Chemistry: electrical and wave energy – Processes and products – Electrostatic field or electrical discharge
Patent
1992-03-27
1994-08-23
Niebling, John
Chemistry: electrical and wave energy
Processes and products
Electrostatic field or electrical discharge
204299R, C25B 700
Patent
active
053404530
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
This invention concerns analysis of carbohydrates.
BACKGROUND TO THE INVENTION
International Publication No. WO88/10422 discloses, inter alia, techniques for analysing carbohydrate structures or distinguishing or separating carbohydrate substances, involving applying carbohydrate substances to an electrophoretic gel and running the gel to cause differential migration of different substances. The carbohydrate substances may be pre-labelled with a fluorescent labelling reagent, e.g. amino fluorescein, to impart a charge to the substance, thereby to enable electrophoretic separation, and to enable visualisation of the substances after running of the gel. In this case, visualisation may be effected with the naked eye, but enhanced sensitivity is obtained by viewing with a charge coupled device (CCD).
The present invention concerns a development of such techniques, and is based on the discovery that unexpectedly good results can be obtained by using naphthalene derivatives as the fluorescent labelling reagent.
SUMMARY OF THE INVENTION
According to the present invention there is provided a method of separating or distinguishing carbohydrate substances, comprising labelling carbohydrate substances with a labelling reagent comprising a fluorescent naphthalene ring structure having as a substituent a reactive group capable of reacting with a reducing sugar to bind thereto and also having at least one substituent group capable of carrying a charge but Which does not react with reducing sugars and does not extinguish fluorescence of the label ling reagents applying the labelled substances to an electrophoretic gel; and running the gel to cause differential migration of different substances.
The structure of naphthalene is shown in FIG. 1, with carbon atoms Where groups may be substituted identified by number (1 to 8) and type (alpha and beta).
The currently preferred reactive group capable of reacting with a reducing sugar is a primary amino group (--NH.sub.2), but other groups such as a hydrazino group (--NH--NH.sub.2) or a secondary amino group (--NHR) may alternatively be used. Further possible groups will be apparent to those skilled in the art. The reactive group my be attached to any of carbons 1 to 8 of the naphthalene. The reactive group may be directly attached to the carbon, or may be indirectly attached via a linker or spacer group such as an aliphatic group. The presently preferred arrangement comprises a primary amino group directly attached to an alpha carbon of the naphthalene ring structure. It is desirable to include only one such reactive group so that only one sugar molecule will bind to each molecule of labelling reagent.
The substituent group capable of carrying a charge preferably comprises a sulphonic acid group (--SO.sub.3 H), which may be initially present in the form of the acid or a salt such as the sodium salt, and which ionises in solution under appropriate conditions to the form --SO.sub.3.sup.-. A wide range of other substituent groups my also be used, including for example, hydroxyl groups (--OH), carboxyl groups (--COOH), tertiary amine groups (--NR.sub.1 R.sub.2) and quaternary ammonium groups (--N.sup.+ R.sub.1 R.sub.2 R.sub.3 X.sup.-). Other possible groups will be apparent to those skilled in the art. Such groups may initially be in the form of salts. Such a group may be attached to one or more (e.g. two or three) available carbons of the naphthalene ring, either directly or indirectly via a linker or spacer group such as an aliphatic group.
The sulphonic acid group is the correctly preferred substituent group capable of carrying a charge. This is because the resulting compounds are stable and are negatively charged over a wide pH range which includes convenient conditions under which to carry out electrophoretic separation.
The preferred labelling reagents are thus aminonaphthalenesulphonic acids, with one, two or three sulphonic acid groups.
One presently preferred labelling reagent is 8-aminonaphthalene-1,3,6-trisulphonic acid (referred to as ANTS for
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Astroscan Ltd.
Delacroix-Muirheid C.
Niebling John
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