Note: Descriptions are shown in the official language in which they were submitted.
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
-- 4 --
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
R3 R4
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 R6
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
-- 10 --
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
53
-12-
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
-13-
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
53
--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
-15-
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
-16-
~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.
7253
-- 19 --
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~
- 22 -
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~
- 24 -
~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
- 26 -
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-
- 28 -
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
- 29 -
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
- 30 -
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~
53
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
53
- 34 -
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
39 -
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
- 41 -
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~
- - 42 -
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
- 43 -
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).
- 44 -
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~
- 45 -
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~
- 46 -
Reagent ~o. Reaction
~mExt/minJ
. . - _
1 85
~ 46
3 76
. . , __ _ _ _ _
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 ~
- 47 -
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~
- 48 -
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
