CA1257253A - Glycosides of resorufin derivatives, processes for the preparation thereof and the use thereof for determining the activities of glycosidases - Google Patents

Abstract

ABSTRACT

Glycosides of resorufin derivatives, processes for the preparation thereof and the use thereof for determining the activities of glycosidases The present invention provides glycosides of resorufin derivatives of the general formulae:- (Ia) (Ib) wherein R is a hydrogen atom, R2, R3 and R5, which can be the same or different, are hydrogen or halogen atoms or lower alkyl radicals, R4 and R6, which can be the same or different, are hydrogen or halogen atoms, cyano groups, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxycarbonyl lower alkyl radicals or carboxamido groups optionally substituted once or twice or radicals of the general formula -COO-(CH2CH2O)n-R7, wherein R7 is a hydrogen atom or a lower alkyl radical and n is a whole number from 1 to 4, and R6 can additionally also represent a sulphonyl or nitro group and Y is a nitrogen atom or the group N ? 0.
The present invention also provides processes for the preparation of these glycosides and diagnostic agents containing them.
Furthermore, the present invention provides starting materials for the preparation of these glycosides.

Description

i257253 The pre~ent invention provide~ glyco~ides of re~orufin derivative~, processes for the preparation thereof and dia~no~tic agent~ containing them and al~o provide~ ~tarting material~ for the preparation 5 of these glyco~ides.
In the human and animal organi3m, glycosida~es fulfil a variety of physiological function~. Thus, for example, ~-n-galactosidase plays an important part in carbohydrate metaboli~m since, by mean3 of it, ~he hydrolysi~ of lactose takes place. Furthermore, ~-D~
galactosida~e represents a key enzyme in the breakdown of glycolipid~, mucopolysaccharides and glycoprotein~.
Fur~her physiologic~lly important glycosidase~ include, - for example, a-D-galacto~ida~e, -D- and ~-D-gluco~id-ase, a~ well as a-D-mannosidase.
Quite apart from their physiological value, in recent years the glyco~idases have achieved importance in the diagno3tic field9 as well a3 in the biotechnical field. Tbu~, for exampl~, thes~ enzyme~ are used to 20 an increa~ing extent as indicator enzymes for enzyme-immunoa~says. In thi~ connection, ~-D-galacto~ida~e i8 espec:ially preferred (~ee" for example, Annals of Clinic~l Biochemistry~ 16" 221-24t)/197g).
The deter~ination oP the activity of the glyco-2S sida~s a~cordingly pl~y~ an increa~ing part not onlyin cli~ical che~istry but also i~ diagnoai~. F~r thi~
purpo~, quit~ ~enerally9 a glycosidas~-cont~ing

- 2 - ~ 3 sample i~ mixed with an appropriate ~ubstrate, this sub~trate being ~plit by the enzyme and one of the fission product~ i8 detected in an appropriate manner.
There can be measured either the glycone or the aglycone liberated by the action of the enzyme. A~
a rule, the latter is determined.
A~ ~ubstrate, there can often be u~ed the natural ~ub~trate of the enzyme to be detect~d. How-ever, it i~ especially preferable to u~e glycosides in which the aglycone i~ a re~idue which can ea~ily be detected.
A number of glyco~idase ~ubstrates are known in the ca~e of which, after ~plit~ing by the glyco~ida~e, the aglycone can be measurçd apsctro~copically in the visible or al~o in UV rang~, a3 w~ll as fluorometric-ally.
Thu~, in Biochem. Z., 333, 209/1960, there i3 de~cribed phenyl-~-D-yalacto~id~, a~ well as ~ome . further derlvative~ substituted in the aromatic ring, fox exampl~ o-nitrophenyl~ and ~nitrophenyl-~-D-galactoaid~, a~ ~ub~trate~ for ~-D-galacto~idas~.
The phenols liberated by th~ hydrolysis can be deter-mined photom~tric~lly in ~he W rang~ or, in the case of the nitrophenol~, in th~ Yhortwave, viaible wave-length rang~. A~ indicator reaction, there can al~obe added an oxidati~e coupling with aminoantipyrin~
(~ee Analytical Blo~hem., 40 J ~81J1~71).

3 1~ 5~

For hi tochemical inve~tigation~, naph~hyl~
galactosides are u~ed, for exa~ple the l-naphthyl compound (Hi3tochemie, 35, 199/1973), the 6-bromo-2-naphthyl derivative (J. Biol. Chem., 195, 239/1952) and naphthol-~-D-galacto~ide (HiRtochemie, 37, 89 1973). For vi~ualisation, the resultant naphthol~
are thereby reacte~ with various diazonium 3alts to give azo dyestuff3.
The determination of enzyme a~tivities wi~h fluorogenic sub~trate~ i9 widely u_ed since, in com-pari-qon with the photometric methoda, the sen~itivity of fluorometric detenminations i9 often increa~ed by everal power~ of ten. In ~ome ca~es, it i9 necessary to wvrk with fluorogenic substrate~, for exa~ple, in th~ ca e of the inve~tigation of enzymatic activity in cells u~ing autom~tic device~ for c~ll different-iation (cytofluoro~etry), aa well as in the case o~
the analy~i~ of immobili~ed enzyme~ with flow-through micrQfluorom~tryO In other caa~, for exa~ple in the 20 ca~s of th~ determinatiosl of th~ enzymatic marking of te~st system~ (enzyn~ immunoa~say~), th~ multiplic:ation effect of the enzymatic cataly~is i~ con~iderably ~trengthened by the U~9 o:lE fluorogenic ~ub~tratea.
q~he hith~rto known fluorogenic ubatrate~ *or 25 ~-~galacto~ida~e and other glycosida~ contain, a~
fluorophore~9 d~riva i~e~ o~ fluoro~c~in~ indoxyl s:~r methyl-umbelliferone. ~ow~v~r, or ~h~ kinetic:

1%5~253 analysis of complex system~ the3e compound~ po~se~8 ~erious disadvantages. The disubstituted derivative3 of fluoro~cein are hydrolysed in a multi~tep reaction ~equence and the mono~ub~tituted fluore~cein glycoside~
already fluoresce them~elve~. Indoxyl derivatiYe~
undergo, after their enzymatic ~plitting, a ~eries of chemical changes which also complicate the kinetic analysi~ Derivatives of methyl-umbelliferone must be activated in UVO The inherent fluorescence of biological or ~ynthetic material~ can hereby disturb.
Furthermore, W excitation, e~pecially in the ca~e of la~er optic3, iY expen~ive. Most fluorogenic 8ub-strate~ give ri~e to reaction product~ which have only a low ~olubiliky 90 that they are not ~uitable for kinetic analysea of enzyme activities, for which a good ~olubility of the ~ub~trate and of the product i~ nece~sary.
Therefore, there i~ ~till a need for ~ubstrate~
with which variou~ glycosidase3 can be determined in a si~ple, rapid and dependable way and which can pvs~ibly be u~ed not only for photom~tric but al~o for fluorometric determination proces~es. It i~ an object of the pre~ent invention to ~ati~fy thi~ need.
problem i~ solved by new glycoside~ o re~orufin derivative ~ihich~ with the h~lp of glyco-sidasss, ca~ bs split into th~ ~ugar moiety and into ~e re~orufin derlvative3~ The latter are readily ~ 5~

wat~r ~oluble compound~ which di~piay a readily mea~urable ab~orption in the vi~ible rang~ and, furthermore, can easily be excited to fluorescence.
Thus, according to the prese~t invention, there are provided glyco~ides of resorufin derivatives of the general fonmulae Ia and Ib * R

glyooside-O ~ O ~ R ~la) Rl R6 R2 ~ Y ~ R5 (Ib) O ~ O ~ O-glycoside wherein Rl i~ a hydrogen atom, R2, R3 and R , which can be the ~ame or differentO are hydrogen or halogen atom3 or low~r alkyl radical3, R4 and R6, which can b~
the same or different~ are hydrogen or halogen atom3, cyano grou~s, lower alkyl, lower alkoxy, cArbo~qrl, lower alkoxycarbonylO carboxy lower al~yl or low~r lS alkoxycarborlyl lower alkyl radicals or carboxa~ido group~ optionally substituted once or twice or radical~
of the general formula -coo-sc~2c~2o~n 0 i~ a hydroge~ ato~ or a low~r xlkyl radical and iJ
a whole numb~r ~r~ 1 to 4, a~d R6 ~an additionally ~ - 6 - l~ S3 alQo repre~ent a sulphonyl or nitro group and Y iq a nitrogen atom or the group ~ -~ O.
The glycoside~ o~ re~orufin derivative3 of the general formula I are new compound~. They can be prepared by method~ known from carbo~ydrate chemi~try.
Preferably, a resorufin derivative of ~he general tautomeric formulae IIa and IIb:
Rl ~6 R (IIa) R ~ ~
J~

R ~ ,y ~ R (IIb) O ~ ~ OH
~3 R

wherein Rl to R6 and Y have the ~ame meanings a~ given above, is reacted in known ma~ er with a mono- OT oligo-saccharide OT a 1-halogeno-derivative thereof, all hydToxvl gTOUpS being 3ub~tituted wnth prote~tive groups known in carbohydrate chemistry~ to giv~ a p~r-O~ub~tituted glyco~ide fro~
which, by splitting off the protective groups in known manner, there i3 obtain~d a glycoside of a resorufin derivative of general formula I.
The reaction of th~ on~pounds of general formula II with the p~r-o-aub~titut2d l-halogeno-saccharides is 20 preferably carried out in the pre~en~:2 of an acid lZ~25~3 acceptor, or example an alkali metal hydroxide or carbonate, in aqueou~ aceto~e or, under pha~e tran~fer conditions, in a water/benzene or water~chlorofonm mixture.
- 5 Furthermore, thi~ proce~s can be carried out by fir~t converting the resorufin derivative~ of general formula II, by mean~ of an alkali metal hydroxide or alcoholate, into the alkali metal salts or, by means of optionally sub~tituted amine~ into the ammonium ~alts and the~e are then reacted in a dipolar aprotic ~olvent, for example acetone, dimethyl sulphoxide, dichloromethane, tetrahydrofuran or dimethylformamide~
with the per-0-sub3tituted l-halogeno-saccharides.
Furthenmore, in the case of the synthesi R of the p~r-0-~ub~tituted glyco~lde~ from the resorufin deriv-ative~ of genQral fonmula II and the per-0-sub~tituted 1-halogeno-saccharides, the addition of individual ~ilver ~alt~ or mixtures of ~ilver ~alt~ ( ~ilver oxide, Yilver carbonat~, ~ilv~r carbonate on Celits* ~ilver triflate or ~ilv~r ~alicylate~ and~or of individual mercury 3alt~s or mixturs~ of mercury salts (msrcury bromida, msrcury ~:yanid~ ~ mercury acetate or mercury c~xide ) ~ optionally with the U8~ of a drying ayent, ~u~h a~ ~:aloium chloride, molec:ular sieve~ or Drierite* in a ~olv~nt, ~or e~ample, m~thylene chloride, chloroform, benzen~, tolue~e or diox~n, ~av~ proved to b~ u~eful.
~or the ~ynthe~s of the a-attached glyco~ides~ a * trade mark - 8 - ~ 3 compound of general fonmula II i~ preferably melted with a s~ccharide, the hyd~xyl ~rou~s ~-fwhich are ~ubstituted with protective group~, e3pecially with acetyl group~, in the pno~ence of a Lewis acid, for S exampls tin tetrachloride, aluminium chloride or preferably zinc chloride ~cf. Chem. Ber., 66~ 378-3R3/
1933, and Method~ in Carbohydrate Chemistry, 2, 345-347/1967). The temperature hereby u~ed iB in the range of from 80 to 150 C. and preferably of from 110 to 130C.
The per-0-~ubstituted glycosides of the resorufin derivative~ of general fonmula II thu~ obtained are al~o new compounds.
The splitting off of the prote~tive groups from the per-0-~ubs~ituted glycoside3 to give the glyco~ide~
of general fonmula I i9 carried out ~ccording to method~
known in carbohydrate chemi~try [see, for exa~le, Advances in Carbohydrate ChemO, 12, 157/19753, for example in the ca~e of acyl protective group~ by mean~
of ~odium methylate or barium m~thylate or ammonia in methanol.
By halogen in the de~initions of R to R is to bæ under~tood fluorine, chlorine, bromine and iodine, preferably chlorine and bromineO
Th~ lower alkyl radieal~ in the definitions of * to R6 contain up to 5 and p~eferably up to 3 car~on ato~3, the methyl radical being e~peclally pre~rred.

