A Concise,Modular Antibody–Oligonucleotide Conjugation Strategy Based on Disuccinimidyl Ester Activation Chemistry

The synthesis of antibody–oligonucleotide conjugates has enabled the development of highly sensitive bioassays for specific epitopes in the laboratory and clinic. Most synthetic schemes to generate these hybrid molecules require expensive reagents, significant quantities of input antibody, and multistep purification routes; thus limiting widespread application. Herein a facile and robust conjugation strategy is reported that involves “plug-and-play” antibody conjugation with succinimidyl-functionalized oligonucleotides, which are high yielding and compatible for use directly after buffer exchange. The succinimidyl-linked oligonucleotides are synthesized with 5′-amine-modified oligonucleotides and disuccinimidyl suberate (DSS), both of which are inexpensive and commercially available. Direct incubation of the resulting stable succinimidyl– oligonucleotide conjugates with commercial antibodies yields conjugates ready for use after benchtop buffer exchange. It is demonstrated that the resulting oligonucleotide–antibody and oligonucleotide–streptavidin conjugates retain potent and specific binding in activity-dependent proximity ligation imaging, and proximity ligation-mediated qPCR detection of endogenous proteins in native cellular contexts down to picogram levels of whole proteome. This DSS conjugation strategy should be widely applicable in the synthesis of protein–oligonucleotide conjugates.     

A Brief Guide to Preparing a Peptide–Drug Conjugate

Peptide–drug conjugates (PDCs) have recently emerged as interesting hybrid constructs not only for targeted therapy, but also for the early diagnosis of different pathologies. In most cases, the crucial step in the PDC synthesis is the final conjugation step, where a specific drug is bound to a particular peptide-/peptidomimetic-targeting unit. Thus, this concept paper aims to give a short guide to determining the finest conjugation reaction, by considering in particular the reaction conditions, the stability of the linker and the major pros and cons of each reaction. Based on the recent PDCs reported in literature, the most common and efficient conjugation methods will be systematically presented and compared, generating a short guide to consult while planning the synthesis of a novel peptide–drug conjugate.     

Acrylonitrile copolymer films: Influence of modification by reaction with ethylene oxide on biological degradation

The biological degradation of acrylonitrile–dimethylaminoethyl methacrylate and acrylonitrile–acrylic acid copolymers has been studied by evaluating copolymer films in a continuous in vitro rumen system. The copolymer films were examined before and after modification by reaction with ethylene oxide gas. The degradation of the acrylonitrile–dimethylaminoethyl methacrylate copolymer followed a pattern similar to that occurring on thermal degradation. Modification by reaction with ethylene oxide gas, which induces C?N conjugation, did not improve the resistance to degradation. The acrylonitrile–acrylic acid copolymer films disintegrated, but on prior modification by reaction with ethylene oxide they remained virtually unaltered. Modification of the acrylonitrile–acrylic acid copolymer results in crosslinking with an absence of C?N conjugation, leading to improved resistance to biological degradation.     

Conjugate Vaccines from Bacterial Antigens by Squaric Acid Chemistry: A Closer Look

By using O‐SP‐core (O‐SPcNH₂) polysaccharide, isolated from Vibrio cholera O1 lipopolysaccharide (LPS) and related synthetic substances, a detailed study of factors that affect conjugation of bacterial polysaccharides to protein carriers through squaric acid chemistry to form conjugate vaccines has been carried out. Several previously unrecognized processes that take place during the squarate labeling of the O‐SPcNH₂ and subsequent conjugation of the formed squarate (O‐SPcNH‐SqOMe) have been identified. The efficiency of conjugation at pH 8.5, 9.0, and 9.5 to bovine serum albumin (BSA) and to the recombinant tetanus toxin fragment C (rTT‐Hc) has been determined. The study led to a protocol for more efficient labeling of O‐SPcNH₂ antigen with the methyl squarate group, to yield a higher‐quality, more potent squarate conjugation reagent. Its use resulted in about twofold increases in conjugation efficiency (from 23–26 % on BSA to 51 % on BSA and 55 % on rTT‐Hc). The spent conjugation reagent could be recovered and regenerated by treatment with MeI in the absence of additional base. The immunological properties of the experimental vaccine made from the regenerated conjugation reagent were comparable with those of the immunogen made from the parent O‐SPcNH‐SqOMe.     

