Phage-display selection of a human single-chain fv antibody highly specific for melanoma and breast cancer cells using a chemoenzymatically synthesized G(M3)-carbohydrate antigen

Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 10(-5) M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.     

Spectroscopy, binding to liposomes and production of singlet oxygen by porphyrazines with modularly variable water solubility

Three novel classes of porphyrazine-like structures were synthesized to form modular structures in which lipophilicity and water solubility can be tuned. Subtle modification of solubility is an important criterion in selecting a compound for biological photosensitization. The general structure takes the form H2[pz(AnB4-n)], where the core is a porphyrazine (pz) group, A is a pyrrole ring with two sulfide linkages (SR moieties) and B is a pyrrole fused with a 4,7-bis(isopropyloxy)benzo group, with n=4, 3 and 2. These molecules possess their longest wavelength absorption band between 700 and 810 nm, hence laser beams of higher tissue penetration depth could be used to illuminate them in photodynamic therapy (PDT). Armed with absorption bands in the far-red and near-infrared (near-IR), and a capability to tune the solubility, these molecules could make for better sensitizers because of optimized uptake by lipidic membranes and better optical properties. We tested several derivatives of the A4, A3B and A2B2 structures for their singlet oxygen quantum yields in methanol and in liposomes, using 9,10-dimethyl anthracene (DMA) as a singlet oxygen target. Singlet oxygen quantum yields in liposomes ranged from 0.01 to 0.44, with the A2B2 group showing the most promise. In the binding assay to find the equilibrium binding constant, Kb, we detected fluorescence changes due to a change in environment. Peripheral long-chain moieties (the R group in the SR moieties) dominate lipid binding. These moieties range in the hydrophobicity that they induce from C8H17 and benzene, which rendered the molecule totally insoluble in water, to polyethylene glycol (PEG) and carboxylate groups, which imparted water solubility. Each molecule had between 4 and 8 such identical chains. Chains bearing an ether or ester link resulted in measurable equilibrium constants, with a higher Kb for ether substituents. Results for Kb ranged from 0.23 to 26.52 (mg mL(-1))(-1). A delicate balance exists between water solubility and good partitioning to membranes. In general, a higher oxygen-to-carbon ratio in the chains improves binding. Fewer chains and a centrally coordinated zinc ion further improve binding and singlet oxygen production.     

Mass spectrometric characterization of lipid-modified peptides for the analysis of acylated proteins

The analysis of acylated proteins by mass spectrometry (MS) has largely been overshadowed in proteomics by the analysis of glycosylated and phosphorylated proteins; however, lipid modifications on proteins are proving to be of increasing importance in biomedical research. In order to identify the marker ions and/or neutral loss fragments that are produced upon collision-induced dissociation, providing a means to identify the common lipid modifications on proteins, peptides containing an N-terminally myristoylated glycine, a palmitoylated cysteine and a farnesylated cysteine were chemically synthesized. Matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF-TOF), electrospray ionization quadrupole time-of-flight (ESI Q-TOF), and electrospray ionization hybrid triple-quadrupole/linear ion trap (ESI QqQ(LIT)) mass spectrometers were used for the analysis. The peptide containing the N-terminally myristoylated glycine, upon CID, produced the characteristic fragments a1 (240.4 Th) and b1 (268.4 Th) ions as well as a low-intensity neutral loss of 210 Da (C14H26O). The peptides containing a farnesylated cysteine residue fragmented to produce a marker ion at a m/z of 205 Th (C15H25) as well as other intense farnesyl fragment ions, and a neutral loss of 204 Da (C15H24). The peptides containing a palmitoylated cysteine moiety generated neutral losses of 238 Da (C16H30O) and 272 Da (C16H32OS); however, no marker ions were produced. The neutral losses were more prominent in the MALDI-TOF-TOF spectra, whereas the marker ions were more abundant in the ESI QqQ(LIT) and Q-TOF mass spectra.     

Optimizing the monomer structure of polyhedral oligomeric silsesquioxane for ion transport in hybrid organic–inorganic block copolymers

Poly(ethylene oxide)-b-polyhedral oligomeric silsesquioxane (PEO–POSS) mixed with lithium bis(trifluoromethanesulfonyl)imide salt is a nanostructured hybrid organic–inorganic block copolymer electrolyte that may enable lithium metal batteries. The synthesis and characteristics of three PEO–POSS block copolymer electrolytes which only differ by their POSS silica cage substituents (ethyl, isobutyl, and isooctyl) is reported. Changing the POSS monomer structure results in differences in both thermodynamics and ion transport. All three neat polymers exhibit lamellar morphologies. Adding salt results in the formation of a disordered window which closes and gives way to lamellae at higher salt concentrations. The width of disordered window decreases with increasing length of the POSS alkyl chain substituent from ethyl to isobutyl and is absent in the isooctyl sample. Rheological measurements demonstrate good mechanical rigidity when compared with similar all-organic block copolymers. While salt diffusion coefficient and current ratio are unaffected by substituent length, ionic conductivity increases as the length of the alkyl chain substituent decreases: the ethyl substituent is optimal for ion transport. This is surprising because conventional wisdom suggests that ion transport occurs primarily in the PEO-rich domains, that is, ion transport should be unaffected by substituent length after accounting for the minor change in conducting phase volume fraction. © 2020 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2020 © 2020 Wiley Periodicals, Inc. J. Polym. Sci. 2020 , 58, 363–371     

The [4Fe-4S](2+) cluster in reconstituted biotin synthase binds S-adenosyl-L-methionine

The combination of resonance Raman, electron paramagnetic resonance and M?ssbauer spectroscopies has been used to investigate the effect of S-adenosyl-l-methionine (SAM) on the spectroscopic properties of the [4Fe-4S]2+ cluster in biotin synthase. The results indicate that SAM interacts directly at a unique iron site of the [4Fe-4S]2+ cluster in BioB and support the hypothesis of a common inner-sphere mechanism for the reductive cleavage of SAM in the radical SAM family of Fe-S enzymes.     