2~5~
g ~ he lower alkoxy radical3 in the definition~ of R4 and R6 contain u~ to 5 and preferably up to 3 carhon atom3, the me~hoxy radical being ~specially preferred.
The lower alkoxy and the lower alkyl moietie~
o the lower alkoxycarbonyl, carboxy lower alkyl and lower alkoxycarbonyl lower alkyl radical~ in the definitions of the ~ub~tituents R and R also contain up to 5 and preferably up to 3 carbon atoms, the methoxy radical and the methylen~ radical being especially pre-ferred.
As protective groups which are conventional incarbohydrate chemistry, there are especially preferred the acetyl, benzoyl, benzyl and trimethylsilyl radicals.
As ~ub~tituents of the carboxamido group, ther~
~y be mentioned alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonylalkyl radicals, the alkyl moietie~
thereby containing up to ~ and pr~ferably up to 3 carbon ato~s~ In thc ca~e of a disubatituted carbvx-amido function, th~ two sub~titu~nt~ can b~ joined to 20 form a ring which can be interrupt~d by hetero atom~
for exa~le oxygen, nitrog~n ~nd sulphur~
A~ glycol3idl~: radical w}lich i g attached to the re30rufin derivatives of geIIeral formula II to give the g~ycosidsa of gener~l formul~ I, there can bs used 25 all mono- and oliQo-saccharides which car~be s~lit off a~ain from ~e re~orufin structure~ by mean~ of ~h~3 a~ropriat~
s~ly~osida~0 As exa~l~ oï glyco~ide~ according to ~% ~ 2 ~3 th~ present invention, there may be mentioned ~-D~
galactopyrano~ide~, a-D-galactopyrano~ide~, ~-D-gluco-pyrano~ides and a-D-glucopyranoside~, a3 well as a-D-mannopyrano~ide~.
As glycosidic radical there can also be used oligosaccharides which can be split by saccharide chain splitting enzymes producing a mono- or oligosaccharide which can be split off from the resorufin structure by means o~ an appropriate glycosidase.
Particularly suitable is an oligosaccharide having 2 to 10 preferably 2 to 7 monosaccharide units.

The preAent invention also provides the n~w co~pounds of the general tautom~ric formula~:
R R6' R5' (II'a) ~0 ~ I O
R3 R4~
1~
Rl' R6' ~ y ~ R (II'b) ~2~X53 wherein * to R6 have the same meaning~ a~ the ~ubstituent~ R to R6, whereby R to R cannot all ~imultaneou~ly be hydrogen atom~, and Y ha~ the above-given meaning.

These compounds are new. They are eqpecially suitable a~ intermediates for the preparation of the glyco~ide~ of resorufin derivative~ of general fonmula I according to the present invention.
The compound3 of general formula II' can ke prepared analogou~ly to proce~se~ ~hich are suitable for the preparation of the ~nown resorufin ~compound of general formula II in which R to R are all hydrogen atom~).
The compound~ of general formula II' are advantageou~ly prepared by reacting nitro~ore~orcinol derivatives of the general formula:-Rl ~
R2' ~ ~ (III) HO ~ OH
R3' with re~orcinol derivative~ of the general fonmul~:-H ~ ~ R (IV~

HO ~ OH
R4' wherein * to R6 have the a~ov~-giv~n meanings, in the presence of pyrolu~ite*and ~ulphuric acid at a low te~peratur~. There are ther~by first formed compounds of general fonmula II' in which Y i~ an ~ -~O group.
The~e compounds can ea~ily b~ converted with zinc pow~er in ths pr~senc~ of ammonia into compounds of general formula II~ in which Y i~ a nitrogen atom~
The reaction of compound~ of g~n2ral formula III
with compounds of general fonmula IV is u~ually carried *manganese dioxide ~ ~ ~ 5 ~ ~ ~3 out at a temperature of from -10 ~o 50C. and prefer-ably of from 0 to 30C. The reaction takes place e~pecially gently when the compounds of general fonmula III and of general formula IV are mixed at about 0 C.
and the reaction mixture i~ subsequently allowed to warm up to ambient temperature. The concentration of the pyrolu~ite i~ preferably from 0.5 to 5 and more preferably fro~ 1 to 2 mol/litre. The sulphuric acid concentration should be from 0.5 to 5 and pr~eferably from 1 to 3 mol/litre.
The reduction of compounds o~ generàl formula II', ~herein Y i~ a N -~ O group, to give compounds of general formula II', wherein Y i8 a nitrogen ato~, i9 preferably carried out in ammoniacal solution with zinc dust (cf. Nietzki et a O, Ber. dt~ch. Chem. Ge~, 22, 3020/1889). A~ ~olvent, there i~ preferably u3ed a water-alcohol mixture and re preferably a mixture of 1 part of wa~er with 0 to 4 parts of me~hanol. Per mole of reducing 3ubstance, t~er~ are added thereto portionwi~s 1 to 20 and preferably 1 to 5 mola of zinc du~t. The tempera~ure o tbe re~ction solution i~
thereby maintained at from -10 to +35C. and preferably fro~ 5 to 10C. The precise maintenance of the temper-ature rang~ ha3 proved to be nece~ary for a ~pecific course of the rsaction. Without cvoling, th~ exothenmal reaction ~iY~8 ri~e to by-product~ w~ich are difficult to separate7 --14~

Under the qelected mild condition~, the reaction ~etween the compound~ of general formula III and of general fo~nula IV proceed~ qpecifically and with good yield. Th~3 ~electPd ~yntheRis route i~ capabl2 5 of variation. ~his open~ up numerou~ po~ibilities of synthe~i~, e~pecially having regard to the preparation of a~ylmnetrically substituted resorufin and also re~azurin derivatives.
Resorufin derivatives of general formula ~I ', 10 wherein R4 and/or R6 are lower alkoxycarbonyl, . optionally mono- or disub3tituted carboxamido groupq or a radical of the general fonnula -COO- ~CR~CH20)n-R
are preferably prepared via the triacylated dihydro-re~orufins of the general formula:

R7 ~1~ ~ ( V ) w~erein Rl, R2, R3 and R have the above-given meaning~, R and R6 ar~ carboxyl groups and R i a hydrogen atom or a lower alkyl radical.
The carboxylic acid function is converted into 20 the acid chloride by proce~se3 known from th~ liter-ature, for ~xampl~ h oxalyl chloride/dimethyl-3L2 ~a~2S3 fonmamide or with thionyl ch}oride/dimethylfonmamlde, from which the correspondingly ~ub~tituted carboxylic acid e~ter or carboxamide derivative~ are obtained by reaction with any de3ired alcohol~ or amines By treatment of the so obtained acetylated derivative~ of general formula III with an aqueou3 ~olution of an alkali metal hydroxide, preferably with 0.1 to 5 M aqueou~ ~odium or potas~ium hydro~ude 801-ution, or with 1 to 15 M aqueous ammonia and an oxidation agent, preferably potaA~ium ferricyanide~
with the addition of a water-~oluble organic ~olvent, for exampl~ 1,4-dioxan or methanol, there are obtained the corre~ponding re~orufin derivative3 of general formula II.
A~ alcohol component, there can, in prin~ipl~, be u~ed all pos.ible alcohol~, especially preferred being diethylen~ glycol monoethyl ether, triethylene glycol monoethyl ether or ~imple alcohol~, for example, methanol or ethanol. ~he amine components can also be ~elected from all po~ible amine~, e~pecially pre-~erred being amine~ wnth a polar group, for example morpholine~ methoxyethylamine or glycinamide, or ammonia or a primary or ~econdary lower alkyl ~ ne.
FurthermoreO th~re can be used ami~ocar~oxylic acids with the carkoxyl function protected in the u~ual ma~er, ~or example glyci~ t~rt.-butyl e~terO gly~i~e benzyl e~t~r or ~a BOC 1Y81B~ ~thyl ~sterO ~ft~r ~ 2 ~7~ 5 ~plitting off the protective groupn, there are thu~
obtained re~orufin~ II or resorufin glyco~ide~ I with an aliphatic carboxylic acid function~
The acetylated dihydrore~orufin~ o~ general fonmula V are obtained from the corresponding resorufin or reRazurin by reaction with a strong reducing agent, for example tin dichloride or chromium acetate, or by electrochemical reduction and ~ub~equent acetylation.
For the reduction, the re~orufin or resazurin is warmed for from 10 minute~ to 1 hour with 2 to 10 and pref~r-ably 2 to 6 equivalents of tin dichloride in 5 to 35%
aqueous hydrochloric acid. Upon cooling, the dihydro compound precipitates out. ~he acetylation take~ place in the u~ual way by reaction with acetic anhydride.
~he compound~ of general formula V are preferably pre-pared in a one-pot proces~ by reductive acetylation.
The corre~ponding resorufin or resazurin i9 heated under reflux wnth 2 to 6 ~quivalents of tin dichloride for 5 minutes to 5 hour~ and preferably for 10 minute~
to 3 hour~ in acetic a~hydride or i~ ~tirred at ambient temperature for from 4 to 16 hour~.
In tha ca~e of the ~ynthe~i3 of glycoside~ of the resorufin derivative3 of g~neral for~ula I1, there ar~ obtained non-fluorescing ~ompound~ from which, by enzymatic ~plitting wnth the h~lp of appropxiate gly~osida~e~ the fluore~cing re~o~ufin d~rivat~ve~
are again liberat~d. In co~pari~on with the pre~iou~ly -17- ~L2S~ 2~3 known glyco~ide~ of other chromogenic or fluorogenic groups, the fluorogenic resorufin derivatives accord-ing to the pxesent invention po3se~s very advantageou~
propertie~. As monosubstituted derivative^~, they di~-play a kin~tic of the enzymatic hydrolysi~ which canbe described ve~y simply by the Michaeli~-~enton relation~hip. For resorufin~ -galactopyranoside, for example, there applies a Michaelis con~tant of ~ = 0.3~ mmole/litre. The inhibition of thi~ hydroly~is by the natural sub~trate lactose i8 competitive 90 that, for cert~in investigations, the specific reaction of glycoside~ of th¢ re30rufin derivatives can be altered in a definite and rever~ible way by the addition of natural glyco3ide~0 such a~ lactose in the ca~e of ~-D-galacto~idase.
The glyco3ide~ of resorufin derivative~ according to the preaent invention are of practically unlimited ~torage ~tability in a~ueou~ ~olution at 4C. The solubility of th~ glycoside~ of the re~orufin basic structure i~ already sufficient for most kinetic pur-po3e~. By the introduction of carboxylic acid residue~, carboxylic acid derivative~ wlth polar groùp~ or sulphonic acid groups, the ~olubility of the corre~-ponding glyco~ides can be ~till ~urther improvedO
The excitation and emi~sion of the co~pound3 according to th~ pr2s~nt invention lie in the ~isible ~pectral range wlth ~ufficient qua~tu~ yield. Th~