Carrier‐bound methotrexate. II. Water‐soluble polyaspartamide methotrexate conjugates with amide links in polymer–drug spacer

In Part I of this series, conjugation of methotrexate (MTX) to water‐soluble polyaspartamide carriers was accomplished through ester link formation between an MTX carboxyl group and a carrier‐attached hydroxyl function. Contrasting with that type of anchoring link, this project utilizes amide formation as the means of drug conjugation. This is achieved through condensation of one of the drug's carboxyl groups with a carrier‐attached primary amine function. Derived from polysuccinimide by a time‐proven nucleophilic ring‐opening process in the presence of aliphatic diamines, polyaspartamide‐type carriers 1–12 comprise subunits equipped with tert‐amine or hydroxyl side group terminals for hydrosolubilization and other subunits equipped with primary amine terminals as drug‐binding sites. MTX conjugation with these carriers is effected in aprotic solvent, the reaction being mediated by 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate. The water‐soluble conjugates are fractionated and purified by size exclusion chromatography and dialysis; they are isolated by freeze‐drying in typical yields of 40–65%. In the molar MTX/NH₂ feed ratios that are chosen (generally 1.2–1.3) and with the mole fractions of drug‐binding subunits restricted to 10 and 20%, drug loading in the resultant conjugates approximates 20–30% by mass. In follow‐on study, conjugates 1‐MTX–12‐MTX thus obtained will be screened in cell culture tests for antiproliferative activity against a number of human cancer lines. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 3415–3424, 2006     

A New SiF–Dipropargyl Glycerol Scaffold as a Versatile Prosthetic Group to Design Dimeric Radioligands: Synthesis of the [18F]BMPPSiF Tracer to Image Serotonin Receptors

A novel SiX–dipropargyl glycerol scaffold (X: H, F, or ¹⁸F) was developed as a versatile prosthetic group that provides technical advantages for the preparation of dimeric radioligands based on silicon fluoride acceptor pre‐ or post‐labeling with fluorine‐18. Rapid conjugation with the prosthetic group takes place in microwave‐assisted click conjugation under mild conditions. Thus, a bivalent homodimeric SiX–dipropargyl glycerol derivatized radioligand, [¹⁸F]BMPPSiF, with enhanced affinity was developed by using click conjugation. High uptake of the radioligand was demonstrated in 5‐HT_1A receptor‐rich regions in the brain with positron emission tomography. Molecular docking studies (rigid protein–flexible ligand) of BMPPSiF and known antagonists (WAY‐100635, MPPF, and MefWAY) with monomeric, dimeric, and multimeric 5‐HT_1A receptor models were performed, with the highest G score obtained for docked BMPPSiF: ?6.766 as compared with all three antagonists on the monomeric model. Multimeric induced‐fit docking was also performed to visualize the comparable mode of binding under in vivo conditions, and a notably improved G score of ?8.455 was observed for BMPPSiF. These data directly correlate the high binding potential of BMPPSiF with the bivalent binding mode obtained in the biological studies. The present study warrants wide application of the SiX–dipropargyl glycerol prosthetic group in the development of ligands for imaging with enhanced affinity markers for specific targeting based on peptides, nucleosides, and lipids.     

13C-NMR-Untersuchung der interannularen Konjugation in substituierten Pyrazolen

¹³C-N.M.R. Investigation of Interannular Conjugation in Substituted Pyrazoles The correlation of the ¹³C-chemical shifts with the Hammet values for substituted 3-anilino-1, 5-diphenylpyrazoles (1a–k) and substituted 3-methyl-1, 5-diphenylpyrazoles (2a–g) provides evidence for extensive interannular conjugation only between the N-benzene-and the pyrazole rings, whereas in the capto-dative disubstituted system 1 extensive conjugation through the whole molecule is possible. The chemical shifts correlate well with the HMO-calculated π-electron densities. From a comparison of the chemical shifts of unsubstituted 1 and diphenylamine it can be concluded that the 1,5 -diphenylpyrazol-3-yl group has the same electronic effect as the phenyl group.     

Palladium‐Mediated Labeling of Nucleic Acids

New applications of Pd‐catalyzed coupling reactions (Suzuki–Miyaura, Sonogashira, and Stille–Migita coupling) for post‐conjugation of nucleic acids have been developed recently. Breakthroughs in this area might now pave the way for the development of sophisticated DNA probes, which might be of great interest in chemical biology, nanotechnology, and bioanalysis, as well as in diagnostic domains.     

