J. Bruce Pitner

Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein.

The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor. By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants. The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation. The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP. Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM). One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5mM. These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices.

Engineering and rapid selection of a low‐affinity glucose/galactose‐binding protein for a glucose biosensor

Periplasmic expression screening is a selection technique used to enrich high‐affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D‐glucose/D‐galactose‐binding protein (GGBP) having low affinity to glucose. Wild‐type GGBP has an equilibrium dissociation constant of 0.2 μM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7–33 mM). To accomplish this a focused library was constructed using structure‐based site‐saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H‐bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site‐specifically at E149C with the fluorophore IANBD (N,N′‐dimethyl‐N‐(iodoacetyl)‐N′‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)ethylene‐diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000‐fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H‐bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure‐based site‐saturation mutagenesis and periplasmic expression screening to discover low‐affinity GGBP mutants having potential utility for measuring glucose in humans.

Multi-day pre-clinical demonstration of glucose/galactose binding protein-based fiber optic sensor.

We report here the first pre-clinical demonstration of continuous glucose tracking by fluorophore-labeled and genetically engineered glucose/galactose binding protein (GGBP). Acrylodan-labeled GGBP was immobilized in a hydrogel matrix at the tip of a small diameter optical fiber contained in a stainless steel needle. The fiber optic biosensors were inserted subcutaneously into Yucatan and Yorkshire swine, and the sensor response to changing glucose levels was monitored at intervals over a 7-day period. Sensor mean percent error on day 7 was 16.4±5.0% using a single daily reference blood glucose value to calibrate the sensor. The GGBP sensors susceptibility to common interferents was tested in a well-plate system using human sera. No significant interference was observed from the tested interferents except for tetracycline at the drugs maximum plasma concentration. The robust performance of the GGBP-based fiber optic sensor in swine models and resistance to interferents indicates the potential of this technology for continuous glucose monitoring in humans.

Spectroscopic studies of YO and YOYO fluorescent dyes in a thrombin-binding DNA ligand

The fluorescent, oxazole yellow dye YO-Pro-1 iodide (YO) and its homodimer YOYO-1 iodide (YOYO) were studied in a thrombin-binding DNA ligand, or aptamer, (tb-ligand) and in an oligomer with the same base composition in a scrambled sequence (s-ligand), both single strands of 15 bases in length. Binding constants for the dye-ligand complexes, assuming 1:1 stoichiometry, were determined to be on the order of 107M−1 for YOYO and 105M−1 for YO, which are approximately 105 smaller than estimated constants for YOYO in double-stranded DNA. In both ligands, YOYO assumes a folded conformation that promotes stability of the dye-ligand complex and excitonic coupling between the two YO groups. The folded conformation provides greater overlap of the YO groups than has been reported for YOYO in double-stranded DNA; the overlap is slightly greater in tb-ligand than in s-ligand. Both dyes exhibit bi-exponential fluorescence decay in the ligands and the lifetimes of YOYO (3–4 ns and 7–8 ns) are longer and more discrete than those of YO (1–3 ns and 4–5 ns). Fluorescence anisotropy of YOYO is a low, constant value in both ligands due to intramolecular energy transfer between the overlapping YO groups. Higher anisotropies are observed for YO, and the value is slightly higher in s-ligand than in tb-ligand. The addition of thrombin to the s-ligand affects the fluorescence intensity and anisotropy of YO but not of YOYO. The absence of intermolecular G-quartet formation of the s-ligand was demonstrated. This suggests that thrombin interacts weakly with the s-ligand but is not sensed by the fluorescence of YOYO, which is dominated by the coupling between the YO groups in the folded conformation of the bound dye. The results of these studies have implications for the application of these dyes for detection of single-stranded DNA ligands and their binding interactions.

