaks and alkaline-labile sites. The extent of the specific cleavage of the target DNA was 15% in the case of a fivefold excess of the conjugates over the DNA duplex. The site-specific triplex-mediated cleavage of a target DNA was shown for the first time to occur predominantly (>90%) with the formation of the direct breaks of both DNA strands. The results show the availability of bleomycin-containing oligonucleotides as antigene compounds.     

Synthesis and biological activities of nucleoside-estradiol conjugates

Nucleosides were coupled to estradiol via a 17alpha-ethynyl spacer group using Pd(II) as a catalyst. The conjugates were evaluated in vitro for estrogen receptor (ER) binding affinity and cytotoxicity against cell lines with and without ER. The highest receptor binding affinities (RBA approximately 3) were observed with conjugates coupled via a relative long spacer group, while none of the conjugates exhibited cytotoxicity against either cell lines.     

Synthesis and anticancer activity of open-resorcinarene conjugates

The first example of conjugation of open-resorcinarenes with chlorambucil, ibuprofen, naproxen and indomethacin are presented. The cytotoxic properties of the obtained conjugates were tested against the cancer cell lines U-251, PC-3, K-562, HCT-15, MCF-7 and SKLU-1. It was found that the conjugate with chlorambucil, naproxen or indomethacin (having 8 moieties) was toxic towards cancer cell lines U-251 and K-562, with no activity against non-cancerous COS-7 cells. The conjugates with naproxen and indomethacin showed high selectivity towards U-251 tumor cells.     

Synthesis and biological activities of phthalocyanine-estradiol conjugates

Phthalocyanine-based photosensitizers, coupled via a 17alpha-ethynyl group to estradiol using Pd(II) as a catalyst, were synthesized and evaluated for their estrogen receptor binding affinity and in vitro photocytotoxicity. The highest receptor binding affinities (RBA=8-13) were observed with lipophilic conjugates coupled via a relative long spacer group while the sulfonated analogues showed little binding affinities (RBA <2). The highest photocytotoxicity was observed with the sulfonated conjugates, the nature of the spacer group did not have a pronounced effect.     

Synthesis of catecholamine conjugates with nitrogen-centered bionucleophiles

The enzymatic (tyrosinase) and chemical (NaIO₄, Ag₂O or Frémys’s salt) oxidation of biologically relevant catecholamines, such as dopamine (DA), N-acetyldopamine (NADA) and the Ecstasy metabolites (α-MeDA and N-Me-α-MeDA) generates the corresponding o-quinone which can be trapped with nitrogen bionucleophiles such as N-acetyl-histidine and imidazole in a regioselective reaction that takes place predominantly at the 6-position of the catecholamine.     

Synthesis and spectroelectrochemistry of N-cobaltacarborane porphyrin conjugates

N-Substituted porphyrins are well-known for the distortion they exhibit of the porphyrin plane through the sp(3) hybridization of one of the pyrrolenic units. They have served as model compounds in investigations of many biochemical processes. In this paper, we developed an efficient route to N-substituted porphyrins, and report the synthesis of a series of new N-substituted cobaltacarborane-porphyrins containing one or two cobaltabisdicarbollide anions linked by (CH(2)CH(2)O)(2) chains to either the core porphyrin nitrogens or to a meso-aminophenyl group. These conjugates show different degrees of distortion of the porphyrin macrocycle, which affect their spectroscopic and electrochemical properties. In particular, the core N-substituted conjugates show significant fluorescence quenching in comparison with the noncore substituted macrocycles. The X-ray structures of two targeted core N-cobaltacarborane porphyrin conjugates are presented. The electrochemical and spectroelectrochemical properties of these porphyrin conjugates were investigated; while the peripheral N-substituted cobaltacarboranylporphyrins undergo three reversible reductions and three reversible oxidations (two attributed to the porphyrin and one to the Co(III) cluster), the core N-substituted porphyrins exhibit complicated electrochemical behavior with coupled chemical reactions.