Synthesis,X-ray crystal structure and optical properties of novel 2,5-diaryl-1,3,4-oxadiazole derivatives containing substituted pyrazolo[1,5-a]pyridine units

A series of pyrazolo[1,5-a]pyridine-containing 2,5-diaryl-1,3,4-oxadiazole derivatives were synthesized and their structures were characterized by IR, ¹H NMR and HRMS spectra. The crystal structure of 3a was determined using single crystal X-ray crystallography. Its spatial structure was found to be monoclinic, and all aromatic rings were approximately coplanar, which allowed conjugation. The absorption results showed that compounds 1a–f presented their absorption peaks ranging from 264 nm to 290 nm, while compounds 3a–f with a larger conjugation system exhibited red-shifted absorption character (absorption maxima between 283 nm and 303 nm) compared to the corresponding absorption of 1a–f. Fluorescence spectra revealed that these compounds exhibited blue fluorescence (421–444 nm) in dilute solutions and showed quantum yields of fluorescence between 0.32 and 0.83 in dichloromethane.     

Fourier transform infrared study of polypyrrole–poly(vinyl alcohol) conducting polymer composite films: Evidence of film formation and characterization

The electrochemical preparation of polypyrrole (PPY)–poly(vinyl alcohol) (PVA) conducting polymer composite films on an indium–tin oxide glass electrode from an aqueous solution containing a pyrrole monomer, a p‐toluene sulfonate electrolyte, and a PVA insulating polymer is reported. The prepared PPY–PVA composite films were characterized by Fourier Transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), and conductivity measurements. The FTIR study showed that the composite of PPY and PVA formed through bond formation between PVA and the p‐toluene sulfonate dopant anion. The conductivity data of PPY–PVA showed that with increasing PVA concentration in the pyrrole solution, the conductivity of the prepared PPY–PVA film increased up to a certain level due to an increase in conjugation length, and later, it decreased with further increases in the PVA concentration in the solution due to a decrease in conjugation length. This was supported by the FTIR band intensity I₁₅₆₀/I₁₄₈₀. The TGA results show that the PPY–PVA polymer composite film was thermally more stable than the PPY film. A shielding effectiveness of 45.6 dB was exhibited by the PPY–PVA composite film in the microwave frequency range. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4107–4113, 2006     

Chemical immobilization of retinoic acid within poly(ε‐caprolactone) nanoparticles based on drug–polymer bioconjugates

All‐trans‐retinoic acid (RA) was chemically conjugated to biodegradable poly(ε‐caprolactone) (PCL₁₀; number‐average M_w ≈ 1250) via an ester linkage. The conjugation was carried out using N,N‐dicyclohexylcarbodiimide and 4‐dimethyl aminopyridine as a coupling agent. The molar ratio of the drug to the polymer was 1.11 as determined by ¹H‐NMR analysis. DSC and WAXD results showed that the formation of crystalline structures of RA was effectively suppressed by conjugation with PCL. The RA–PCL conjugates were formulated into nanoparticles by a spontaneous phase‐inversion technique. Morphological characteristics of the resultant nanoparticles and drug‐loading efficiencies were compared with those of free RA‐loaded nanoparticles. The drug‐loading efficiency of RA–PCL conjugates was almost 100%, while that of free RA was only ～12%. The majority of unconjugated RA was found to form undesirable free‐drug crystals out of nanoparticles, as observed by TEM analysis. This study demonstrates that the conjugation approach of RA to PCL can be an effective means to immobilize and encapsulate RA within nanoparticles for pharmaceutical applications. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1631–1637, 2003     

Morphology and thermal properties of nylon copolymers containing dimer acid,adipic acid,and hexamethylenediamine

The effects of the molar ratio of adipic acid to total diacid on the phase morphology and thermal properties of nylon copolymers containing dimer acid, adipic acid, and hexamethylenediamine were investigated. A significant structural transition from the γ form to α and β mixture forms occurred with increasing molar ratio. Meanwhile, the content of the crystals increased markedly. A σ–π* hyper conjugation effect was present in all of the as‐prepared samples. A strong σ–π* hyper conjugation effect could decrease the average distance between interchain amide groups and suppress the scission of the C? N bond through the formation of highly combining aza double‐bond C?N groups, which would, consequently, make the nylon copolymer much more stable. Differential scanning calorimetry–thermogravimetric analysis results indicate that the low thermal stability of the nylon copolymers with higher molar ratios of adipic acid to total diacid was simply due to the formation of cyclopentanone and caprolactam at 350–420°C, whereas for the nylon copolymers with lower molar ratios, the scission of the C? C bond at 430–500°C directed the pyrolysis of the main chain. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Carrier‐bound methotrexate. I. Water‐soluble polyaspartamide–methotrexate conjugates with ester links in the polymer–drug spacer