Evaluation of the relative stability of liganded versus ligand‐free protein conformations using Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method

Many proteins change their conformation upon ligand binding. For instance, bacterial periplasmic binding proteins (bPBPs), which transport nutrients into the cytoplasm, generally consist of two globular domains connected by strands, forming a hinge. During ligand binding, hinge motion changes the conformation from the open to the closed form. Both forms can be crystallized without a ligand, suggesting that the energy difference between them is small. We applied Simplicial Neighborhood Analysis of Protein Packing (SNAPP) as a method to evaluate the relative stability of open and closed forms in bPBPs. Using united residue representation of amino acids, SNAPP performs Delaunay tessellation of the protein, producing an aggregate of space‐filling, irregular tetrahedra with nearest neighbor residues at the vertices. The SNAPP statistical scoring function is derived from log‐likelihood scores for all possible quadruplet compositions of amino acids found in a representative subset of the Protein Data Bank, and the sum of the scores for a given protein provides the total SNAPP score. Results of scoring for bPBPs suggest that in most cases, the unliganded form is more stable than the liganded form, and this conclusion is corroborated by similar observations of other proteins undergoing conformation changes upon binding their ligands. The results of these studies suggest that the SNAPP method can be used to predict the relative stability of accessible protein conformations. Furthermore, the SNAPP method allows delineation of the role of individual residues in protein stabilization, thereby providing new testable hypotheses for rational site‐directed mutagenesis in the context of protein engineering. Proteins 2004.

Bioconjugatable, PEGylated hydroporphyrins for photochemistry and photomedicine. Narrow-band, near-infrared-emitting bacteriochlorins

Synthetic bacteriochlorins absorb in the near-infrared (NIR) region and are versatile analogues of natural bacteriochlorophylls. The utilization of these chromophores in energy sciences and photomedicine requires the ability to tailor their physicochemical properties, including the incorporation of units to impart water solubility. Herein, we report the synthesis, from two common bacteriochlorin building blocks, of five wavelength-tunable, bioconjugatable and water-soluble bacteriochlorins along with two non-bioconjugatable benchmarks. Each bacteriochlorin bears short polyethylene glycol (PEG) units as the water-solubilizing motif. The PEG groups are located at the 3,5-positions of aryl groups at the pyrrolic β-positions to suppress aggregation in aqueous media. A handle containing a single carboxylic acid is incorporated to allow bioconjugation. The seven water-soluble bacteriochlorins in water display Qy absorption into the NIR range (679-819 nm), sharp emission (21-36 nm full-width-at-half-maximum) and modest fluorescence quantum yield (0.017-0.13). Each bacteriochlorin is neutral (non-ionic) yet soluble in organic (e.g., CH2Cl2, DMF) and aqueous solutions. Water solubility was assessed using absorption spectroscopy by changing the concentration ∼1000-fold (190-690 µM to 0.19-0.69 µM) with a reciprocal change in pathlength (0.1-10 cm). All bacteriochlorins showed excellent solubility in water, except for a bacteriochlorin-imide that gave slight aggregation at higher concentrations. One bacteriochlorin was conjugated to a mouse polyclonal IgG antibody for use in flow cytometry with compensation beads for proof-of-principle. The antibody conjugate of B2-NHS displayed a sharp signal upon ultraviolet laser excitation (355 nm) with NIR emission measured with a 730/45 nm bandpass filter. Overall, the study gives access to a set of water-soluble bacteriochlorins with desirable photophysical properties for use in multiple fields.

Bioconjugatable, PEGylated hydroporphyrins for photochemistry and photomedicine. Narrow-band, red-emitting chlorins

Chromophores that absorb and emit in the red spectral region (600-700 nm), are water soluble, and bear a bioconjugatable tether are relatively rare yet would fulfill many applications in photochemistry and photomedicine. Here, three molecular designs have been developed wherein stable synthetic chlorins - analogues of chlorophylls - have been tailored with PEG groups for use in aqueous solution. The designs differ with regard to order of the installation (pre/post-formation of the chlorin macrocycle) and position of the PEG groups. Six PEGylated synthetic chlorins (three free bases, three zinc chelates) have been prepared, of which four are equipped with a bioconjugatable (carboxylic acid) tether. The most effective design for aqueous solubilization entails facial encumbrance where PEG groups project above and below the plane of the hydrophobic disk-like chlorin macrocycle. The chlorins possess strong absorption at ~400 nm (B band) and in the red region (Qy band); regardless of wavelength of excitation, emission occurs in the red region. Excitation in the ~400 nm region thus provides an effective Stokes shift of >200 nm. The four bioconjugatable water-soluble chlorins exhibit Qy absorption/emission in water at 613/614, 636/638, 698/700 and 706/710 nm. The spectral properties are essentially unchanged in DMF and water for the facially encumbered chlorins, which also exhibit narrow Qy absorption and emission bands (full-width-at-half maximum of each <25 nm). The water-solubility was assessed by absorption spectroscopy over the concentration range ~0.4 μM - 0.4 mM. One chlorin was conjugated to a mouse polyclonal IgG antibody for use in flow cytometry with compensation beads for proof-of-principle. The conjugate displayed a sharp signal when excited by a violet laser (405 nm) with emission in the 620-660 nm range. Taken together, the designs described herein augur well for development of a set of spectrally distinct chlorins with relatively sharp bands in the red region.