-18- 12~72S3 maximum fluore~ent inten~ity of the re~orufin i~
achi~ved at pH value above 7.0 and only decreases slowly at lower p~ value~. In aqueous qolution, ~he glycosides of resorufin are mostly yellow coloured ( AmaX at about 470 nm). After the enzymatic reaction~
the product~ mostly display a red colour ( ~max a~
about 570 nm~ ~o that the substance3 are also out~tand-ingly ~uitable for photometric detenmination~ and non-in~trumental vi~ual processes.
The pre~ent invention i9 al~o concerned with the use of the new glyco~ide3 of resorufin deri~ative~ of general fonmula I for the determination of the activity of corre ponding glycvsidases, for example of a-D- and ~-D-galacto~idase and of a-D- and ~-D-glu~o~ida-qe~, aa well as of a-D-mannosidase.

Glycosides of resorufin derivatives of general formula I, wherein the glycoside radical is a oligosaccharide chain, can especially be used for the determination of saccharide chain splitting enzymes, for example of ~-amylase. The oligosaccharide is split: by the enzyme to be determined in a characteristic way if necessary by means of additional enzymes producing preferably a monosaccharide which can be split off from the resorufin structure by means of an appropriate glycosidase.

In particular, in the determination of a saccharide chain splitting enzyme, the oligosaccharide is split by the enzyme in a characteristic way. The product of the splitting is a derivative of resorufin with less monosaccharide units than the original oligosaccharide; and this derivative is then split by a glycosidase to the aglycone ~resorufin derivative) and the sugar moiety.
Before enzymatically splitting with a glycosidase, the resorufin oligosaccharide derivative obtained as a result of the first step employing the chain splitting enzyme, it may be appropriate to degrade the derivative to one with a still smaller oligosaccharide chain or a derivative with only one monosaccharide unit. This may be achieved by means of additional appropriate enzymes.
The present invention also provides diagnostic agents for the determination of the activity of glucosidases which contain the new glycosides of resorufin derivatives of general formula I. The use of the glycosides of resorufin derivatives of general formula I as substrates for the glycosidases leads to markedly more sensitive test systems than have hitherto been known, The new substrates can be advantageously used for the determin~tion of the activity of glycosidases not ~nly ln the biochemical and biotecnnical fields but also in the clinical-chemical fields. They are more sensitive from which result several advanta~es:

- 20 - 12~72~

a) ~maller enzyme activitie~ can be measured, b) ~maller amounts of ample can be u~ed, c) the determination of the activity can take place in a con~iderably ~horter period of time, d) the ~mall sample usage ana the favourable wave-length also reduce the su-~ceptibility o~ the method to di~turbance~ ~y other components in the samples and e) the reaction can be measured in carrier matrice~
with immobilised enzyme.
We have found that the new substrate~ according to the pre~ent inventio~ can be used for the determin-ation of the activity of glyco~idase of any origin.
~he diagno~tic agents according to the present invention containing ~ubstrates of general fonmula I react markedly mor~ sen~itively than the previously known test agents.
The glyco~lde3 of resorufin derivatives of general fon~ula I can also ~e used for immunological methods of determination in which glycosida~es are u~ed as indicator enzymes, the activity of which mu~t ke determin~d by carrying out the immunological reaction. Such immunological mæthods o~ determination with enzy~atic indicator reaction~ are known a~ enzym~
2s immunoas3ays. The~e methods ~erve for the detenmin ation of ~he conc~ntration of protei~0 poly~accharides~
hor~one~, pharmaceuti~ and o~her low ~olecular we~h~

- 21 - ~ 5~

E~ubstance~ in the rarlge of f rom lO 5 to ~ 2 mole/litre. Depending upon the requirement of phase separation ~t~p~, a differentiation i~ made between a homogeneou and a heterogenou~ carrying out of the test. A further subdivi~ion into competitive and non-compstitive te~t principle~ can al~o be made.
~ owever, all test principles work wi~h enzyme~
antigen or enzyme-antibody ~onjugate~0 me enzymatic indicator reaction is com~on to all enzyme immunoa~ays.
Indicator enzyme-q which can be used for ~uch purpose~
include, for example, glyco3ida~es, especially ~-D-galàctosida~e. The determination of the glycosidase~
in such enzyme immunoas-~ay~ usually take~ place by adding an appropriate ~ub3trate which i~ split enzymat-ically and measured photometrically or also fluoro-metrically in the u-~ual way.
~ n improvement of th~ glyco3idase te~t ~ystem thu~ also result~ in con~iderable advantages in the case of ~uch en~ymæ immunoa~say~:
1. A higher ~en3itivity here also makes po3sible a further lowering o~ the limits of detection, shorter reaction time-~ and ~mall~r amount~ of 3ample and thus also le~ser di~turbance~ by other co~ponent~ o~ ~h~
~ample~
~5 2. The mors favourable m~a~sur~enlt wavelength lower8, in the ca~ o~ c~in ~r~yR of carrying out the deter~ni~tion~ ~he ~ug~cept~bili~y to di~turbanc~s 725~

of the method by in~oluble component~, for example by turbiditie~.
Be~ide~ one or more ~ubstrates of general formula I, the diagnostic agent~ according to the pre~ent invention contain an appropriate buffer sy~tem, as well as po~sible further appropriate additive9 conventionally employed for ~uch diagno~tic agentq, for example wetting agent~, ~tabili~ers and the like. The diagnostic agent can be in the fonm of a solution, lyophilisate, powder mixture or reagent tablet or can be appl.ied to an ab orbent carrier.
The diagnostic agents according to the pre~ent invention in th~ form of a ~olution preferabl~ contain all ~he reayents required for the test. A3 solvent, there can be u3ed water or mixtur~ of water wqth water-solubl~ organic ~olvent~, for example methanol, ethanol, aceton~ or dimethylformamide. For reason~ of ~torage ~tability, it can be advantageou~ to divide up the reagent3 r~quired for the ts~t into two or more ~olutions which are only mixed when actually carryi~g out the inve~ti~ation.
For the preparation of th~ diagno~tic agent in the form of a lyophili~ate with a total weight of, in each ~a~e, fro~ about 5 to 20 mgO ~ preferably o about 10 mg.~ a ~olution i~ dried which, b~sides all the reagent~ raguired for th~ t~stO contain~ co~ventional ~tructure form~r~, for ~xam~l~ pol~vinylpyrrolido~, 12~2S~

and possibly further adjuvants~ for example mannitol, qorbitol or xylitol.
A diagnostic agent in the fonm of a powder mix~ure or reagent tablet can be prepared by mixing the compon-ents of the test with conventional galenical additive~and then granula~ing. Additiveq of thiR kind include, for example, sugar alcohol~, ~uch a~ ~annitol, ~orbitol or xylitol, or other ~oluble inert compound~, such as polyethylene glycol~ or polyvinylpyrrolidone. The powder mixtures or reagent tablets generally have an end w2ight of about 30 to 200 mg. and pref~rably of from 50 to 80 mg.
For the production of the diagno~tic agent in the form of a t~t ~trip, an absorb~nt carrier~ prefer-ably filter paper or cellulo3~ or ~ynthetic re3in fibrefleece, i8 impregnated with solutions of the nece~sary reagents conventionally u~ed for the production of test strip~ in readily vol~tile solvents, for example water, methanol, e~hanol or acetonQ. This can take place in one impregnation step. ~owever, it i3 often desirable to carry out th~ impregnation in ~everal step~, ~ol-ution~ being used each o which contain only a part of the components of the diagnostic agent~ Thu~ t for exa~ple, in a fir~t ~tep, i~pregnation can b~ carried out wnth an agu~ou~ ~olution which contains buffer and oth~r w~ter-~olubl~ additiv~ and then, in a ~e~ond ~tep, with a solution w~lch contain~ th~ glyco~da~e s~

~ub~trate. The fini hed te3t paper can be u~ed as such or, in known manner, can }:e ~tuck on to handles or preferably ~ealed between ~yntheti~ resin3 and fine meshe~ according to Federal Republic of Germany Pat~t Specification No. 2118455.
The following ~xample3 illu~trate ~ome of the numerous proce~ variants which can be used for the synthesi~ of the compound~ a~cording to the pre~ent invention, a~ well as, by way of example, the use of the new glycoside~ of resorufi~ derivative~ for the determination of the activity of glycosida3e~.
Example 1.
~.
a) ~ .
Re~orufin iB prepared from its oxidation product, resazurin, which i commercially obtainable in good purity, using the method descrikRd by Nietzki ~t alO
~Ber. dt3ch. Ghem. Ges., 22, 3020-3038/1889). For thi~ purpo~e, 10 g. (43 mmole) resazurin (obtainable 20 :Ero~ Fluka, Buch3, Switzerland) is heated for 30 minuta~
on a boiling waterba~h in a glas~ beaker with 50 ml. of 25% ammonia ~olution, 25 mlO 37% sodium hydroge~ ~ulphite ~olution and 50 ml. water. A further 20 ml. of ~he ammonia ~olution~ 7 ml. of the hydrogen ~ulphite ~ol-ution and 20 ml. water ara ~dded thereto and heaking~o~tinued for a :l~urth~r 30 minute~D The conversion ca~ be obs~rv~d by the c~olour chan~e from blue to rad 5;~

or by thin layer chromatography. After the reaction ha~ proceeded to completion, 10 ml. 3~% hydrochloric acid ~olution are added thereto until a p~ of 5 is reached. The reaction 801ution i 9 then left to ~tand for a day in a refrigerator. Thereafter, tha browni~h precipitate i~ filtered off with uction, wa~hed with ice-~old hydrochloric acid (p~ 4) and dried at llo&.
The yield i~ 7.8 g. (36.7 mmole: 85% of theory).
b) GlYco~ylation of re~orufin to gLve reqorufin-~-D-galactopvrano3ide.
The glycoaylation of the phenolic hydroxyl groups taXe3 place via the intenmediate stage of the tetra-acetate using the following proce~. 2.13 g. (10 mmole~
of finely powder~d re~orufin, 4.12 g. (10 mmole) aceto-bromo-a-D-galactose (Sigma, Munchen), 1.16 g. (5 mmol~) ~ilver oxide (Fluka~ Buch~, Sw~tzerland), 5 g. calcium ~ulphate hemihydrate (CaS04.1/2H20: Drierite), a few grain~ of iodine and 50 ~1. quinoline are 3tirred for 40 hours 3t ambient emperature in 50 ml. methyl~ne chlorid~, 4~% of the ra~o~ufin th~reby bsing converted int~ resorufin-~-D-galactopyrano~ide tetra~cetate. m~
monitoring of the reaction can again be carried out by thin layer chrom~tography. After separation of ~he ~olid co~ponent~ by mean~ of a fluted filter and centri~uging, the m~thylene chloride i~ ~trippe~ off on a rotavapor. The re~orufin~ galactopyrano~ide tetraac~tat0 remain~ b~ind a~ ~ bro~ y~llo~ ~yrup.

