CA2099542A1 - Photochemical labelling of nucleic acids with europium chelate reagents and their use in gene probe test systems - Google Patents
Photochemical labelling of nucleic acids with europium chelate reagents and their use in gene probe test systemsInfo
- Publication number
- CA2099542A1 CA2099542A1 CA 2099542 CA2099542A CA2099542A1 CA 2099542 A1 CA2099542 A1 CA 2099542A1 CA 2099542 CA2099542 CA 2099542 CA 2099542 A CA2099542 A CA 2099542A CA 2099542 A1 CA2099542 A1 CA 2099542A1
- Authority
- CA
- Canada
- Prior art keywords
- alkyl
- labelling
- alkoxy
- dna
- independently
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
- G01N2458/40—Rare earth chelates
Abstract
Photochemical labelling of nucleic acids with europium chelate reagents and their use in gene probe test systems Abstract The present invention concerns photochemical labelling reagents comprising a lanthanide ion-chelating structure and a furocoumarin derivative bound via a spacer. The labelling reagent can be used in gen diagnostic.
Le A 29 093
Le A 29 093
Description
2~99~2 Gene probe diagnostics is a method for the sequence-specific detection of DNA/RNA sequences. It is based on the hybridisation of the gene probe sequence with comple-~entary sequence regions of the DNA~RNA to be detected [J.A. Matthews, L.J. Kricka, Analytical Biochemistry 169, 1-25 (1988); U. Landegren, R. Kaiser, C.T. Caskey, L. Hood~ Science 242, 229 (1988)].
Gene probe diagnostics makes possible the detection of infectious diseases and genetic defects. Prerequisites for the broad application of gene probe diagnostics are adequate sensitivity of detection, simplicity in perform-ance and the avoidance of radioactivity.
One variant of gene probe diagnostics proceeds by way of the direct photochemical labelling of the DNA/RNA to be detected; subsequently hybridisation occurs to gene probes with complementary nucleic acid sequences [N. Dattagupta, P.M.M. Rae, E.D. Huguenel, E. Carlson, A. Lyga, J.S. Shapiro, J.P. Albarella, Analytical Bio-chemistry 177, 85 (1989); J.P. Albarella, R.L. Minegar/
W.L. Patterson, N. Dattagupta, E. Carlson, Nucleic Acids Research 17, 4293 (1989)].
Furocoumarins which are linked to biotin by way of suitable spacer molecules have been shown to be very suitable for the photobiotinylation of nucleic acids.
After hybridisation to a gene probe with a complementary Le A 29 093 - 1 -, .. .
:
' ~
~" ~
, 2~99~L2 nucleic acid sequence, and a separation step, detection -takes place, for example by addition of a complex of antibiotin-antibody or avidin or streptavidin with alkaline phosphatase. For the cletection, a colour reac-tion, which is elicited by alkaline phosphatase, iscarried out in an additional step [J.J. I.eary, D.J. Brigati, V.C. Ward, Proc. Natl. Acad. Sci. USA 80, 40~5-~059 (1983)].
A disadvantage of the detection system using biotin is the wide distribution of biotin in biological systems.
A possible alternative would be direct photolaballing of the DNA/RNA to be de~ected, for example using a fluore-scent dye. However, this has been found not to be prac-ticable under the conditions of the photoreaction, because of preferential energy wasting. In addition, a suitable label would have to be photoinert.
Surprisingly, lanthanide chelates which are linked to suitable furocoumarins by means of a spacer have been found to be suitable.
Lanthanide chelates, in particular europium chelates, are already being routinely employed in immunodiagnostics [P. Degan, A. Abbondandolo, G. Montangnoli, J. of Bio-luminescence and Chemiluminescence 5, 207 (1990)]. A
particular advantage of using them is the possibility of time-resolved measurement of fluorescent light. Their application in gene probe diagnostics has now also been Le A 29 09-3 - 2 -:~, ' ~ :
-.
2~99~2 described for the first time [A. Oser, W.K. Roth,G. Valet, Nucleic Acids Res., 3, 1181 (1988)], though in this work labelling with europium chelate reagents takes place using a costly procedure. In addition, the use of S europium chelate primers for the PCR reaction has been described [P. Dahlen, A. Iitia, V.-M. Mukkala, P. Hurs-kainen, M. Kwiatkowski, Molecular and Cellular Probes 5, 143-149 (1991)].
According to the invention, a labell.ing reagent of the general formula Ln-S-Fu is synthesised, where:
Ln = a lanthanide ion-chelating structure, S = a spacer molecule and 5 Fu = a furocoumarin derivative as a photochemically linkable structure.
The lanthanide ion-chelating structure (Ln) is a pyridine derivative of the formula /~ --C02R
~-X-~ ,N
~, ~co2R
- N
\~02R
where Le A 29 093 - 3 -~ j, 1 . ~ . . ~ . .
.
.
