Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Patent
1992-09-28
1995-04-11
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
435 71, 435815, 435175, 436501, 436547, 436548, 436819, 536 41, C12Q 148, C12Q 100, G01N 33566, C07G 300
Patent
active
054057531
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The subject of the present invention is a new process for the preparation of CMP-activated (cytidine-5'-monophosphate), fluorescing or absorbing as well as new CMP-activated fluorescing or absorbing sialic acids.
Sialic acids (also called acylneuraminic acids) are components of many bacterial and animal glycoproteins and glycolipids. In the case of inflammations, tissue-destroying processes and certain tumour diseases, higher concentrations of sialic acids, especially of 5-N-acetylneuraminic acid, and increased activities of so-called sialyl transferases are also found in the blood plasma. The latter incorporate in a physiological manner the sialic acid intracellularly into glycoconjugates; it is a question of glycoprotein enzymes which have a definite specificity for the glycan sequence of the glycoconjugate acceptor into which the sialic acid is incorporated and for the glycosidic binding type in which the incorporated sialic acid is chemically bound. Not only the detection of the sialic acid concentration in the blood and tissues, especially also the sialylation pattern on the cell membrane surface, but also the detection of the sialyl transferase activity and specificity in cells or body fluids is, to an increasing extent, interesting for medical diagnosis.
It is problematical in the case of the detection of such enzymes that they are normally present in extraordinarily low activity so that direct detection methods are difficult. Therefore, according to the state of the art, radioactively-labelled CMP-activated sialic acids are used as substrate in order to determine sialyl transferase-catalysed reactions (cf. Beyer et al., Adv. Enzymol. Relat. Areas Mol. Biol., 52, 23-175 (1981)). Apart from the fact that, in many places, it is difficult to handle radioactive substances, these processes are time-consuming and expensive.
Therefore, there is a need for a simpler labelling substance which is problem-free to handle with which also small amounts of reaction products of the sialyl transferase reaction can be detected certainly, simply and sensitively.
Therefore, in Eur. J. Biochem., 177,583-589 (1988), a process is described of coupling 9-amino-5-N-acetyl-neuraminic acid with fluoresceinyl isothiocyanate which, according to the process described by Gross and Brossmer, Glycoconjugate J., (1987), Volume 4, 145-176, and Eur. J. Biochem., (1987) 168, 595-602, can be coupled with CMP. The fluorescein-labelled CMP-sialic acid prepared in this way proves, as indicator, to be superior to the known radioactively-labelled CMP-sialic acids. Not only the preparation of the fluoresceinyl-N-acetyl-neuraminic acid but also the subsequent CMP activation proceed, on the basis of the poor reaction and the numerous purification steps, only with a yield of about 5%, referred to the initially used 9-amino-5-N-acetyl-neuraminic acid. Therefore, there is a need to prepare this substance in a simpler way and with higher yields and thus to make it available more economically.
SUMMARY OF THE INVENTION
Surprisingly, it has been found that one can also prepare this compound as well as other hitherto not known CMP-activated, fluorescing sialic acid derivatives in that one first couples 9-amino-5-N-acetylneuraminic acid with CMP as this is described in the literature (cf. Gross et al., Eur. J. Biochem. (1987) 168,595-602). This reaction proceeds with high yield (95%) and also the purification of the end product is comparatively simple. Instead of the preferred 5-acetyl group, another acyl group can also be present. Free 5-amino groups are not suitable since these compounds are not stable.
According to the invention, the so-obtained product can now be coupled with an activated, fluorescing compound, for example with fluorescein isothiocyanate. Surprisingly, this reaction also proceeds with very high yield and comparatively simple purification of the end products, although it was to have been assumed that numerous side reactions would occur due to the large number of possible reactive centres and that a con
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patent: 5059535 (1991-10-01), Mazid et al.
patent: 5173407 (1992-12-01), Uemura et al.
patent: 5180674 (1993-01-01), Roth
Gross et al, European Jour. of Biochem, vol. 177, No. 3 (1988).
Gross et al., "A Highly Sensitive Fluorometric Assay for Sialytransferase Activity Using CMP-9-fluroresceinyl-NeuAc as Donor" Analytical Biochemstry, 186:127-134 (1990).
Gross et al., "Enzymatic Introduction of a Fluorescent Sialic Acid into Oligosaccharide Chains of Glycoproteins", Eurpoean Journal of Biochemistry, 177:583-89 (1988).
Chemical Abstracts, 77:(9) 57834u.
Chemical Abstracts, 109:(13) 108046C.
Brossmer Reinhard
Gross Hans J.
Leary Louise N.
Wityshyn Michael G.
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