~L~5~2~3 The yield is about 2 g. ~35% of theory)0 2 g. of re30rufin-~-D-galactopyrano~ide tetra-acetate are, wnthout further purification, taken up in 80 ml. dry methanol. 0.5 g. Sodium in the form of ~all piece~ i~ di~solved in 20 ml. dry methanolO while cooling in an ice-bath. While stirring in an ice-bath, 6 to 8 ml. of the methoxide solution are in~roduced in portions of 1 ml. within the course of 30 mi~ute~, into the resoruin-~-D-galactopyrano~ide tetraacetate ~ol-ution and the deacetylation is obqerved via thin layerchromatography. The precipitated, orange-coloured resorufin-~-D-galactopyranoside i~ filtered off with suction and washed wnth a little ice-cold methanol.
Yield: about 1 g. of cr~de ~ubstance ~70% of theory)O
Recry~talli~ation from methanol, with sub~equent drying (not above 60C.),-gives 0.2 g. of pure product. All thin layer chromatographic inve3tigation~ for the monitoring of the ~our~e of the reaction can be carried out with the sy~tem ~ilica gel 60 (~erck, Danm~tadt) and elution agent methylene chloride/methanol (9/1 vJv~.
The following Rf value~ apply for thi~ sy~tem:
resorufin 0.4 re3azurin 0035 re~orufin~ galactopyrano~ide~
tetraacetate 0.9 re30rufin-~-D-galactopyrano~id~ 0.1 - 27 - 125~
.

Resorufin-~-~qlucoside .
2~13 g. (10 mmole) resorufin (prepared according to Example la), 4.11 g. (10 mmole) acetobromo-a-D-glucose and 3.64 g. ~10 mmole~ hexadacyltrimethyl-ammonium bromide are heat~d under reflux for 4 hour~ in a mixture of 11.5 ml. lN aqueous ~odium hydroxide ~ol-ution and 50 ml. chlorofonm. Thereafter, the reaction mixture iq evaporated to drynes~ and dige~ted with ethyl acetate. The ethyl acetate solution iq again evapor~ted to dryne~ and the re~idue obtained i~
taken up in 20 ml. ethanol. The tetraac~tylr~orufin-~-D-gluco3ide precipitated out of this ~olution i~
recrystalli~ed from methanol, with the addition of active charcoal.
For daacetylation, it is taken up in 10 ml.
methanol7 After stirring for 2 hours at ambient temp-erature, the precipitated product is filtered off with suction and dri~d. Yield: 50 mg.
lH-~MR ~[D~6-~MSQ): ~ o 3.15 - 3080 (m~ 6H) 4.5 - 6.5 (broad, 4~), 5012 (d, J 6.9 ~z, lH),o 6.29 (do J
2.0 Hz, lH~, 6.80 (dd, J ~ 9.6 and 2.0 Hz, lH), 7.12 ~dd, J ~ 8.5 and 2.0 Hz, lH): 7~16 (d, J - 2.0 Hz~
lH)o, 7.55 (d~ J ~ 906 Hz, lH), 7080 (d, J 3 8~5 ~z, lH) ~ .
_~ _O

50 g. Nitrosoresorcinol, 53.3 g. methyl 3,5-dihydro~ybenzoate and 28.0 g. pyrolu~i~e are dis~olved or suspended in 500 ml. methanol. 3404 ml. concentrated sulphuri~ acid are add~d dropwnqe thereto, while cooling with ice to 5 to 10C ~f~r removal of t~e ice-bath, ~tirring i~ contin~ed for 2 hour~. Thereafter, 200 ml.
aqueous ammonia solution are added, while cooling.
The precipitate obtained i~ filtered off throu~h a gla~ fibre filter. 10 g. of zinc powder are added portionwnse to the filtrate at 5 to 10C. Stirring i8 continued at ambient temperature until the reduction . .
i8 complete (TLC monitoring using, as elution agsnt, ethyl acetate/methanol (4:1 v/v), reaction time about 1.5 hour~ he volum~ i~ then reduced to about one third on a rotary eva~orator at a bath temperature of 25C. The desired product is precipitated out by acidifying, while cooling, with concentrated hydro-chloric acid until a colour change takes place. After wa~hing with dilute hydrochloric acid and drying in a vacuum over anhydrous calcium chloride, there i~
obtained methyl re~orufin-l-carboxylate. The yield i3 30.9 g. (3~% of theory)O
lH-~MR: ([D36-DMSo3: ~ - 3.98 ~8, 3~), 6.47 (d, J -2.7 ~z, lH), 6~85 - 6.95 (m, 2H) 7.09 (d, J -2.2 ~z, lH), 7.50 (d, J ~ 9.6 ~z, lH).
Fluores~ence: ab~o~ptio~ ~maK 3 570 n~
emi~ion ~ 588 ~.

27~3 In an analogou3 manner, there is obtained fromu a) 3,5-dihydroxybenzoic acid and nitro~or~sorcinol, via resazurin-l-carboxylic acid, re~orufin-l-carb~vlic id, W /VIS (0.1 M pota~ium phssphate buffer P~ 7 5~ ~ max - 569 nm b) methyl 4-0-methylgallat~ and nitro~oresorcinol, via methyl 4-methoxyresazurin~ arboxylate, ~hY~

4-methoxvre~oru5lscjLI~Eb~y_ ~" W/VIS ~0.1 M
potaqsium phosphate buff~r pH 7 5): ~ax ~ 592 n~
c) methyl 3,5-dihydroxybenzoate and 4-chloro-6-nitro~o-resor~inol, via methyl 8-chlorore~azurin-1-carboxylate, methYl 8-chlorore~orufin-1-carbox~late, d) methyl 4-0-methylgallat~ and 4-bro~o-6-nitroso-reqorcinol, via methyl 8-bromo-4-m~thoxyresazurin-l-carboxylat~, methyl 8-bromo-4-m~thoxyre~orufin-1-carboxylato _ e) 5-nitrosoresorcinol and nitro~orosorcinol, via l-nitror~zurin, I_ ~
) re~orcinol-5-~ulphonic acid ~nd nitro~ore~orcinol, via re~azurin l-~ulphonic acld~
ulphonic acld.

., 1.60 g (10.5 mmole3 Nitrosore~Qrcinol, 1.55 g.
(10~0 ~mole) 2,6-dihydroxybenzoic a~id and 0.86 g.
(10 m~ole~ pyrolu~i~e are tak~ up in 20 ~lo m~ha~ol and cooled to O~C~ 1.06 ~1. ~once~trated ~ulph~ri~

2~3 acid are added dropwQ~e thereto. The reaction mixture i~ then ~tirred for a further 2 hours wQthout cooling.
The pre~ipitated red product i~ filtered off, washed with methanol and dried. Yield 2.3 g. ( 85% of theory ) .
W /VIS (0.1 M pota~sium phosphate ~uffer, pH 7.5):
a 614 nm ( 48 cm2 mol l)o, after acidifi~ation:

~max ~ 522 nm ( = 32 cm2 mol~l).
Example 5.
Re~orufin-4-carboxy~lic acid.
2.3 g~ Resazurin-4-carboxylic acid (prepared according to ~xample 4) are dis~olved i~ 20 ml. water and S ml. of 2~ ammonia solution. 5 g. of zinc dust are added, while cooling, to the blue solution. There-after, the ice cooling is removed 90 that the solution gradually wanm~ up to ambient temperature. The reduction can b~ readily recogni~ed by a colour change from blu~ to dark violet or with the help of thi~ layer chromatography (elution agent: methanol/ethyl acetate 1:1 v/v). ExcesJ zinc pow~er i8 filtered off and the filtrat~ i~ ac~dified with 5 ml. glacial acetic aci~
and concentrat~d hydrochloric acid. The precipitated product i~ filtered of~, wa~hed wQth dilute hydrochloric acid and dri~d in a vacuum over anhydrou3 calcium chlorid~. Yield- 1.8 g. ~8~X o~ theory).
W~VIS: (Ool M pota89ium phosphate buff~r pH 7.5~:
~ max ~ 579.4 nm ( -- 48.6 cm2 mol 1~ after ac:idi-ficatio~l ~ma~c ~ 485.9 D~ ( ~9 34-7 ~:m2 mol~l~-- 31 - ~ 2~

Fluore~cence: ab~orptio~ ax ~ 579 nm emi~ion ~max ~ 593 nm.