2~9~9L2 X represents C5- to C14-arylene, optionally containing ~ hetero atom grouping, or C1- to Cz4-alkylene containing hetero atom groupings [N, O, S ~lx, more than once)], Y and optionally X ~ Y represents N-oxysuccinimido, N-maleimido, NH2~ OH, COCH2-halogen, halogen, NCO, NCS, CHO, COOH, SH, CO-halogen, COOCOR1, CH=CHC02R1, ~ , - S- S ~ CH2--CH2 o ~ 503H
-N J -C- N
o where R1 represents hydrogen,- a saturated or unsatu-rated C1- to C20-alkyl radical, optionally substitu-ted by a phenyl gxoup, or a phenyl group, R represents, in each case independently of the others, hydrogen, ammonium or an equivalent of an alkali metal or a 1/2 equivalent of an alkaline earth metal.
The synthesis of the pyridine derivative Ln takes place according to methods which are known per se [see, e.g., F. Vogtle and C. Ohm, Chem. Ber. 117, 849 to 854 (1984);
.:
Le A 29 09~ _ 4 _ ,.
!
' ;' . . . ' ` ` ' .
~ ' ' . ' ' ' ' ~';'" ' ' ' ' ' ' ' ' ' 2099~42 R. Singh and G. Just, J. Org. Chem. 54, 4453 (1989)].
The spacer is a polyalkylamine, a polyethylene glycol or a combination of these.
Polyalkylamines have the follo~ing general formula:
~ R
-N--L - t CH2 ) X N--]~f where R represents H, Cl-C7-alkyl, aryl (such as, e.g., phenyl, naphthyl or anthracyl ), hydroxyl or C~-C7-alkoxy;
x represents a number between 2 and 7;
y represents a number between 3 and 10.
R can occur differently in the possible variants mentioned above, i.e. it must not be identical for each R
repetition of the -(CH2)~-N- unit in the spacer. The same J iS also the case for x, i.e. x must not be identical for each repetition of the (CH2)X- unit in the spacer.
.
Preferably the Rs, independently of each other, = H, Cl-C4-alkyl (e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, tert-butyl); x = 2, 3, 4 or 5; and Le A 29 0~3 - 5 -: .. : , . ... .
~ , - -~, , . .. . : ~ . .
'' : ' . :
2~99~2 y = 3, 4, 5 or 6.
Particularly preferred are N-4,N-9-dimethylspermine derivative~ of the formula H ¦ 1 3 -N-~CH2)3-N-(C~)4-N-~cH2)3 N
Polyethylene glycols have the fc)llowing general formula -o-[-(cH2)x-~)-]
where x is = 2, 3, 4 or 5 and y is = 3, 4, 5 or 6.
Preferred are polyethylene glycols with x = 2, 3, 4 or 5;
y = 3, 4, 5 or 6. Particularly preferred are polyethylene glycols with x = 2 and y = 4, 5 or 6.
Spacer molecules with combined amine/glycol structures have the following general formula:
-Zl ~ (CH2)X-~2 3y-1 ~CH2)X-?.3-where Zl, Z2 and Z3, independently of each other, represent O
or NR, Le A 29 093 - 6 -'~
2099~2 R represents H, Cl-C7-alkyl, aryl (such as, e.g., phenyl, naphthyl or anthracyl), hydroxyl or Cl-C7-alkoxy;
x represents a number between 2 and 7;
y represents a number between 3 and 10.
Preferred are spacer structures with ZZ = O and zl, Z2 =
NR where the Rs = H, Cl-C4-alkyl (e.g. mel:hyl, ethyl, n-propyl, n-butyl, i-butyl, tert-butyl); x = 2, 3, 4 or 5; and y = 3, 4, 5 or 6.
Particularly preferred are structures with ZZ = O, zl z3 = NR, R = H, methyl, ethyl, x = 2, y = 6-Suitable photochemically linkable structures are inparticular furocoumarins, such as, for example, angelicin (isopsoralen) or psoralens as well as derivatives of these which react photochemically with nucleic acid.
Le A 29 093 - 7 -.. . .
` `
:
' .
~: :
2099~i~2 Angelicin deri.vatives hava the followi.ng general formula:
R2 Rl O ~r R~
where Rl, R2 and R3, independently of each other, represent H or Cl-C7-alkyl, and R4 represents H, Cl-C7-alkyl or a lower alkyl with hydroxyl, Cl-C7-alkoxy, amino, halo or N-phthalimido substituents.
Particularly preferred are angelicin derivatives which contain the following Rl-R4 groupings:
Rl R2 R3 R4 __ ___________________________________________ H H H H
~H3 H CH3 CH20CH3 H H CH3 CH2Cl H H C~I3 C~2 -O
Le A 29 093 - 8 -,.. .,.: ~ .
, . - . . .
- . ., :
, : ~ - , ~ ,: . :
- :
. :;:- . . ..
