Antibodies are engineered by replacing one or more amino acids of a parent antibody with non cross-linked, highly reactive cysteine amino acids. Antibody fragments may also be engineered with one or more cysteine amino acids to form cysteine engineered antibody fragments (ThioFab). Methods of design, preparation, screening, and selection of the cysteine engineered antibodies are provided. Cysteine engineered antibodies (Ab), optionally with an albumin-binding peptide (ABP) sequence, are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered antibody-drug conjugates having Formula I: Ab-(L-D)p I where p is 1 to 4. Diagnostic and therapeutic uses for cysteine engineered antibody drug compounds and compositions are disclosed.

Cysteine engineered antibodies and conjugates

Small-molecule fluorophores manifest the ability of chemistry to solve problems in biology. As we noted in a previous review (Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–155), the extant collection of fluorescent probes is built on a modest set of “core” scaffolds that evolved during a century of academic and industrial research. Here, we survey traditional and modern synthetic routes to small-molecule fluorophores and highlight recent biological insights attained with customized fluorescent probes. Our intent is to inspire the design and creation of new high-precision tools that empower chemical biologists.

Bright Building Blocks for Chemical Biology

Cholesterol and sphingomyelin form together a highly ordered membrane phase, which is believed to play important biological functions in plasma membranes of mammalian cells. Since sphingomyelin is ...

Switchable nile red-based probe for cholesterol and lipid order at the outer leaflet of biomembranes.

The present invention provides a microfluidic system for fast, accurate and low cost electrophoretic analysis of materials in the fields of chemistry, biochemistry, biotechnology, molecular biology and numerous other fields. Light from periodically spaced regions along a channel in the microfluidic system are received by a photodetector. The intensity of light received by the photodetector is modulated by the movement of species bands through the channel under electrophoretic forces. By Fourier analysis, the velocity of each species band is determined and the identification of the species is made by its electrophoretic mobility in the channel.

Fourier detection of species migrating in a microchannel

Benzophenoxazine-based fluorescent dyes for labeling biomolecules

A series of novel functionalised benzophenoxazinones,
analogues of Nile Red, is prepared and their fluorescence
properties evaluated. The ring system is prepared by reaction of
5-diethylamino-2-nitrosophenol with 1,6-dihydroxynaphthalene
followed by alkylation of the 2-hydroxy group with
6-bromohexanoic acid derivatives. Subsequent ester cleavage under
a variety of conditions gives the acid 6. Replacement of the
9-diethylamino group with the
N-ethyl-(3-sulfonylpropyl)amino group is carried out to
increase water solubility and the resulting dye 14 has similar
fluorescence properties. The acid 6 is coupled to a range of
amino compounds.

Synthesis of functionalised fluorescent dyes and their coupling to amines and amino acids

A method of separating or distinguishing carbohydrate substances, by the use of a fluorescent labelling reagent which has a positive charge when bound to a carbohydrate, involves separating or distinguishing labelled carbohydrate substances, e.g. by performing electrophoresis to cause differential migration of different labelled carbohydrate substances, or by isoelectric focusing in a pH gradient. Preferably the fluorescent labelling reagent is a cyanine dye.

Analysis of carbohydrates

Disclosed is a method for increasing the fluorescence of a Cyanine dye molecule comprising at least one NO2 group characterised by the reduction of the at least one NO2 group to NHOH or NH2 by the action of a nitroreductase. The cyanine dye molecule comprising at least one NO2 group can be used as a substrate for detecting nitroreductase enzyme activity in a composition and allows for the use of a nitroreductase enzyme in an enzyme-reporter system for the detection of analytes, binding reactions or gene expression.

Fluorescent detection method and reagent

Methods employing increased dye-fluorescence upon nitroreduction

Deprotonation of 2-phenylthiotetrahydrothiopyran 10 and addition of the epoxide 14 gave the 2-alkyl-tetrahydrothiopyrans 16a, b. Deprotection, followed by cyclisation using silver nitrate and triethylamine in aqueous acetonitrile, gave the monothiospiroacetals 18 and 19(93%), ratio 67:33. The monothiospiroacetals 29 and 30 were similarly obtained from 2-phenylthiotetrahydrothiopyran 26. The formation of dithiospiroacetals from 2-phenylthiotetrahydrothiopyrans was inefficient. However, the 2H-3,4-dihydrothiopyran 25 was alkylated using epoxides 14 and 56, and the products converted into the dithiospiroacetals 48 and 50, and 62 and 63, respectively. The 2H-dihydrothiopyran 65 was similarly alkylated using the epoxide 14, and the product taken through to provide the dithiospiroacetal 71. In each case the major product was that expected on the basis of the anomeric effect. The structures of (7RS,11RS)-1,4,8-trioxa-13-thiadispiro[4.1.5.3]pentadecan-11-ol 18 and the p-nitrobenzoyloxydithiospiroacetal 49, were established by X-ray crystallography.

Synthesis of thiospiroacetals. X-Ray crystal structure determinations for (7RS,11RS)-1,4,8-trioxa-13-thiadispiro[4.1.5.3]pentadecan-11-ol and (4RS,6SR)-4-p-nitrobenzoyloxy-1,7-dithiaspiro[5.5]undecane

Mark Samuel Jonathan Briggs

Papers

Synthesis of functionalised fluorescent dyes and their coupling to amines and amino acids

Analysis of carbohydrates

Fluorescent detection method and reagent

Methods employing increased dye-fluorescence upon nitroreduction

Synthesis of thiospiroacetals. X-Ray crystal structure determinations for (7RS,11RS)-1,4,8-trioxa-13-thiadispiro[4.1.5.3]pentadecan-11-ol and (4RS,6SR)-4-p-nitrobenzoyloxy-1,7-dithiaspiro[5.5]undecane