~.
840 mg. 2,6-Dihydroxy-4-methylbenzoic acid, 760 mg. nitrosoresorcinol, 430 mg. pyrolu~ite and 0053 ml. sulphuric acid are reacted analogously to kxample 4 to give 1-methylresazurin~4-carboxylic acid.
Yield: 0.8 g.
The ~o obtained l-~ethylresazurin-4-carbQxylic acid i 8 reduced analogou~ly to Example 5 to give 1-methylre30rufin-4-carboxylic acid. Yield: 0.4 g.
UV/YIS: (0.1 M pota3~ium phosphate buffer, pH 7.9):

5 S71 nm.
max ~ .
Re orufin-4-carboxylic aci ~ ~
1) N~0~0-Triacetyldihydroresorufin-4-carboxylic acid.
~.
S g, (19.4 Emole) Re~oruin-4-carbo~ylic acid or 5.3 g. (1~.4 mmole) re~azurin-4-carboxylic acid are heated under reflux for 30 minutes in 100 ml. of 1~%
hydrochloric acid wi~h 7 g. (38 mmole~ tin dichloride.
The ~olution thereby ~ecome~ gree~ coloured. It i~
allowed to cool, the precipitated dihydrore~orufin-4-carboxylic acid i8 filtared off u~der an ab~o~p~ere of nitrogen and then dried in a ~acu~ over pho~phoru~
pentoxid~. ~he cr~de produc~ thu~ obtai~d ~ hea~ed - 32 ~ 2 ~

under reflux for 30 minutes wQth 30 ml. acetic anhydride and 20 mg. sodium acetate. The reaction mixture i~ introduced into 200 ml. of ice wat~r and ~tirred for 14 hours. T~e pre~ipitate obtained i8 filtered off and recry~tallised from aqueou~ e~hanol.
There are obtained 4.8 g. (65% of theory~ of the desired compound, m.p. 197 - 199C. TLC (silica gel, elution agent: chloroform/meth~nol/glac~al acetic acid 9:1:0.1 v/v/v~, Rf - 0.33 }0 Variant b)~
5.1 g. (20 mmole) Re~orufin-4-carboxylic acid are stirred for 1 hour at 80C, in 20 ml. acetic anhydride with 11 g. (60 mmole) tin dichloride. The reacti.on mixture is introduced into 230 ml. ice water, ~tirred for 1 hour, filtered and then wor~ed up analogou31y to Variant a). Yield: 5.4 g. ~71% of theory).
2) ~S~0-Triacet ~ rboxylic acid chloride.
3.85 g. (10 m~ol~ 00O-triacetyldihydro-re~orufin-4-carboxylic acid are mixed with 5~4 ml.
(60 mmole) oxalyl chloride and cooled to -10C. A
drop of dimethylformamide i~ added thereto and the r~action mixture allsw~d to warm up to amblent t2m~-erature, while stirring, th~ ~duct thereby di~solvi~g with th~ evolution of gas. Aft~r ending of th~ ga~
evolution, stirring i~ continued for 30 ~inut~ th~

evaporated, tak~n up 3 time~ with 20 ml, amounts of dry methylene chloride and evaporated to dryne~s.
~here ar~ thu~ obtained 4 g. of crude product which i9 further worked up witlhout further purification.
TLS ~silica gel, elution agent chloroform~
methanol/glacial a~etic acid 9:1:0.1 v/v/v): R
O.42, colourle~ spot which becomes red coloured after a few hour~.
3) ~!O!O-TriacetYldihydrore~oruf i 4-carbGxylic acid morpholideO
11.3 g. (31.4 mmole) of the crude acid chloride are di~olved in 150 ml. dry methylene chloride.
8.7 ml. ~63 mmole) Tri~thylamine are added dropwQse thereto, followed ky 3.3 ml. (37.7 mmole~ morpholine.
~he reaction mixture iQ further stirred for 2 hour~, the 801ution i~ wa~hed wnth 1% aqueoua citric acid ~olution, aqueous sodium bicarbonate ~olution and water and th~ organic phase i 8 dried over anhydrous magnesium sulphate and evaporated. The re~idue i8 cry~talli~ed from ethanol. Yieldo 8.1 g. (63% of theory), m.p. 133 - 135C. (decomp.).
4) ~ .
3-7 g~ t9 mmole~ Triac~tyldihydrore~orufin 4-~arboxylic acid are taken up in 250 ml. me~hanol and 250 ml. wat~r. 36 ml. 1 ~ aqu~ou~ ~odiu~ hydroxide 301ution and 6~0 g. (18 mmol~) potas~ium ferriey~nide are added ther~to and ~he react~o~ mixtuxe i~ ~tirred for 14 hour~ at ambient temperature. After acidifying wnth hydrochloric acid to pH 3, the solution i~
evaporated to dryne~ and the re~idue i3 dige~ted wit~
~cetone. q~he dyestuff ~olution i~ filterad over 500 ~1. ~ilica gel 3 using acetone as elution age~t.
After e~aporating th~ dye~tuff-containing eluate~
ther~ ar~ obtained 2.3 g. (8~% of theory) of the desired product.
W ~VIS (0.1 M potas~ium phosphate buffer, pR 7.5):

~ ~ 575 nm, - 55,000 cin2 mol~l.
max TL~ (elution agent, see under 2) above), R~ = 0.52~
lH-~MR (~D]6-DMSO): ~ - 3.3 - 3.8 (m, 8H), 6.50 (d, J - 2 Hz, ~H), 6.64 (do J - 10 Hz, 1~, 6.76 ~dd, J , 10 and 12 Hz, lH~, 7.44 and 7.51 ~in each ca~e d, J _ 10 ~, 2~). .
~.
Methyl tetraacet~lre~orufin-l-carboxylate ,8-~

6.8 g. Methyl re30rufin-l-carboxylate ~prepared according to Bxample 3), 5.75 g. ~ilver oxide, 6.75 g.
silver carbonate, 15 gO ~olecular ~ieve 4 ~ and 10 g.
a bro~otetraacetylgalactose are ~tirred for 4 hours at a~bient ~perature in 250 ml. anhydrous chloroform.
25 After the addition of a further 5 g. a~bro~otetraacetyl-galacto~e, sti rring i 8 continued oYernight O ~h8 re~ctlo~ mixture i8 filtered ~hrough a gla~ f~br~

2~3 . - 35 -filter and the filtrate is evaporated. The oily crude product i~ chromatographed on 2 litres of ~ilica gel with chlorofonm/ethyl acetate (2:1 v/v) a~ elution agent. 2.5 g. of a yellow fraction are eluted with Rf = 0.2~ (HPTLC ~ilica gel, wnth the same el~tion agentt. After stirring with methanolO there i~
obtained methyl tetraacetylresorufin-9-carkoxylate ~-D-galactopyranoside in the form of orange-coloured cry~tals. Yield: 1.5 g.
l~_~MR ([D]6-DMSO): ~= 1.95, 2.03, ~.04 and 2.14 (in each case ~, 12H), 3.88 ~, 3H), 4.11 (d, J = 7 Hz, 2H), 4.51 (t, J - 7 Hz, 2H), 5.24 (m, 2~) 5.36 (m, lH~, 5.69 (m, l~)o, 6.31 (d, J = 2 Hz, lH), 6.97 (d, J = 2 H2, lH), 7.04 (dd, J _ 8.8 and 2,4 Hz, lR3; 7.14 (d, J = 2.4, lH), 7.78 (dd, J _ 8.8 H~, lH).
Thereafter, there i~ eluted 1.6 g. of a fraction with Rf - 0.24 which i3 also yellow. After recrystall-i~ation from methanol, there i~ obtained m~thyl tetra-acetylr~sorufin-l-carboxylate ~-D-galactopyranoside in 20 the fonn of yello~orange cry~tals~, Yield 1. 2 g.
MR ([D]6-l~ 1.95, 2.029 2.04 a~d 2.14 ~in each ca~e ~, 12H), 3.90 (~, 3H), 4.09 (m, 2~; 4.51 (m, lH3, 5~24 (d, J L 7 Rz, lH), S.25 (mO lH); 5~.36 (m, lH~, 5.71 (m, lH), 6~50 ~do J :~ ~ Ha:, lH), 6.83 (dsl, J = 10 and 2 ~I;c, lH), 7.20 and 7024 (ir~ each ca~.
d, J -- 2 Hz, 2H~ ,o 7.47 (d, J -- 10 ~Iz" lH3u - 36- ~ 253 setween the two pure product~, there c:an also be eluted a mixed fraction from which, by recryAtall-i~tion from methanol" there can be obtained 2.5 g.
of cry3tals of a mixture of the two i~o~eric compound~.
In analogou~ manner~ there i8 obtained from a-bromotetraacetylgalactoqe and:
a) methyl 4-methoxyresorufin-1-carboxylate, meth~l tetraacetyl-4-methoxyresorufin-1-carbox~late ~D-qalactop~r,anosid,e and methYl tetraacet~l-6-methoxy-resorufin-9-carb_xy~te ~ ~ .
b) methyl 8-chlororesorufin l-carboxylate, methy~
tetraacetYl-8-chlo-ror~e~-o~rufin-l=carbox~late ,~
,aalactopYranoside and methyl,_tetraacet~yl-2-chloro-~.
1~ ~a~-Tetraacety~lre ~ rufin-4 carboxYlic acid rpholide ~.
3.26 g~ ~10 mmole) Re~oruf~n-4-carboxylic ac~d morpholid~ ar~ gal~cto~idat~d ~nalogou~ly to kxample 8.
The crude product i 8 chroMatographed on 1 litre of ~ilica gel with ethyl acetat~/acato~e (3:1 v/v) as elution agent. There ar~ thu~ obtained 0. 9 g~ tetra-a~:etylr~orufin-6-carboxylic acid morpholide ,B-D-25 galac!topyranosid~, 1~ (3ilica yel, elution agent 8Bxampl~ 7), R~ . 0.71, and 0.4 yO ta~raac~ylreson 4-carboxylic acid m~rpholide ~ galaetopyr~o~ide?, ~2~2~;~3 TLC (silica gel~ the 3ame elution agent~ Rf = O.76, as well as 1.2 g. of a mixed fraction of the two i~omer~.
Example 10.
Methy~ orufin-1-carboxylate ~-D-qal topyxano~ide.
1.2 g. Methyl tetraacetylre~orufin-9-carboxylate ~-D-galactopyransside are deacetylated with ~odium methylate/methanol analogou31y to kxample 2. Yield ~.8 g.
W /VIS (0.1 M potas~ium pho~phate buffer, pH 7.5):
max = 464 nm ( ~ _ 21.8 cm2 mol~l).
After ~plitting with ~-galactosida~e, ther~ iq obtained the anion of methyl re30rufin-l-carboxylate:

= 572 nm ( ~ 65.4 cm2 mol~l).
max lN-NMR t[D~6-DMSO): ~ ~ 3.20 - 3.80 (m, 6H), 3.91 (8, 3H), 5.09 (d, J - 7.5 Hz, lH), 6.30 (d, J =
2.1 Hz, lH), 6.81 (dd, J ~ 9.8 and 2.1 Hz, lH~:

7.30 (m, 2H) 7~51 (d, J 3 0.8 Hz, lH), OH protons very broad at 5.
In an analogou~ ma~ner, by deacetylation of the corresponding tetraacetat~, there are obtained:
a) methyl r~sorufin-g-carboxylat~ ~-D-galactopyrano3ide 1~-~MR (ED]6 DMSO) s - 3.4 - 3.7 (m, 6H~, 5.09 ~d, J 7.5 HZ, 1H), 6.34 (d, J ~ 2 HZ, 1H~: 6097 (d, J Y 2 ~z, lH), 7.08 - 7.17 (m, 2H), 7075 ld, J = 10 ~z, lH), OH: very broad at 5 pp~.

.

