CMP-activated, fluorescing sialic acids, as well as processes for their preparation
OwnerReinhard Brossmer, Hans J. Grossinventors
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Abstract
A process for preparing CMP-activated fluorescence indicator-labelled sialic acids is disclosed. According to the process, a 5-acylamido-9-amino-3,5,9-tridesoxy-.beta.-D-glycero-D-galactonoulosonic acid or 5-aminoacylamido-3,5-didesoxy-.beta.-D-glycero-D-galactonoulosonic acid is reacted with cytidine phosphate in the presence of a CMP-sialic acid synthase which is then reacted with a fluorescing compound to give a CMP-activated fluorescing sialic acid. Also disclosed are novel CMP-activated fluorescing sialic acids.
Claims22
Process for the preparation of cytidine-5'-monophoshate-activated (CMP-activated) fluorescence indicator-labelled sialic acids of the formula IV ##STR4## wherein either R.sup.3 signifies the group Fl--Sp--X'--NH-- and R.sup.4 an acyl group or R.sup.3 a hydroxyl or acylamino group and R.sup.4 the group Fl--Sp--X'--NH-acyl, Fl signifies a fluorescing group, Sp a valency or a coupling spacer group and X' a --CO--, --CS--, --SO.sub.2 -- or triazinyl group, comprising reacting a 9-amino-5-N-acyl- or 5-aminoacyl-neuraminic acid of the formula I ##STR5## in which R.sup.1 represents an amino group and R.sup.2 an acyl group or R.sup.1 a hydroxyl or acylamino group and R.sup.2 an aminoacyl group, with cytidine triphosphate (CTP) in the presence of the enzyme CMP-sialic acid synthase (E.C. 2.7.7.43) to give CMP-activated sialic acids of the formula II ##STR6## in which R.sup.1 and R.sup.2 have the above meaning, and reacting the CMP-activated sialic acids of formula II with a fluorescing compound of the formula III
- Fl--Sp--X (III)
- in which Fl signifies a fluorescing group, Sp a valency or a coupling spacer group and X an activated carboxyl, thiocarbinyl, triazinyl or sulphonic acid group, to give CMP-activated, fluorescing sialic acids of the formula IV ##STR7## wherein R.sup.3 and R.sup.4 have the above meaning.
CMP-activated fluorescence indicator-labelled sialic acid of the formula IV' ##STR8## wherein either R.sup.3 is the group Fl--Sp--X'--NH-- and R.sup.4 is an acyl group or, alternatively, R.sup.3 is a hydroxyl group or acylamino group and R.sup.4 is the group Fl--Sp--X'--NH-acyl in which Fl is a fluorescing group and Sp is a valency or a coupling spacer group and X' signifies a --CO--, --CS-- or --SO.sub.2 -- or triazinyl group, wherein R.sup.3 is not a fluoresceinyl group.
CMP-sialic acid derivatives selected from the group consisting of
- CMP-9-tetramethylrhodaminylamino-NeuAc
- CMP-9-rhodaminylamino-NeuAc
- CMP-9-eosinylamino-NeuAc
- CMP-9-xanthenerhodaminyl-NeuAc
- CMP-9-(4-N,N'-dimethylaminoazobenzo-4)-amino-NeuAc
- CMP-9-fluoreseinyl-(5,6)-carboxamido-NeuAc
- CMP-9-rhodaminyl-(5,6)-carboxamido-NeuAc
- CMP-9-resorufinylamino-NeuAc
- CMP-9-(7-amino-4-methylcoumarinylacetamido)-NeuAc
- CMP-9-N-(fluoresceinylaminochlorotriazinyl)-amino-NeuAc
- CMP-9-dansylamino-NeuAc
- CMP-9-(Texas-red)-amino-NeuAc
- CMP-5-fluoresceinylaminoacetyl-Neu
- CMP-5-tetramethylrhodaminylaminoacetyl-Neu
- CMP-5-rhodaminylaminoacetyl-Neu
- CMP-5-eosinylaminoacetyl-Neu
- CMP-5-xanthenerhodaminylaminoacetyl-Neu
- CMP-5-(4-N,N'-dimethylaminoazobenzo-4')-aminoacetyl-Neu
- CMP-5-fluoresceinyl-(5,6)-carboxamidoacetyl-Neu
- CMP-5-rhodaminyl-(5,6)-carboxamidoacetyl-Neu
- CMP-5-resorufinylacetyl-Neu
- CMP-5-(7-amino-4-methylcoumarinylacetamido)-acetyl-Neu
- CMP-5-fluoresceinylaminochlorotriazinylaminoacetyl-Neu
- CMP-5-dansylaminoacetyl-Neu and
- CMP-5-(Texas-red)-aminoacetyl-Neu.
A method of measuring sialyl transferase activity, comprising the steps of:
- incorporating into an acceptor molecule, by means of a sialyl transferase, a CMP-activated fluorescence indicator-labelled sialic acid of the formula IV ##STR9## wherein either R.sup.3 signifies the group Fl--Sp--X'--NH-- and R.sup.4 an acyl group or R.sup.3 a hydroxyl or acylamino group and R.sup.4 the group Fl--Sp--X.sup.1 --NH-acyl, Fl signifies a fluorescing group, Sp a valency or a coupling spacer group and X' a --CO--, --CS--, --SO.sub.2 -- or triazinyl group;
- and
- measuring acceptor-bound fluorescence.
A method of measuring differences in acceptor substrate specificity of different sialyl transferases, comprising the steps of:
- incorporating, by means of a first sialyl transferase, a cytidine-5'-monophosphate-activated (CMP-activated) fluorescence indicator-labelled sialic acid of the formula IV, ##STR10## wherein either R.sup.3 signifies the group Fl--Sp--X'--NH-- and R.sup.4 an acyl group or R.sup.3 a hydroxyl or acylamino group and R.sup.4 the group Fl--Sp--X'--NH-acyl, Fl signifies a fluorescing group, sp a valency or a coupling spacer group and X' a --CO--, --CS--, --SO.sub.2 -- or triazinyl group, into a first acceptor molecule;
- incorporating, by means of a second sialyl transferase, a CMP-activated fluorescence indicator-labelled sialic acid of the formula IV into a second acceptor molecule;
- and
- measuring and comparing the level of fluorescence of said first and second acceptor-bound sialic acids.
A method of labelling a cell surface, comprising the steps of:
- bringing a solution of cells into contact with a cytidine-5'-monophosphate-activated (CMP-activated) fluorescence indicator-labelled sialic acid of the formula IV, ##STR11## wherein either R.sup.3 signifies the group Fl--Sp--X'--NH-- and R.sup.4 an acyl group or R.sup.3 a hydroxyl or acylamino group and R.sup.4 the group Fl--Sp--X'--NH-acyl, Fl signifies a fluorescing group, Sp a valency or a coupling spacer group and X' a --CO--, --CS--, --SO.sub.2 -- or triazinyl group, in the presence of sialic transferase;
- washing away excess indicator-labelled sialic acid;
- and
- measuring surface-bound fluorescence.
Dependencies are inferred from the claim text.