Chaperone/Polymer Complexation of Protein-Based Fluorescent Nanoclusters against Silica Encapsulation-Induced Physicochemical Stresses.

Silica encapsulation under ambient conditions is commonly used to shield protein-based nanosystems from chemical stress. However, encapsulation-induced photo- and structural instabilities at elevated temperatures have been overlooked. Using bovine serum albumin-capped fluorescent gold nanoclusters (BSA-AuNCs) as a model, we demonstrated that chaperone/polymer layer-by-layer complexation can stabilize the template to resist encapsulation-induced fragmentation/reorganization and emission increases at 37 °C or higher temperatures. We first wrapped BSA-AuNCs with α-crystallin chaperones (α-Crys) to gain the highest thermal stability at a 1:50 molar ratio and then enfolded BSA-AuNC/α-Crys with thermoresponsive poly-N-isopropylacrylamide (PNIPAM) at 60 °C to shield silica interaction and increase the chaperone-client protein accessibility. The resulting BSA-AuNC/α-Crys/PNIPAM (BαP) was encapsulated by a sol-gel process to yield BαP-Si (∼80 ± 4.5 nm), which exhibited excellent structural integrity and photostability against chemical and thermal stresses. Moreover, targeted BαP-Si demonstrated prolonged fluorescence stability for cancer cell imaging. This template stabilization strategy for silica encapsulation is biocompatible and applicable to other protein-based nanosystems.

Synthesis of Multisubstituted 1,2,3-Triazoles: Regioselective Formation and Reaction Mechanism.

A synthetically useful approach to functionalized triazoles is described via the reaction of ß-carbonyl phosphonates and azides. 1,4- and 1,5-disubstituted and 1,4,5-trisubstituted triazoles can be regio- and chemoselectively accessed under mild conditions in good to excellent yields (31 examples, up to 99%). A mechanism is proposed that rationalizes the avoidance of the 4-phosphonate byproducts, which is aligned with crystallographic and experimental evidence.

Dimethyl Labeling Coupled with Mass Spectrometry for Topographical Characterization of Primary Amines on Monoclonal Antibodies.

Site-specific solvent accessibility of the primary amines (mainly lysine or the N-termini) on proteins is of great interest in many research areas because amines are an important functional group for protein conjugation. In this study, we coupled dimethyl labeling via reductive amination with liquid chromatography-mass spectrometry (LC-MS) to fully characterize the solvent accessibility of lysine residues and the N-termini on human immunoglobulin G (IgG). Circular dichroism (CD) and fluorescence spectroscopy revealed that dimethyl labeling did not alter the conformation of the native IgG molecule. Based on intact protein measurements, up to 28 (light chain) and 66 (heavy chain) dimethyl tags, covering all lysine residues and the N-termini, were sequentially incorporated into IgG molecules in 1000 s. All labeled sites were identified and quantified by a bottom-up proteomics approach. Some highly exposed hot-spots (for example, the N-termini of both the heavy and the light chains) and some buried sites (for example, K415 in the heavy chain and K39 in the light chain) were unambiguously revealed. This method was also used to characterize aggregation-induced structural changes in IgGs by increasing the temperature. Substantial changes in the labeling percentage of many lysine sites were observed, indicating a non-native aggregation triggered by thermal stress. Due to high labeling yields and the van der Waals surface of the labeling reagents being comparable to that of water, dimethyl labeling is a highly promising technique for probing the amine's surface topography of proteins. It can also be used as a complementary approach to other methods for resolving the higher-order structure of proteins by LC-MS.

A short synthesis of (±)-antroquinonol in an unusual scaffold of 4-hydroxy-2-cyclohexenone.

Antroquinonol, which was first isolated from a mushroom, Antrodia cinnamomea, found in Taiwan, is an anticancer compound with a unique core structure of 4-hydroxy-2,3-dimethoxycyclohex-2-enone carrying methyl, farnesyl and hydroxyl substituents in the 4,5-cis-5,6-trans configuration. A short synthesis of (±)-antroquinonol is accomplished in seven steps from 2,3,4-trimethoxyphenol, which is oxidized in methanol to a highly electron-rich substrate of 2,3,4,4-tetramethoxycyclohexadienone and then a Michael reaction with dimethylcuprate is performed as the key step, followed by alkylation, reduction and epimerization to incorporate the required substituents at three contiguous stereocenters.

Conversion of azides into diazo compounds in water.

Diazo compounds are in widespread use in synthetic organic chemistry but have untapped potential in chemical biology. We report on the design and optimization of a phosphinoester that mediates the efficient conversion of azides into diazo compounds in phosphate buffer at neutral pH and room temperature. High yields are maintained in the presence of common nucleophilic or electrophilic functional groups, and reaction progress can be monitored by colorimetry. As azido groups are easy to install and maintain in biopolymers or their ligands, this new mode of azide reactivity could have substantial utility in chemical biology.

Tuning the regio- and stereoselectivity of C-H activation in n-octanes by cytochrome P450 BM-3 with fluorine substituents: evidence for interactions between a C-F bond and aromatic π systems.