- 38 - 125~5:~

b) methyl 4-me~hoxyre~o~ufin-1-carboxylate ~-D-galactopyrano~ide c) m~thyl 6-methoxyresorufin-9-carboxylata ~-D-galactopyranosiae d) methyl 8-chlororesorufin-1-carboxylate ~-D-galactopyrano-~ide e) methyl 2-chlorore~orufin-9-carboxylate ~-D-galactopyranoside f) re30ntfin-6-carboxylic acid morpholide ~-D-galactopyrano~ideR~ ~ethyl acetate/i30propanol/water 9:4:2 v/Y/Y): 0.3 After ~plitting with ~-galactosidase:
W /VIS (0.1 M pota~ium phosp~ate hufferg pH 7.5):

~; = 574.6 nm max Floure~cence emis~ion: ~ ~ax 593 nm g) re90rufin-4 carboxylic acid morpholide ~-D-galactopyrano~ide Rf (ethyl acetate/isopropanol/water 9:4:2 v/v/v): 003 After ~plitting with ~-galacto~idase.
~0 W /VIS ~0.1 M pota~sium phosphate buffer, p~ 7.5):
~ aX ~ 574.6 ~m Flourescence emi~ion: ~max - 593 ~m.

trieth lammonium ~altO
~ .
266 ~g~ Methyl r~soru in-9-c~rboxylat~ ~-D-yalactopyrano~id~ ~r~ taken up i~ 50 ~1. watsr and 1~5~253 20 ml4 1,4-dioxan. 5 ml. O.lN aqueous sodium hydroxide Qolution are added thereto in portions of 0.5 ml., the pH value of the ~olution not keing allowed to exceed 12.50 The reaction mixture i8 then applied to 6 ml.
DEAB-Sephadex*in ~he carbonate form. It i~ wa~hed with 180 ml. water. Ther~after, the product i~ eluted with 0.1 M triethylammoni~m carbonate buffer (pH 7.5). The eluate i3 evaporated in a vacuum. Thereafter, it i8 evaporated ~everal time3 with ethanol. Yield: 110 mg.
resorufin-9-carboxylic acid ~-D-galactopyrano~ide triethylammonium salt.
W /VIS (0.1 M pota~ium pho~phate buf~er: pH 7.5):

= 465 nm max After splitting with ~-galacto~id~e:
lS ~ max = 570 n~.

Ex~ele 12.
Re~azurin ~-D-qalactopyrano~id~.
12.6 g. of ths sodium ~alt of resazurin are di~solved in 65 ml. 1~ aqu~ou~ sodium hydroxide ~olution and 80 ml. wat~r. 20~6 g. Acetobromo-a-D-galactose and 18.2 g. hexadecyltrimethylam~oniu~ bromide in 150 ml.
chlorofonm ar~ add~d thereto. ~he mixture i3 then hea~ed under reflux for 3 hours. Thereafter, it i3 evaporated to dryness and the r~idue i9 dige~ted with 2S ethyl acetateO ~he ethyl acetate solution i~ agai~
evaporated and chromatographed on S00 g. silica gel (eluent: methylene ch~oride/ethyl ac~tat~ 3:1 v/v~.

* trade mark - 40 ~ 53 ~here ar~ obtained 2.13 g. tetraacetylresazurin-~-D-galactopyrano~ide with Re = . 5 (HP~LC silica gel, ~he same elution aç~en~ ) .
For deacetylation, 2~13 g. of the tetraacetyl 5 derivative are 3tirred wi~h 0.1 g. ~odium methylate in 100 ml. anhydrou~ methanol for 1 hour at ~l~bient te~perature. The precipitated product is filtered off with ~uction and dried in a vacuu~ over anhydrou~
calcium chloride. Yield 1.0 g.
R~ ~ 0.62 (HPTLC ~ilic~ gel: ethyl acetate/i opropanol/
water 9:4:2 v/v/v) ~_~MR ([D]6-DMSO)~ ~ - 3.30 - 3.90 tm, 6H), 4.54 (br. d, J ~ 4.4 Hz, lH), 4.67 (br~ t, J = 4.4 Hz, lH), 5.07 ~d, J ~ 7 Hz, lH), 5.27 ~br. d, J = 4.4 Hz, lX) 6.15 (d, J ~ 2 ~z, lH), 6.63 (dd, J ~ 9.6 and 2 ~z, 1~) 7.10 (dd, J ~ 9 and 2 Hz~ lH), 7.2 (m, 2H), 7.96 (d, J ~ 9.6 ~z, lH), 8.07 (d, J ~ 9 Hz, lH).

8-Ch ~ .
4.3 g. ~30 mmol~) 4-~hlorore~orci~ol are di~-solved in 20 ml~ ethanol. After th~ addition of 2.4 g~
pot~-~ ium hydroxide, the mixture i~ cooled to 5~.
Whil~ cooling, 2.81 ml. isopentyl nitrite are addçd ther~to dropwis~. Thereafter, the reaction mixture i8 furth~r stirr~d overnight at ambient temperature. ~he precipitate obtained i8 ~iltered offO di~olved in wat~r and a~idifi~d. The y~llow pr~cipitate obtained 5~3 i3 again filtered off and dried in a vacuum at 40~C.
Yieldo 2.5 g. (4~% of theory) 8-chloro-6-nitroso-resorcinol.
Rf: 0.28 (HPTLC, ~ilica g~l, elution agent: methanol/
ethyl acetate 1:3 v/v).
l.B3 g. 4-Chloro-6-nitrosore~orcinol, 1.S5 g.
2,6-dihydroxybenzoic acid~ 0.86 g. pyrolu~ite and 1.06 ml. sulphuric acid are introduced into 20 ml.
methanol and stirred for 2 hour~ at 0C. and for 14 hours at ambient temperature. A red precipitate i~
obtained which i~ filtered off and dried. Yield:
2.45 g. (8~% of theory) 8-chlororesazurin-4-carboxylic aci~.
W /VIS ~0.1 M pota~ium phosphate buffer, pH 7.5~:

A = 62105 nm.
max In an analogous manner, there is obtained from:
a) 2-methylresorcinol, via 2-methyl-6-nitro~oresorcinol, 6-methylr~sazurin-4-carboxylic acid b) 4-methylre~orcinol, via 4-me~hyl-6-nitrosoresorcinol~
8-methylresazurin-4-carboxylic acid c) 4-bromoresorcinol, via 4-bromo-6-nitrosoresorcinol, 8-bro~ore~azurin-4-carboxylic acid.
.

~.
2 g. 8-Chlorore~azurin-4-carboxylic acid (prepar~d according to ~xample 10) are dissolv~d i~ 20 ~1~ w~t~r and 5 ml. 25% aqueou~ ammonia ~olution. Zinc powd~r i~

added thereto, wnth ice cooling, until co~plete cQlour change to red-violet. ~x~e~ zinc i~ filtered off.
After acidification9 the precipitated product i8 filtered off and dried in a vacuum at 40C. o~er anhydrou~ calcium chloride. Yield 1.5 g. (7gX of theory3 8-chlororeYorufin 4-carboxyli~ acid.
W/YIS (0~1 M pota~sium phoqphate buffer, p~ 7.5):
aX ~ 585.5 nm In an analogouR manner, there is obtained fro~
a) 6-methylresazurin 4-carboxylic acid, 6-methyl-re~oxufin-4-carboxylic a~id b) 8-methylre~azurin-4-carboxylic acid, 8-methyl-resorufin-4-carboxylic acid c) 8-bromore azurin-4-carboxylic acid, 8-bromo-re~orufin-4-carbo~ylic acid.
EX~.
Methyl re~orufin-9-carboxylate a-D~alactop~ranoside.
3.9 g. Pentaacetylgalacto~ are mix2d with 9.1 g.
anhydrou~ zinc chloride and h¢at~d to 125C. Th~
mixture is stirred for 20 minute~ at 10 m~.Hg. 1 g.
Methyl resorufin-l-carboxylat~ i~ added to the ~elt.
The mi~ture i~ ~tirred for 1 hour at 41C. and there~
after chromatographed on ~ilica gel with chloroform/
ethyl ~etat~ ~2:1 v/v) in ths mann~r d~3crib~d in : 25 ~xample 7. Yield: 25 ~g. m~thyl tetraacetylr~orufin-

9-carboxylate a-D-galactopyranoside (yellow oil)0 Rf - 0~22 ~PTL~ ca g~l~ ~h~ e ~lution aga~t).

l ~ ~2~ ~ ~ 3 The deacetylation to give methyl resorufin-9-carboxylate -D-galactopyrano~ide ia carried out analogously to Example lO.
Example l6.
Resorufin-9-carbo~ylic acid ~3,6=dioxaoct~l~ ester ~-D-qalacto~ o~ide.
0.6 g. Methyl tetraacetylresorufin-9-carboxylate ~-D-galactopyrano~ide i~ mixed with 50 ml. die~hylene glycol monoethyl ether and a ~patula tip o~ sodium hydride and stirred for 15 minute~ at ambient temper-ature. Thereafter, lO0 ml. acetone are added theretoO
The precipitate obtained i~ filtered off and dried.
Yield: 56 mg~ resorufin-9-carboxylic a~id (3,6-dioxa-octyl~ ester ~-D-galactopyrano~ide.
R~: 0.54 tsilica gel HPTLC, elution agent: ethyl acetate/isopropanol 9:4 v/v)0 Example 17 Resorufin-maltoheptaoside In addition to a hydrolytic and cyclising action amylase, zo for example amylase ~rom Bacillus macerans, (E.C.2.4.1.19) also has glycosyl-transferring properties which can be utilised for the synthesis of oligosaccharides and derivatives thereof (see Methods in Carbohydrate Chemistry 11 ~1963) p.347).

680 mg.amylase from Bacillus macerans DSM 24 (lyophilisate; 0.46 U/mg,weighed amount, protein content of the wei~hed amoun~ 28.5 ~0).
500 mgOResorufin-glucoside 3.5 g.~-cyclodextrin 70 ml,Soerensen phosphate buf~e~ (pH 6.2; 0.01 M) are mixed.
The batch is incubated for 24 hours at 37 C. For purification, ~cyclodextrin and formed ~-cyclodextrin are first separated off by means of the tetrachloroethylene inclusion compound.
After chromatography on cross-linked dextran ~"Sephadex"*LH 20), there are obtained 50 mg. of lyophilisate of Resorufinyl-maltoheptaoside, which is highly active in the amylase assay.