Fluorescence Resonance Energy Transfer Glucose Sensor from Site-Specific Dual Labeling of Glucose/Galactose Binding Protein Using Ligand Protection

Background: Site-selective modification of proteins at two separate locations using two different reagents is highly desirable for biosensor applications employing fluorescence resonance energy transfer (FRET), but few strategies are available for such modification. To address this challenge, sequential selective modification of two cysteines in glucose/galactose binding protein (GGBP) was demonstrated using a technique we call “ligand protection.” Method: In this technique, two cysteines were introduced in GGBP and one cysteine is rendered inaccessible by the presence of glucose, thus allowing sequential attachment of two different thiol-reactive reagents. The mutant E149C/A213C/L238S was first labeled at E149C in the presence of the ligand glucose. Following dialysis and removal of glucose, the protein was labeled with a second dye, either Texas Red (TR) C5 bromoacetamide or TR C2 maleimide, at the second site, A213C. Results: Changes in glucose-dependent fluorescence were observed that were consistent with FRET between the nitrobenzoxadiazole and TR fluorophores. Comparison of models and spectroscopic properties of the C2 and C5 TR FRET constructs suggests the greater rigidity of the C2 linker provides more efficient FRET. Conclusions: The ligand protection strategy provides a simple method for labeling GGBP with two different fluorophores to construct FRET-based glucose sensors with glucose affinity within the human physiological glucose range (1–30 mM). This general strategy may also have broad utility for other protein-labeling applications.

Synthesis and stereoselective reduction of (±)-, (+)- and (–)-6-substituted-6-azabicyclo[3.2.1]octan-3-one

Starting with 6-oxabicyclo[3.2.1]oct-3-en-7-one 6, a three step, general synthetic route to both racemic and optically active 6-substituted 6-azabicyclo[3.2.1]octan-3-ones has been developed. Opening of the lactone ring of 6 with amines gave amides which were reduced with lithium aluminium hydride to amino alcohols. Allylic oxidation of amino alcohols 8a, 8b, 12 and 13 with manganese dioxide provided the bicyclic ketones 1b, 1c, 14 and 15, respectively, without isolation of the intermediate monocyclic ketones. Methods for stereoselective reduction of the bicyclic ketones to the corresponding 6-substituted 6-azabicyclo[3.2.1]octan-3α-ols and -3β-ols have been developed. Displacement of the R-α-methylbenzyl chiral auxiliary from the diastereomeric alcohols 16, 17, and 20, 21 by catalytic debenzylation followed by reductive amination provided the optically active 6-methyl-6-azabicyclo [3.2.1]octan-3-ols 1d–1e, respectively. The absolute stereochemistry of all reported optically active compounds has been established by comparison of diastereoisomers 10 and 11 with the R-(+)-methylbenzylamine amides derived from optically enriched lactone 6.

A concise synthesis of (±)-, (+)-, and (–)-6-methyl-6-azabicyclo[3.2.l]octan-3α-ol

(±)-6-Methyl-6-azabicyclo[3.2.1]octan-3-one (1c) was prepared in three steps from 6-oxabicyclo[3.2.1]oct-3-en-7-one, and stereoselective reduction of (1c) provided (±)-6-methyl-6-azabicyclo[3.2.1]octan-3α-ol (1a); adaptation of the sequence provided the first synthesis of (+)- and (–)-(1a).