We employed the water-soluble cytochrome P450 BM-3 to study the activity and regiospecificity of oxidation of fluorinated n-octanes. Three mutations, A74G, F87V, and L188Q, were introduced into P450 BM-3 to allow the system to undergo n-octane oxidation. In addition, the alanine at residue 328 was replaced with a phenylalanine to introduce an aromatic residue into the hydrophobic pocket to examine whether or not van der Waals interactions between a C-F substituent in the substrate and the polarizable π system of the phenylalanine may be used to steer the positioning of the substrate within the active-site pocket of the enzyme and control the regioselectivity and stereoselectivity of hydroxylation. Interestingly, not only was the regioselectivity controlled when the fluorine substituent was judiciously positioned in the substrate, but the electron input into the iron-heme group became tightly coupled to the formation of product, essentially without abortive side reactions. Remarkable enhancement of the coupling efficiency between electron input and product formation was observed for a range of fluorinated octanes in the enzyme even without the A328F mutation, presumably because of interactions of the C-F substituent with the π system of the porphyrin macrocycle within the active-site pocket. Evidently, tightening the protein domain containing the heme pocket tunes the distribution of accessible enzyme conformations and the associated protein dynamics that activate the iron porphyrin for substrate hydroxylation to allow the reactions mediated by the high-valent Fe(IV)=O to become kinetically more commensurate with electron transfer from the flavin adenine dinucleotide (FAD)/flavin mononucleotide (FMN) reductase. These observations lend compelling evidence to support significant van der Waals interactions between the CF(2) group and aromatic π systems within the heme pocket when the fluorinated octane substrate is bound.

Novel N-transfer reagent for converting α-amino acid derivatives to α-diazo compounds.

A novel universal N-transfer reagent for direct and effective transformation of α-amino ketones, acetamides, and esters to the corresponding α-diazo products under mild basic conditions has been developed. This one-step synthetic approach not only allows for generation of α-substituted-α-diazo carbonyl compounds from α-amino acid derivatives but also permits preparation of α-diazo dipeptides from N-terminal dipeptides (32 examples, up to 91%).

Selective and predicable amine conjugation sites by kinetic characterization under excess reagents.

The site selectivity for lysine conjugation on a native protein is difficult to control and characterize. Here, we applied mass spectrometry to examine the conjugation kinetics of Trastuzumab-IgG (Her-IgG) and α-lactalbumin under excess linker concentration ([L]0) based on the modified Michaelis-Menten equation, in which the initial rate constant per amine (kNH2 = Vmax/NH2/KM) was determined by the maximum reaction rate (Vmax/NH2) under saturated accessible sites and initial amine-linker affinity (1/KM). Reductive amination (RA) displayed 3-4 times greater Vmax/NH2 and a different panel of conjugation sites than that observed for N-hydroxysuccinimide ester (NHS) chemistry using the same length of polyethylene glycol (PEG) linkers. Moreover, faster conversion power rendered RA site selectivity among accessible amine groups and a greater tunable range of linker/protein ratio for aldehyde-linkers compared to those of the same length of NHS-linkers. Single conjugation with high yield or poly-conjugations with site homogeneity was demonstrated by controlling [L]0 or gradual addition to minimize the [L]0/KM ratio. Formaldehyde, the shortest aldehyde-linker with the greatest 1/KM, exhibited the highest selectivity and was shown to be a suitable probe to predict conjugation profile of aldehyde-linkers. Four linkers on the few probe-predicted hot spots were elucidated by kinetically controlled RA with conserved drug efficacy when conjugated with the payload. This study provides insights into controlling factors for homogenous and predictable amine bioconjugation.

Camphor sulfonyl hydrazines (CaSH) as organocatalysts in enantioselective Diels-Alder reactions.

Cyclic sulfonyl hydrazine was demonstrated for the first time as a new functionality for organocatalysis. A series of six-membered cyclic hydrazines derived from camphor sulfonic acid were synthesized. With trichloroacetic acid as cocatalyst, they are efficient organocatalysts for enantioselective Diels-Alder reactions with good chemical yields and up to 96% ee. The reactions took place in brine at 0 degrees C to room temperature.

Polyene cyclization promoted by the cross-conjugated alpha-carbalkoxy enone system. Observation on a putative 1,5-hydride/1,3-alkyl shift under Lewis acid catalysis.

Polyene cyclization of compounds 3 and 4 under catalysis with AlCl3 and/or SnCl4 gave rise to complex bicyclic products 8 and 9, structures of which were highly unexpected, and X-ray analyses were invoked for unambiguously structural identification. Mechanistically, a tandem sigma-bond rearrangement process, including an unusual through-space 1,5-hydride or 1,3-alkyl shift as a key operation, is proposed.

Polyene cyclization promoted by the cross-conjugated alpha-carbalkoxy cyclohexenone system. An unusual 1,2-hydride shift under Lewis acid catalysis.

Polyene cyclization of the titled compounds under catalysis with AlCl(3)/SnCl(4) gave rise to the corresponding polycyclic products, many of which were structurally highly unexpected, and thus, individual X-ray analysis was required to finalize the structural identification. Mechanistically, an unusual 1,2-hydride shift is proposed to elucidate the product formation.