~.
15 Determinatio the activity~ y~o3ida~3e.
a) Preparation of th~ ~olutions u~ed:

H~PES 100 mmole~litre 90diUI~ chloride154 mmole~'litre 20 magne~ium I~a~3partate 2 n~nole/li$re bovin~ ~rum al~umin10 gO /litre q~e~* 20 0.5 g./litre pH valu~ ~adjust~d with aqueou~
~odium hydroxide 801UtiOJ1~ 7.3 ~37Co) * trade mark ~2~7Z~

e~u~
0.8 mmole/litre resorufin ~-D-galactopyrano~ide are dis~olved in the above de~cribed buf~er ~olution.
Reaaen _~olution 2:
305 mmole/litre re30rufin-9-carboxylic acid ~-D-galactopyrano~ide are dissolved in the above-described buffer ~olution~
eaaent solution 3:
1.O mmole/litre methyl resorufin-9-carboxylate ~-D-galactopyrano~ide are dissolved in the above-de~cribed buffer ~olution.
En~yme solution:
Co~mercially available ~-D-galacto~idase from E~cherichia coli is di-Ysolved in th~ above-described buffer ~olution. The activity o~ thi~ ~olution i~ about 0.08 U/ml. (referred to the statements of the producer~0 b) Carryin~ out of the measurements:
The mea3urement take~ place photometrically at 579 nm.
950 ~1. of reagent are mixed in a 1 cm. cuvette at 37Co wqth 50 ~1. enzyme ~olution. As a mea~ure for the reaction, th~re i~ determined the extinction increase per unit ti~e in ~mExt/min~ Calculation is made from the mea~ured extinction by division by the reaction time.
In ~he following Table are ~et out the m~a~ur~-m~nt values found:

~2~2~

Reagent ~o. Reaction ~mExt/minJ
. . - _ ~ 46 . . , __ _ _ _ _ Exampl~ 1 9.
Detecti~ of ~=~yalac:to-Yidase with the help of an indicator f i lm .
~e following component~ are worked up to give

10 a homogeneous mas~:
5 mlO of a~ aqueous solution with 0O SM potas~ium phosphate and 0.05M magneElium chlorid~ (p~ 7. 3) 0.13 g. sodium alginate 15 8 g. of a 50% di~per~ion of polyvinyl propionate 10 g. ~iliCZI g~l 12 ml. wat~r 0.4 g. ~riton* X-lV0 50 mg. re~orufin ~ galactopyranosid~, di~solved in 20 S ml. ~thanol.
Thi8 ma~ applied to ~ 0.1 n~n. thick pvly carbonate fil~ (Pokalon*, LonYa ~G) with a ~trip br~adth of 0~, 2 mm~ coating ~ ~ drled at 50C. and t}ler~-after cut up into piec~ dimen~ion~ of 6 ~c 25 6 mm. With ~h~ h~lp of an ad~es~ strip, th~ ns ~tuclc o~ to a 400 ~LM. thick poly~tyrane fil * trade mark 5 ~

The te9t strip~ ~o obtained are dipped for a ~hort time into the ~-D-galactosida~e-containing solution to be te~ted. After a waiting time of 2 minute~
at ambient temperature, a red reaction colour has fonmed, 5 the inten~ity of which dependq upon the concentration of the ~-D~galacto~ida3e in the te~t 801ution. With the help o~ te3t ~olution~ ~ith a definite, known content of ~-D-galacto idase, a colour ~cale can be obtained on the basis of which the unknown content of ~-D-galacto3ida~e in a ~ample can ba determined.
The above-de~cribed te~t strips can al~o be u~ed kinetically to determine the ~-D-galactosidase activity pre~ent in a sample with the help of reflection photo-metry. For thi~ purpo~e, too, i~ the u~ual way, on the ba~i9 of sample3 with knvwn ~-D-galactosida~e activitie~ there can be produced a calibration curve with the help of which an unknown ~-D-galactosidase activity of a s~mple can be determined.
~xample 20_.

~.
a) ~ on of the ~olution~ u~ed:
Buffer ~olution:
H~P~S 100 mmole/litre ~odium chlorid~154 mmole/litr~
magnesiu~n L-~partat~2 msl~le/litre bovin~ ~eru~ alblL~inlt) g O /litr~

~2~2~

Twee~ 20 0.5 g./litre p~ value (ad~u~ted wnth an aqueous ~olution of ~odium hydroxide3 7~3 (37 C.) eaqen~t ~olution:
0O8 mmole/litre re~orufin ~-D-galactopyrano3ide i~ di~olved in the above-de~cribed bu~fer.

Commercially available ~-D-galactosida~e from ~cherichia coli i-q dis-qolved in buffer. The activity of thi~ ~olution is about 0.08 U/ml. (referred to the ~tatement of the producer).
Enzyme coniuqate ~olution:
A ~-D-galacto3ida~e-antibody preparation i~ used.
The preparation of such enzyme-antibody conjugate~ i~
known. It iq de~cribed, for example9 in Biochem.
Biophy3. Acta, 6 , 40-49/1980. The preparakion i~
diluted with buffer in such a manner that there i3 obtained an activity approximately comparable with ~h~
above de~cri~ed enz~me solution.
b) ~
The measurement is carried out photometrically at 578 nm. 950 ~lo reayent 301ution are, in each case, mixed in a 1 cm. cuvette at 37C. with 50 ~1. of enzym~
solu ion o~ with 50 ~1. enzyme-conjugate ~olution. A~
a m~asure for th~ reaction, there i~ determi~ed the exti~ction increa~e psr unit tim~ ln [~kxt/min]~

~2~~2S:~
- 4~ -For the reaction wqth the free ~D-galactosida9e, there i~ mea~ur~d 85 mExt/min and for ~he reaction with ~-D-galacto~idase-antibody conjugate 92 mExt/min.
Both mea~urement valu~-~ show that not only wQth the free but al90 with the conjugated ~-D-galacto~idase, there ~ found a very readily mea~urable extinction difference.
It follow~ from thi~ tha~ the described compounds ~an be u~ed as ~ubstrates not only for free glyco~ida~e~
but al~o for glycosida~e conjugates in the ~am~ way.
Thu~, the new ~ub~trates can be used not only as diagno~tic agent~ for the determina~ion of free glyco-~ida~e~ but they can al90 be used in an advantageou~
manner in the case of immunological method~ of determin-ation in which a glyco~ida~e i 8 used as indicatorenzyme.

Example 21 ation of the activit of ~-am lase Determln y y Filter paper is impregnated with a solution of resorufin maltoheptaoside (produced according to example 17) in a citrate buffer solution, p~ 6. To the impregnated test paperare successively added the probe containing ~ amylase as well as ~- und ~-glucosidase. After a few minutes the development of a red colour can be observed, The intensity Of this colour is proportional to the concentration of ~-amylase in the pro~e.

- ~ 50 ~ ~ ~ 5~

Measuring p~obes with known concentrations of ~-amylase a calibration cu~ve can ~e pTepared. By means of this calibration curve unknown concentrations of ~-amylase in probes can be determined.

In the preceding Examples, the following abbrevi~tion~ have been u~ed:
HEP~S - 2-[4-(2-hydroxyethyl)-l-piperazinyl)-ethane-sulphonic acid Twee~ 20 - polyoxyethylene(20) sorbitan monolaurate.
The German Patent Specification referred to herein is more particularly identified below:
Federal Republic of Germany~ Patent 2,118,455, issued April 12, 1973, Hans Lange et al., assigned to Boehringer Mannheim GmbH.

* trade mark

Claims (39)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Glycosides of resorufin derivatives of the formulae;
(Ia) (Ib) wherein R1 is a hydrogen atom; R2, R3 and R5, which can be the same or different, are hydrogen or halogen atoms or lower alkyl radicals; R4 and R6, which can be the same or different, are hydrogen or halogen atoms, cyano groups, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxy carbonyl lower alkyl radicals or carboxamido, mono-substituted carboxamido and di-substituted carboxamido, the substituent of said mono-substituted carbox-amido being selected from alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonyl radicals, in which the alkyl moieties con-tain 1 to 5 carbon atoms, and the substituents of said di-substituted carboxamido being selected from alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonylalkyl radicals, in which the alkyl moieties contain 1 to 5 carbon atoms, or said two sub-stituents being joined together to form a ring uninterrupted or interrupted by hetero atoms selected from oxygen, nitrogen and sulphur, or radicals of the formula -COO-(CH2CH2O)n-R7, wherein R7 is a hydrogen atoms or a lower alkyl radical; and n is a whole number from 1 to 4; and R6 can additionally also represent a sulphonyl or nitro group; and Y is a nitrogen atom or the group N?O, the O-glycoside being other than a neuraminic acid moiety.

2. Glycosides according to claim 1, wherein the glycoside residue is a .beta.-D-galactopyranoside, ?-D-galacto-pyranoside, .beta.-D-glucopyranoside, ?-D-glucopyranoside or ?-D-mannopyranoside residue.

3. Glycosides according to claim 1, wherein the glycoside residue is an oligosaccharide with 2 to 10 mono-saccharide units.

4. Methyl 4-methoxyresorufin-1-carboxylate .beta.-D-galacto-pyranoside.

5. Methyl 6-methoxyresorufin-9-carboxylate .beta.-D-galacto-pyranoside.

6. Resorufin-.beta.-D-galactopyranoside.

7. Resorufin-.beta.-D-glucoside.

8. Methyl tetraacetylresorufin-1-carboxylate .beta.-D-galactopyranoside.

9. Methyl tetraacetylresorufin-9-carboxylate .beta.-D-galactopyranoside.

10. Tetraacetylresorufin-4-carboxylic acid morpholide .beta.-D-galactopyranoside.

11. Tetraacetylresorufin-6-carboxylic acid morpholide .beta.-D-galactopyranoside.

12. Methyl resorufin-1-carboxylate .beta.-D-galactopyranoside.

13. A compound selected from the group consisting of methyl 4-methoxyresorufin-1-carboxylate .beta.-D-galacto-pyranoside, methyl 6-methoxyresorufin-9-carboxylate .beta.-D-galactopyranoside, methyl 8-chlororesorufin-1-carboxylate .beta.-D-galactopyranoside, methyl 2-chlororesorufin-9-carboxylate .beta.-D-galactopyranoside, and resorufin-6-carboxylic acid morpholide .beta.-D-galactopyranoside.

14. Resazurin .beta.-D-galactopyranoside.

15. Methyl resorufin-9-carboxylate ?-D-galactopyrano-side.

16. Resorufin-9-carboxylic acid (3,6-dioxaoctyl)ester B-D-galactopyranoside.

17. Resorufin-maltoheptaoside.

18. A process for the preparation of glycosides of resorufin derivatives of the formulae:
(Ia) (Ib) wherein R1 is a hydrogen atom; R2, R3 and R5, which can be the same or different, are hydrogen or halogen atoms or lower alkyl radicals; R4 and R6, which can be the same or different, are hydrogen or halogen atoms, cyano groups, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxycarbonyl lower alkyl radicals, or carboxamido, mono-substituted carboxamido and di-substituted carboxamido, the substituent of said mono-substituted carbox-amido being selected from alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonyl radicals in which the alkyl moieties contain 1 to 5 carbon atoms, and the substituents of said di-substituted carboxamido being selected from alkyl, alkoxyalkyl, carboxy-alkyl and alkoxycarbonylalkyl radicals, in which the alkyl moieties contain 1 to 5 carbon atoms, or said two substituents being joined together to form a ring uninterrupted or inter-rupted by hetero atoms selected from oxygen, nitrogen and sulphur, or radicals of the formula -COO-(CH2CH2O)n-R7, wherein R7 is a hydrogen atom or a lower alkyl radical; and n is a whole number from 1 to 4; and R6 can additionally also rep-resent a sulphonyl or nitro group; and Y is a nitrogen atom or the group N?O, the O-glycoside being other than a neuraminic acid moiety, comprising:
reacting a compound of the tautomeric formulae:

(IIa) (IIb) in which R1 to R6 and Y are as defined above, with a mono-or oligo-saccharide or a 1-halogeno-derivative thereof, all the hydroxyl groups of which are substituted by protective groups of carbohydrate chemistry, to give the corresponding per-0-substituted glycoside from which the protective groups are split off to give a glycoside of a resorufin derivative of formula (Ia) or (Ib).

