Travelling‐wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N‐glycans

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1‐acid glycoprotein and thyroglobulin were measured with a travelling‐wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man₃GlcNAc₃ from chicken ovalbumin and Man₃GlcNAc₃Fuc₁ from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision‐induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high‐mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high‐mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility‐m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.     

Evidence for a SN2-type pathway for phosphine exchange in phosphine-phosphenium cations, [R2P--PR'3]+

Abstraction of a Cl(-) ion from the P-chlorophospholes, R4C4PCl (R=Me, Et), produced the P--P bonded cations [R4C4P--P(Cl)C4R4]+, which reacted with PPh3 to afford X-ray crystallographically characterised phosphine-phosphenium cations [R4C4P(PPh3)]+ (R=Me, Et). Examination of the 31P-{1H} NMR spectrum of a solution (CH2Cl(2)) of [Et4C4P-(PPh3)]+ and PPh3 revealed broadening of the resonances due to both free and coordinated PPh3, and importantly it proved possible to measure the rate of exchange between PPh3 and [Et4C4P-(PPh3)]+ by line shape analysis (gNMR programmes). The results established second-order kinetics with DeltaS( not equal)=(-106.3+/-6.7) J mol(-1) K(-1), DeltaH( not equal)=(14.9+/-1.6) kJ mol(-1) and DeltaG( not equal) (298.15 K)=(46.6+/-2.6) kJ mol(-1), values consistent with a SN2-type pathway for the exchange process. This result contrasts with the dominant dissociative (S(N)1-type) pathway reported for the analogous exchange reactions of the [ArNCH2CH2N(Ar)P(PMe3)]+ ion, and to understand in more detail the factors controlling these two different reaction pathways, we have analysed the potential energy surfaces using density functional theory (DFT). The calculations reveal that, whilst phosphine exchange in [Et4C4P(PPh3)]+ and [ArNCH2CH2N(Ar)P(PMe3)](+) is superficially similar, the two cations differ significantly in both their electronic and steric requirements. The high electrophilicity of the phosphorus center in [Et4C4P]+, combined with strong pi-pi interactions between the ring and the incoming and outgoing phenyl groups of PPh3, favours the SN2-type over the SN1-type pathway in [Et4C4P(PPh3)]+. Effective pi-donation from the amide groups reduces the intrinsic electrophilicity of [ArNCH2CH2N(Ar)P]+, which, when combined with the steric bulk of the aryl groups, shifts the mechanism in favour of a dissociative SN1-type pathway.     

Enzyme mediated incorporation of zirconium-89 or copper-64 into a fragment antibody for same day imaging of epidermal growth factor receptor

Identification of tumors which over-express Epidermal Growth Factor Receptor (EGFR) is important in selecting patients for anti-EGFR therapies. Enzymatic bioconjugation was used to introduce positron-emitting radionuclides (⁸⁹Zr, ⁶⁴Cu) into an anti-EGFR antibody fragment for Positron Emission Tomography (PET) imaging the same day as injection. A monovalent antibody fragment with high affinity for EGFR was engineered to include a sequence that is recognized by the transpeptidase sortase A. Two different metal chelators, one for ⁸⁹Zr^IV and one for ⁶⁴Cu^II, were modified with a N-terminal glycine to enable them to act as substrates in sortase A mediated bioconjugation to the antibody fragment. Both fragments provided high-quality PET images of EGFR positive tumors in a mouse model at 3 hours post-injection, a significant advantage when compared to radiolabeled full antibodies that require several days between injection of the tracer and imaging. The use of enzymatic bioconjugation gives reproducible homogeneous products with the metal complexes selectively installed on the C-terminus of the antibody potentially simplifying regulatory approval.

Enzymatic bioconjugation to introduce positron-emitting radionuclides (⁸⁹Zr, ⁶⁴Cu) into an anti-EGFR antibody fragment allows same day imaging with positron emission tomography.     

Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional μ/δ-Opioid Agonist–Neurokinin Antagonist Peptidomimetics

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic μ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂) and the dual μ/δ opioid agonist H-Dmt-d-Arg-Aba-βAla-NH₂ (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range μ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.     

Combined Subcutaneous Fat Aspirate and Skin Tru-Cut Biopsy for Amyloid Screening in Patients with Suspected Systemic Amyloidosis

Screening for systemic amyloidosis is typically carried out with abdominal fat aspirates with varying reported sensitivities. Fat aspirates are preferred for use in primary screening instead of organ biopsies as they are less invasive and thereby minimize the potential risk of complications. At Odense Amyloidosis Center, we performed a prospective study on whether the combined use of fat aspirate and tru-cut skin biopsy could increase the diagnostic sensitivity. Both fat aspirates and skin biopsies were screened with Congo Red staining, and positive biopsies were subsequently subtyped using immunoelectron microscopy and mass spectrometry. Seventy-six patients were included. In total, 24 patients had systemic amyloidosis (11 AL, 12 wtATTR, 1 AA), and 6 patients had localized amyloidosis. Combined fat aspirate and skin biopsy were Congo Red-positive in 15 patients (overall sensitivity (OS) 62.5%). Fat aspirates were positive in 14 patients (OS 58.3%), and the skin biopsy was positive in 5 patients (OS 20.8%). In only one patient did the skin biopsy add extra diagnostic information. The sensitivity differed between AL and ATTR amyloidosis—81.8% and 41.7%, respectively. Using skin biopsy as the only screening method is not recommended.     

Inertial focusing of microparticles,bacteria, and blood in serpentine glass channels

Early detection of pathogenic microorganisms is pivotal to diagnosis and prevention of health and safety crises. Standard methods for pathogen detection often rely on lengthy culturing procedures, confirmed by biochemical assays, leading to >24 h for a diagnosis. The main challenge for pathogen detection is their low concentration within complex matrices. Detection of blood-borne pathogens via techniques such as PCR requires an initial positive blood culture and removal of inhibitory blood components, reducing its potential as a diagnostic tool. Among different label-free microfluidic techniques, inertial focusing on microscale channels holds great promise for automation, parallelization, and passive continuous separation of particles and cells. This work presents inertial microfluidic manipulation of small particles and cells (1–10 μm) in curved serpentine glass channels etched at different depths (deep and shallow designs) that can be exploited for (1) bacteria preconcentration from biological samples and (2) bacteria-blood cell separation. In our shallow device, the ability to focus Escherichia coli into the channel side streams with high recovery (89% at 2.2× preconcentration factor) could be applied for bacteria preconcentration in urine for diagnosis of urinary tract infections. Relying on differential equilibrium positions of red blood cells and E. coli inside the deep device, 97% red blood cells were depleted from 1:50 diluted blood with 54% E. coli recovered at a throughput of 0.7 mL/min. Parallelization of such devices could process relevant volumes of 7 mL whole blood in 10 min, allowing faster sample preparation for downstream molecular diagnostics of bacteria present in bloodstream.     

An Aldehyde Responsive,Cleavable Linker for Glucose Responsive Insulins

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.     

Dual Electroactivity in a Covalent Organic Network with Mechanically Interlocked Pillar[5]arenes

The synthesis and characterization of a polyrotaxanated covalent organic network (CON) based on the association between the viologen and pillar[5]arene (P[5]OH) units are reported. The mechanical bond allows for the irreversible insertion of n-type redox centers (P[5]OH macrocycles) within a pristine structure based on p-type viologen redox centers. Both redox units are active on a narrow potential range and, in water, the presence of P[5]OH greatly increases the electroactivity of the material.     

Measurability in modules

In this paper we prove that in modules, MS-measurability (in the sense of Macpherson–Steinhorn) depends on being able to define a measure function on the p.p. definable subgroups. We give a classification of abelian groups in terms of measurability. Finally we discuss the relation with \({\mathbb{Q}[t]}\) -valued measures.