The antifolate‐type anticancer drug methotrexate (MTX) has for many years, in numerous laboratories, been a “workhorse” drug for conjugation with natural and synthetic macromolecular carriers for the purpose of enhancing bioavailability and lowering toxic side effects. In the project here described the polymer–drug conjugation strategy is utilized for the preparation of water‐soluble polyaspartamide–methotrexate conjugates in which the drug is carrier‐anchored through short spacers containing ester groups as biofissionable links. To this end, polyaspartamide carriers 1, poly‐α,β‐D,L ‐N‐(2‐hydroxyethyl)aspartamide, and 2, poly‐α,β‐D,L ‐N‐[2‐(2‐hydroxyethoxy)ethyl]aspartamide, are treated with MTX in DMF solution in the presence of a carbodiimide coupling agent and 4‐(dimethylamino)pyridine catalyst. The molar MTX/OH feed ratios, 0.28 and lower, are chosen in these coupling reactions so as to provide conjugates featuring drug‐loading levels in the approximate range of 3–16 mol % MTX, roughly corresponding to 6–28% by mass. The water‐soluble product polymers are purified by aqueous dialysis, collected in the solid state by freeze‐drying, and structurally characterized by ¹H–NMR spectroscopy. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 1844–1849, 2001     

CYCLIC CONJUGATION IN FLUORANTHENE AND ITS BENZO-DERIVATIVES. PART 1. CATACONDENSED SYSTEMS

Continuing our investigations of π -electron properties of fluoranthene-type polycyclic conjugated compounds [Gutman, ?ur?evi?, Balaban, Polycyclic Aromatic Compounds 29 (2009): 3–11], we studied the energy effects of individual rings in fluoranthene and its catacondensed benzo-derivatives (i.e., benzo-annelated fluoranthenes possessing exactly one internal carbon atom). A number of general regularities were observed: (a) the PCP effect (six-membered rings connected by a single bond to the five-membered ring increase in it the intensity of cyclic conjugation), (b), the retro-PCP effect (the five-membered ring increases the intensity of cyclic conjugation in the six-membered rings to which it is connected by a single bond), (c) the linear effect (six-membered rings, linearly annelated to fluoranthene, decrease the cyclic conjugation in the five-membered ring), (d) the magnitude of the PCP effect differs, depending on whether the six-membered ring belongs to the “male” or “female” benzenoid fragment, and (e) the pattern of cyclic conjugationin of isoarithmic benzofluoranthenes is essentially identical.     

Recent Chemical Approaches for Site-Specific Conjugation of Native Antibodies: Technologies toward Next-Generation Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs), which consist of three components, antibody, linker, and payload, can function as “magic bullets”. These conjugates offer the ability to target drug delivery to specific cells, based on cell-specific recognition and the binding of an antigen by a monoclonal antibody (mAb). In particular, by delivering a cytotoxic payload to cancer cells, ADCs are expected to provide a breakthrough in oncology treatments by providing a way to increase efficacy and decrease toxicity, in comparison with traditional chemotherapeutic treatments. The development of ADC therapeutics has dramatically progressed in the past decade and two ADCs have been approved and used as anticancer drugs in the clinic. However, several critical issues regarding the performance of ADCs are still being discussed and investigated. Indeed, in the past few years, several groups have reported that, changing the number and position of the drug payloads in the ADCs, affects the pharmacokinetics, drug release rates, and biological activity. The use of conventional heterogeneous conjugation methods for ADC preparation results in the drug/antibody ratio and connecting position of the payload having stochastic distributions. Therefore, it is important to investigate how these potential problems can be circumvented through site-specific conjugation. Herein, various site-specific chemical conjugation strategies with native mAbs that are currently used for the production of ADCs, including residue-selective labeling for generating ADCs, disulfide rebridging, and affinity-peptide-mediated site-specific chemical conjugation technologies, are reviewed and described.     