19. A method for determining the activity of a glyco-sidase in a fluid, comprising splitting a glycoside of formula (I), as defined in claim 1, wherein the glycoside moiety corresponds to said glycosidase, with said glycosidase, in a sample of said fluid, and evaluating the resulting resorufin derivative residue as a measure of the glycosidase activity of said fluid.

20, A method according to claim 19, wherein said evaluating comprises a photometric determination of said residue.

21. A method according to claim 19, wherein said evaluating comprises exciting said residue to fluorescence and measuring the fluorescence.

22. A method for the determination of the activity of a saccharide chain splitting enzyme in a fluid comprising:
splitting a first glycoside of formula (I), as defined in claim 3, having a first saccharide chain, with a saccharide chain splitting enzyme to be determined in a sample of the fluid to produce a second glycoside of formula (I), as defined in claim 3 with a second saccharide chain having less monosaccharide units than said first chain, splitting the resulting glycoside with a glycosidase, and evaluating the resulting resorufin derivative residue as a measure of the enzyme activity of the fluid.

23. A method according to claim 22, including a step of degrading said second glycoside to produce a third glycoside of formula (I), with a shorter saccharide chain prior to splitting with said glycosidase

24. A diagnostic agent for the detection of glyco-sidases containing at least one substrate selected from chromogenic, fluorogenic, and chromogenic and fluorogenic substrates, and a buffer substance, wherein the substrate is a glycoside of a resorufin derivative of formula (I), as defined in claim 1.

25. An agent according to claim 24, further including adjuvants selected from wetting agents, galenical additives and stabilizers.

26. A diagnostic agent for the detection of saccharide chain splitting enzymes containing at least one substrate selected from chromogenic, fluorogenic and chromogenic and fluorogenic substrates, a buffer substance, and a glycosidase wherein the substrate is a glycoside of a resorufin derivative of formula (I), as defined in claim 1.

27. A diagnostic agent according to claim 26, further including an adjuvant selected from wetting agents, galenical additives and structure formers.

28. A diagnostic agent according to claim 26 or 27, wherein said derivative of formula (I) has a glycoside residue comprising an oligosaccharide with 2 to 10 mono-saccharide units, and further including an enzyme effective to degrade said residue to a lesser number of monosaccharide units.

29. Resorufin derivatives of the tautomeric formulae:
(II'a) (II'b) wherein R1' is a hydrogen atom; R2', R3' and R5' which can be the same or different, are hydrogen or halogen atoms or lower alkyl radicals; R4' and R6', which can be the same or different, are hydrogen or halogen atoms, cyano groups, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxy carbonyl lower alkyl radicals, or carboxamido, mono-substituted carboxamido and di-substituted carboxamido, the substituent of said mono-substituted carboxamido being selected from alkyl, alkoxy-alkyl, carboxyalkyl and alkoxycarbonyl radicals in which the alkyl moieties contain 1 to 5 carbon atoms, and the substi-tuents of said di-substituted carboxamido being selected from alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonylalkyl radicals, in which the alkyl moieties contain 1 to 5 carbon atoms, or said two substituents being joined together to form a ring uninterrupted or interrupted by hetero atoms selected from oxygen, nitrogen and sulphur; or radicals of the general formula-COO-(CH2CH2O)n-R7, wherein R7 is a hydrogen atom or a lower alkyl radical; and n is a whole number from 1 to 4, with a proviso that R1' to R6' cannot all simultaneously be hydrogen atoms; and Y is a nitrogen atom or the group N?0, provided that when Y is a nitrogen atom, R2', R3', R4' and R5' is bromine, then R6' is other than a hydrogen atom, and provided that when Y is a nitrogen atom, R2' and R5' are methyl and R3' and R4' are hydrogen atoms, then R6' is other than a hydrogen atom, and provided that when Y is a nitrogen atom, R2' and R5' are methyl and R3' and R4' are bromine, then R6 is other than a hydrogen atom, and provided that when Y is a nitrogen atom and R2', R3', R4' and R5' are all bromine atoms, then R6' is other than a hydrogen atom, and provided that when Y is a nitrogen atom, R1', R4' and R5' are hydrogen atoms and R2' and R3' are methyl, then R6' is other than a carboxyl radical.

30. A resorufin derivative of formula (Xa) or (Xb)- (Xa) (Xb) wherein R11 is a hydrogen atom; R12 , R13 and R15, which can be the same or different, are hydrogen or halogen atoms or lower alkyl radicals; R14 and R16, which can be the same or different, are hydrogen or halogen atoms, cyano groups, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxy carbonyl lower alkyl radicals, or carboxamido, mono-substituted carboxamido and di-substituted carboxamido, the substituent of said mono-substituted carboxamido being selected from alkyl, alkoxy-alkyl, carboxyalkyl and alkoxycarbonyl radicals, in which the alkyl moieties contain 1 to 5 carbon atoms, and the sub-stituents of said di-substituted carboxamido being selected from alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonylalkyl radicals, in which the alkyl moieties contain 1 to 5 carbon atoms, or said two substituents being joined together to form a ring uninterrupted or interrupted by hetero atoms selected from oxygen, nitrogen and sulphur; or radicals of the general formula -COO-(CH2CH2O)n-R7, wherein R7 is a hydrogen atom or a lower alkyl radical; and n is a whole number from 1 to 4; Y is a nitrogen atom or the group N?O; and X is hydroxyl or a glycoside-O- radical other than a neuraminic acid moiety;
provided that when X is hydroxyl R11 to R16 are not all hydrogen atoms; and provided that when X is said glycoside-O- radical R16 can also represent a sulphonyl or nitro group, and provided that when Y is a nitrogen atom, X is a hydroxyl, and R12, R13, R14 and R15 are bromine, then R16 is other than a hydrogen atom, and provided that when Y is a nitrogen atom, X is hydroxyl, R12 and R15 are methyl and R13 and R14 are hydrogen atoms, then R16 is other than a hydrogen atom, and provided that when Y is a nitrogen atom, X is hydroxyl, R12 and R15 are methyl and R13 and R14 are bromine, then R16 is other than a hydrogen atom, and provided that when Y is a nitrogen atom, X is hydroxyl, and R12, R13, R14 and R15 are all bromine atoms, then R16 is other than a hydrogen atom, and provided that when Y is a nitrogen atom, X is hydroxyl, R11, R14 and R15 are hydrogen atoms and R12 and R13 are methyl then R16 is other than a carboxyl radical.

31. Methyl tetraacetyl-4-methoxy-resorufin-1-carboxylate .beta.-D-galactopyranoside.

32. Methyl tetraacetyl-6-methoxy-resorufin-9-carboxylate .beta.-D-galactopyranoside.

33. Methyl tetraacetyl-8-chlororesorufin-1-carboxylate .beta.-D-galactopyranoside.

34. Methyl tetraacetyl-2-chlororesorufin-9-carboxylate .beta.-D-galactopyranoside.

35. Methyl resorufin-9-carboxylate .beta.-D-galactopyranoside.

36. Resorufin-4-carboxylic acid morpholide .beta.-D-galacto-pyranoside.

37. Resorufin-9-carboxylic acid .beta.-D-galactopyranoside triethylammonium salt.

38. Resorufin derivatives of the tautomeric formulae:
(II'a) (II'b) wherein R1' is a hydrogen atom; R2', R3' and R5' which can be the same or different, are hydrogen or halogen atoms or lower alkyl radicals; R4' and R6', which can be the same or different, are hydrogen or halogen atoms, cyano groups, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxy carbonyl lower alkyl radicals, or carboxamido, mono-substituted carboxamido and di-substituted carboxamido, the substituent of said mono-substituted carboxamido being selected from alkyl, alkoxy-alkyl, carboxyalkyl and alkoxycarbonyl radicals in which the alkyl moieties contain 1 to 5 carbon atoms, and the substi-tuents of said di-substituted carboxamido being selected from alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonylalkyl radicals, in which the alkyl moieties contain 1 to 5 carbon atoms, or said two substituents being joined together to form a ring uninterrupted or interrupted by hetero atoms selected from oxygen, nitrogen and sulphur; or radicals of the general formula-COO-(CH2CH2O)n-R7, wherein R7 is a hydrogen atom or a lower alkyl radical; and n is a whole number from 1 to 4, with a proviso that R1' to R6' cannot all simultaneously be hydrogen atoms; and Y is a nitrogen atom or the group N?O, and provided that at least one of R4' and R6' is selected from the group of cyano, lower alkoxy, lower alkoxy carbonyl, carboxy lower alkyl, lower alkoxycarbonyl lower alkyl, carboxamide or carboxamide substituted once or twice by alkyl, alkoxyalkyl, carboxyalkyl or alkoxycarbonyl alkyl where the alkyl moieties contain 1 - 5 carbon atoms or in the case of two substituents said two sub-stituents can also form an uninterrupted ring or a ring inter-rupted by a heteroatom selected from the group of oxygen, nitrogen or sulfur, or is a radical of the general formula -COO-(CH2CH2O)n-R7 wherein R7 is a hydrogen atom or a lower alkyl radical and n is 1 to 4.

39. A resorufin derivative of formula (Xa) or(Xb)- (Xa) (Xb) wherein R11 is a hydrogen atom; R12 , R13 and R15, which can be the same or different, are hydrogen or halogen atoms or lower alkyl radicals; R14 and R16, which can be the same or different, are hydrogen or halogen atoms, cyano groups, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxy carbonyl lower alkyl radicals, or carboxamido, mono-substituted carboxamido and di-substituted carboxamido, the substituent of said mono-substituted carboxamido being selected from alkyl, alkoxy-alkyl, carboxyalkyl and alkoxycarbonyl radicals, in which the alkyl moieties contain 1 to 5 carbon atoms, and the sub-stituents of said di-substituted carboxamido being selected from alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonylalkyl radicals, in which the alkyl moieties contain 1 to 5 carbon atoms, or said two substituents being joined together to form a ring uninterrupted of interrupted by hetero atoms selected from oxygen, nitrogen and sulphur; or radicals of the general formula -COO-(CH2CH2O)n-R7, wherein R7 is a hydrogen atom or a lower alkyl radical; and n is a whole number from 1 to 4; Y is a nitrogen atom or the group N?Q; and X is hydroxyl or a glycoside-0- radical other than a neuraminic acid moiety;
provided that when X is hydroxyl R11 to R16 are not all hydrogen atoms; and provided that when X is said glycoside-0- radical R16 can also represent a sulphonyl or nitro group, and provided that at least one of R14 and R16 is selected from the group of cyano, lower alkoxy, lower alkoxy carbonyl, carboxy lower alkyl, lower alkoxycarbonyl lower alkyl, carboxamide or carboxamide substituted once or twice by alkyl, alkoxyalkyl, carboxyalkyl or alkoxycarbonyl alkyl where the alkyl moieties contain 1 - 5 carbon atoms or in the case of two substituents said two sub-stituents can also form an uninterrupted ring or a ring inter-rupted by a heteroatom selected from the group of oxygen, nitrogen or sulfur, or is a radical of the general formula -CCO-(CH2CH2O)n-R7 wherein R7 is a hydrogen atom or a lower alkyl radical and n is 1 to 4.