Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection.

N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.

Chromatographic evidence of silyl ether formation (SEF) in supercritical fluid chromatography.

In this article, we propose that silyl ether formation (SEF) is a major contribution to retention and selectivity variation over time for supercritical fluid chromatography (SFC). In the past, the variations were attributed to instrumentation, but high performance SFC systems have shed new light on the source of variation. As silyl ethers form on the particle surface, the hydrophilicity is decreased, significantly altering the retention and selectivity observed. SEF is expected to occur with any chromatographic particle containing silanols but is slowed on hybrid inorganic/organic particles. The SEF reaction is between alcohols on the particle surface and in the mobile phase solvent. We have found that storage conditions of a column are paramount, which can either prevent or accelerate the process. Because SEF exists as an equilibrium between the liquid phase and the particle surface, the process is also reversible. The silanols can be hydroxylated (regenerated) to their original state upon exposure to water. The next generation of stationary phases will either advantageously utilize SEF or effectively mitigate its effects. Mitigation of SEF would be a significant improvement in SFC that has the potential to vault their performance to levels of similar reproducibility and reliability observed for high performance liquid chromatography (HPLC). Further research in SEF may lead to a better understanding of the mechanism of interaction between the solutes and chromatographic surface.

Functionalization of divinylbenzene/N-vinylpyrrolidone copolymer particles: ion exchangers for solid phase extraction.

A series of four-mixed mode ion exchangers for SPE, consisting of either weak or strong cation or anion exchangers, have been synthesized by functionalization of spherical, porous particles made from a copolymer of N-vinylpyrrolidone and divinylbenzene. These materials are able to selectively retain and release acidic and basic solutes through the judicious choice of wash solvent pH, as shown through the use of SPE recovery tests.

Photohydration and photosolvolysis of biphenyl alkenes and alcohols via biphenyl quinone methide-type intermediates and diarylmethyl carbocations.

Evidence is presented for the photochemical generation of novel biphenyl quinone methide (BQM)-type intermediates on photolysis of hydroxybiphenyl alkenes 7 and 8 and hydroxybiphenyl alcohols 9 and 10. Mechanistic investigations utilizing product, fluorescence, and nanosecond laser flash photolysis (LFP) studies indicate two distinct pathways for the formation of these BQMs depending upon the functional groups of the progenitor. Formal excited-state intramolecular proton transfer (ESIPT) between the phenol and the alkene led to BQMs upon irradiation of the hydroxybiphenyl alkenes 7 and 8, while excited-state proton transfer (ESPT) to solvent followed by dehydroxylation was responsible for BQM formation from the hydroxybiphenyl alcohols 9 and 10. Photolysis of 7 and 8 in aqueous CH(3)CN gave photohydration products via attack of water on the respective BQMs, while photolysis of the analogous methyl ethers (of the phenolic moiety) gave only carbocation intermediates. Hydroxybiphenyl alcohols 9 and 10 yielded the corresponding photomethanolysis products in aqueous methanol, through attack of CH(3)OH on the respective BQMs. Although no evidence was found for BQM formation in LFP studies of 8 and 10, due to its suspected short lifetime, the respective diaryl carbocation (lambda(max) 420 nm, tau = 8.5 micros) has been observed upon irradiation of 8 in 2,2,2-trifluoroethanol. A BQM (lambda(max) 580 nm) was observed for 9 but not for 10, the latter having more complex chemistry on laser excitation, resulting in a transient that appears to mask any BQM absorption. Significant quenching of fluorescence from the hydroxybiphenyl alkenes at low water content implies that H(2)O is directly involved in reaction from the singlet excited state. The decrease in fluorescence intensity of 8 was found to depend on [H(2)O](3); however, the distance required for ESIPT in these systems is too large to be bridged by a water trimer. The nonlinear quenching has been attributed to deprotonation of the phenol by two water molecules, with concerted protonation at the alkene by another molecule of water. Fluorescence quenching of the hydroxybiphenyl alcohols required much higher water content, implying a different mechanism of reaction, consistent with the proposal of ESPT (to solvent water) followed by dehydroxylation.

Two-photon excited intramolecular energy transfer and light-harvesting effect in novel dendritic systems.

Two-photon absorption excited intramolecular energy transfer and light-harvesting effects are demonstrated in three novel dendritic systems. These systems contain both an antenna structure that can effectively absorb two-photon energy at approximately 800 nm and emit fluorescence at approximately 515 nm and a core moiety that can absorb one-photon energy at approximately 520 nm and emit at approximately 590 nm. Covalently combining the core and antenna functionalities intrinsically changes the optical behavior of the component pieces. The two-photon energy absorbed by the antenna structure is resonantly transferred to the core, where the core's emission intensity is enhanced by 8, 20, and 34 times for the three dendritic systems.

Fluorescence resonance energy transfer in a novel two-photon absorbing system.

A novel fluorescence resonance energy transfer (FRET) system containing a two-photon absorbing dye and a nile red chromophore has been synthesized. Upon two-photon excitation by laser at 815 nm this molecule displays efficient energy transfer from the two-photon absorbing dye to the nile red moiety, with an 8-fold increase in emission compared to the model compound. Similarly, single-photon excitation of the two-photon absorbing moiety at 405 nm results in >99% energy-transfer efficiency, along with a 3.4-fold increase in nile red emission compared to direct excitation of the nile red chromophore at 540 nm. This system provides an effective way to use IR radiation to excite molecules that, by themselves, have little or no two-photon absorption.

A FRET-based ultraviolet to near-infrared frequency converter.

The synthesis and photophysical characterization of a generation 1 dendrimer containing four coumarin 2 laser dyes at the periphery and a perylenebis(dicarboximide) derivative at the core are described. It was found that 99% of the UV light absorbed by the peripheral coumarin 2 donor chromophores is transferred through fluorescence resonance energy transfer (FRET) to the core acceptor. Emission from the core via FRET is observed in the near-infrared with a 6.2-fold amplification relative to direct excitation.

Cascade energy transfer in a conformationally mobile multichromophoric dendrimer.

A conformationally flexible, generation-2,3 poly(aryl ether) dendrimer favors quantitative cascade fluorescence resonance energy transfer without the appearance of undesired chromophore self-quenching interactions such as excimer formation.