Effect of thermal annealing in atmosphere on photoluminescence of BTEO–PPV films

The photoluminescence (PL) spectra of poly[2,5‐bis‐(tri‐ethoxy)‐1,4‐phenylene vinylene] (BTEO–PPV) films are blue‐shifted with increasing thermal annealing temperature. It is known from the UV–vis absorption spectra that thermal annealing decreases the conjugation length of the polymer. For BTEO–PPV films, unlike with MEH–PPV films, the symmetric triethoxy side groups further block aggregation of the polymer chains. The absorption Fourier transfer infrared spectra showed that thermal annealing in atmosphere destroyed the chain structure of BTEO–PPV film by thermal oxidation to form aldehyde groups, which resulted in low PL efficiency of the annealed films. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006     

Erweiterte Konjugation in unsymmetrischen Stilbenylsquarainen

Extended Conjugation in Stilbenylsquaraines Unsymmetrical 1-aryl-3-stilbenylsquaraines 2a – e and 1,3-bis(stilbenyl)squaraines 3a – c were prepared by CC coupling reactions of the corresponding substituted arenes with derivatives of squaric acid (Schemes 2 and 3). Dialkylamino and alkoxy groups enhance the solubility of these dyes and enlarge the intramolecular charge transfer of these donor–acceptor–donor systems. The extended conjugation of the stilbene units – in comparison with arene building blocks – leads to significant bathochromic shifts in the Vis/NIR absorption spectra.     

Self‐aggregation behavior of amphiphilic polyaspartamide derivatives containing cholesterol moieties

Biodegradable amphiphilic copolymers were successfully synthesized by the conjugation of various densities of hydrophobic biocompatible cholesterol (Chol) moieties onto poly(2‐hydroxyethyl aspartamide) and poly(N‐isopropylaminoethyl‐co‐2‐hydroxyethyl aspartamide). These were obtained from polysuccinimde, the thermal polycondensation product of L‐aspartic acid, via a ring‐opening reaction with multifunctional pendant groups, including ethanolamine and N‐isopropylethylenediamine (NIPEDA). Copolymers containing 5–30 mol % Chol showed self‐aggregation behavior in aqueous solution, as evidenced by the dynamic light scattering measurement of their particle size distribution. The average particle size of these copolymers increased linearly with increasing Chol content. Moreover, the presence of secondary amine groups in the poly(2‐hydroxyethyl aspartamide)–NIPEDA system made the conjugation more efficient; however, these also seemed to accelerate the degradation of the copolymers in an aqueous medium. The degradation behavior and pH dependence of the particle size of these copolymers in aqueous solution were also examined. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Reactivity of temperature‐sensitive,protein‐conjugating polymers prepared by a photopolymerization process

This study was carried out to characterize the reactivity of temperature‐sensitive, protein‐conjugating polymers prepared by a photopolymerization process. Polymers were based on N‐isopropylacrylamide (NiPAM) and N‐acryloxysuccinimide (NASI). A photoinitiator, 2,2‐dimethoxy‐2‐phenyl‐acetophenone, and monomers at desired ratios were polymerized in a glass flask using an UV source. Polymers were characterized for composition, molecular weight (MW), cloud point temperature (CPT), hydrolysis and aminolysis rates (using ethanolamine as a model compound), and protein conjugation. The monomer feed ratio was found to effectively control the composition of the synthesized polymers. The polymer MWs were between 10 and 20 kD, depending on the polymerization solvent. The CPT of NiPAM/NASI polymers did not depend on NASI content (≤5.6%), nor did the hydrolysis and aminolysis rates. Compared to NASI monomer, the polymerized NASI exhibited a 6‐ and 120‐fold slower rates of hydrolysis and aminolysis, respectively. Although hydrolysis and aminolysis rates were higher at higher pHs, the relative aminolysis : hydrolysis rate was highest at a pH of 7.4, which also gave the most effective protein conjugation. We conclude that characterizing the polymer reactivity is useful for predicting the optimal conditions for protein conjugation and may facilitate the design of polymers with improved protein conjugation kinetics. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 583–592, 2000     

An efficient synthesis of stable sulfur-containing phosphoranes derived from 2-mercapto-1-methylimidazole and 2-thiazoline-2-thiol

Stable crystalline phosphorus ylides were obtained in excellent yields from the 1:1:1 addition reaction between triphenylphosphine and dialkyl acetylenedicarboxylates in the presence of strong SH-acids, such as 2-mercapto-1-methylimidazole and 2-thiazoline-2-thiol. These stable ylides exist in solution as a mixture of two geometrical isomers as a result of restricted rotation around the carbon–carbon partial double bond resulting from conjugation of the ylide moiety with the adjacent carbonyl group.