Amphipols can support the activity of a membrane enzyme

Amphipathic polymers ("amphipols") were introduced several years ago (Tribet, C.; Audebert, R.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047-15050) as an alternative method for solubilizing integral membrane proteins in stable, nativelike conformations. However, direct maintenance of full membrane protein functionality in amphipol solutions has not previously been demonstrated in the absence of added lipid or detergent. In this contribution, the first zwitterionic amphipol "PMAL-B-100" is introduced. PMAL-B-100 not only maintains membrane protein structure and solubility, but also supports the full catalytic activity of an integral membrane enzyme, diacylglycerol kinase, in the complete absence of additional lipid or detergent. All of the roles which a lipid bilayer normally plays in maintaining diacylglycerol kinase's structure and in facilitating catalysis are satisfied by the environment and interactions supplied by PMAL-B-100.     

Synthesis of (r)- and (s)-1-formyl-6,7,8,9-tetrahydro- N,N-(dipropyl)-3H-benz[e]indol-8-amines: Potent and orally active 5-ht1a receptor agonists

An efficient synthesis of the potent and orally active 5-HT_1A agonists, (R)-(+)- and (S)-(-)-1-formyl-6,7,8,9-tetrahydro-N,N-dipropyl-3H-benz[e]indol-8-amines 1a and 1b , is described. This synthesis was accomplished in twelve steps from commercially available 1,5,6,7-tetrahydro-4H-indol-4-one ( 5 ). The key step involved a regio-controlled Friedel-Crafts acylation of 1-(p-toluenesulfonyl)indol-4-acetyl chloride with ethylene to yield a versatile synthon, 3-(p-toluenesulfonyl)-6,7,8,9-tetrahydro-3H-benz[e]indol-8-one ( 10 ). Subsequent coupling of this ketone with chiral α-methylbenzylamine under reductive amination conditions yielded a mixture of diastereomers. These diastereomers were efficiently separated by either chromatography or fractional recrystallization of the derived hydrochloride salts. Debenzylation of the pure diastereomers was followed by alkylation and formylation to yield (R)-(+)- and (S)-(-)-enantiomers 1a and 1b with >99% purity.     

Structural identification of SAR-943 metabolites generated by human liver microsomes in vitro using mass spectrometry in combination with analysis of fragmentation patterns

SAR-943 (32-deoxo rapamycin) is a proliferation signal inhibitor via interaction with the mammalian target of rapamycin (mTOR). Most importantly, SAR-943 has improved chemical stability compared to rapamycin (sirolimus) and is currently under investigation as a drug coated on coronary stents. It was the goal of this study to identify the SAR-943 metabolites generated after incubation with human liver microsomes using high-resolution mass spectrometry (MS) and MS/iontrap (MS(n)) and comparison of fragmentation patterns of the metabolites with those of SAR-943 and other known rapamycin derivatives. Our study showed that SAR-943 is mainly hydroxylated and/or demethylated by human liver microsomes. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl SAR-943; hydroxylated metabolites: hydroxy piperidine SAR-943, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 46-hydroxy and 49-hydroxy SAR-943; didemethylated metabolites: 16,39-O-didesmethyl and 27,39-O-didesmethyl SAR-943; demethylated-hydroxylated metabolites: 39-O-desmethyl, 23- or 24-hydroxy and 39-O-desmethyl, hydroxy piperidine SAR-943 and dihydroxylated metabolites: 12-,23- or 24-dihydroxy SAR-943. In addition, several other demethylated-hydroxylated and dihydroxylated metabolites were detected. However, their exact structures could not be identified.     

Evaluation of direct analysis in real time mass spectrometry for onsite monitoring of batch slurry reactions

Batch slurry reactions are widely used in the industrial manufacturing of chemicals, pharmaceuticals, petrochemicals and polymers. However, onsite monitoring of batch slurry reactions is still not feasible in production plants due to the challenge in analyzing heterogeneous samples without complicated sample preparation procedures. In this study, direct analysis in real time mass spectrometry (DART-MS) has been evaluated for the onsite monitoring of a model batch slurry reaction. The results suggested that automation of the sampling process of DART-MS is important to achieve quantitative results. With a sampling technique of manual sample deposition on melting point capillaries followed by automatic sample introduction across the helium beam, relative standard deviation (RSD) of the protonated molecule signals from the reaction product of the model batch slurry reaction ranged from 6 to 30%. This RSD range is improved greatly over a sampling technique of manual sample deposition followed by manual sample introduction where the RSDs are up to 110%. Furthermore, with the semi-automated sampling approach, semi-quantitative analysis of slurry samples has been achieved. Better quantification is expected with a fully automated sampling approach.