2099~2 Other compounds with different R's may also be synthes-ised by processes known from the literature.
Suitable psoralens have the following general formula:
~2 R
1 '~
R~:~R6 R5~o where Rl, R3 and R~, independently o:E each other, represent H
or Cl-C7-alkyl, R4 represents H, Cl-C7-alkyl or Cl-C7-alkyl with hydroxyl, Cl-C7-alkoxy, amino, halo or N-phthalimido substituents, R2 and R5, independently of each other, represent H, hydroxyll carboxyl, carbo-Cl-C7-alkoxy or Cl-C7-alkoxy.
-Angelicin deri~ati~es are ad~antageous in comparison with psoralens because of the monoadduct formation.
The sequence of the binding of the lanthanide ion chela-ting agent, the spacer and the furocoumarin is arbitrary.
It is thus possible, inter alia, first to link the chelating agent Ln wi~h the spacer S and subsequently to ~, . .
. .
Le A 29 093 - 9 - ~
::
~: . , , ' ':
:
:~ - : :
20995~2 react the product with the furocoumarin Fu. Conversely, Fu-S may first be constructed and then reacted with Ln.
The linking of the moieties is effected in a manner known per se.
Examples Example la !
Preparation of 2,6-biR[N,N-bis(t-butoxycarbonylmethyl)-; amino-methyl]-4-(5-hydroxypent-1-inyl)pyridine (1):
--C02t Bu --N 1n = 3 ~02t~u HO----~CH2~n~ ~----C02tBu 2= 9 ~2 Bu 6 g (10 mmol) of 2,6-bis[N,N-bis(t-butoxycarbonylmethyl)-amiino-methyl3-4-bromopyridine (prepared as described by H. Tokalo, P. Pasanen and J. Kaukare in Acta Chem. Scand.
Ser. B 42, (1988) 373) are dissolved in a mixture of freshly distilled tetrahydrofuran, 15 ml, and 15 ml of trie~hylamine. The solution is degassed and 1 g (12 mmol) of 5-hydrox~pent-1-ine is introduced. The catalyst, consisting of a mixture of 280 mg (0.4 mmol) of bis(tri-phenylpho~phine)palladium(II) chloride, 840 mg (3.2 mmol) of ~riphenylphosphine and 117 mg (0.61 mmol) of Cu(I) iodide, is added at room temperature and with stirring.
The reaction is complete according to TLC after 7 hours Le A 29 093 - 10 -,~ ~
.: .
, of refluxing. After cooling to room temperature and subsequent filtration, the solution is concentrated in vacuo and chromatographed over silica gel (eluent: ethyl acetate, R~ = 0.61).
4.6 g (68% of theory) are obtained of a slightly yellow-ish solid with a melting point of 90C.
Example lb) Preparation of 2,6-bis[N,N-bis(t-butoxycarbonylmethyl)-amino-methyl-4-(11-hydroxyundec-1-inyl)pyridine (2):
6 g (10 mmol) of 2,6-bis[N,N-bis(t-butoxycarbonyl-methyl)amino-methyl]-4-bromopyridine are reacted with 1.93 g (12 mmol) of l-undecin-10-ol under the action of Pd catalysis in analogy with Example la)~ After chromato-graphy on silica gel (eluent: ethyl acetate, R~ = 0.52), 7 g (77% of theory) are obtained of a yellow solid with a melting point of 57 to 59C.
Example 2a) Preparation of 2,6-bislN,N-bis(t-butoxycarbonylmethyl)-amino-methyl]-4-(5-hydroxypentyl)pyridine (3):
/--C02~BU
~--N 3 n = 5 ~C ~02tBu HO--(CH2)n ~ ~--co2~Bu 4 = 11 ~--C2 Bu Le A 29 093 - 11 -- :: :~ . ~ :
.
::
~099~42 472 mg (0.7 mmol) of the compound 1 described in Example 1 are dissolved in 20 ml of abs. ethano:L and 2~ mg of 10%
Pd/C are added. The solution is ~igorously stirred at 45 to 50C under positive hydrogen pressure. The reaction is complete within 1 hour (accordin~ to TLC). After cooling and removal of the catalyst, the solution is concentrated in vacuo and the residue is chromatographed on silica gel (eluent: ethyl acetate, RE = 0.52). 242 mg (51% of theory) are obtained of a slightly yellowish oil.
An improvement of the yield (56% of theory) i9 achieved if PtO2 is used as the catalyst under the same reaction conditions.
Example 2b) Preparation of 2,6-bis~N,N-bis(t-butoxycarbonylmethyl)-amino-methyl-4-(11-hydroxyundecyl)pyridine (4):
1.0 g (1.32 mmol) of the compound 2 described in Example lb) is hydrogenated with PtO2 catalysis (100 mg) in analogy with Example 2a). After chromatography on silica gel (eluent: chloroform/ethanol 15 : 1, R~ ~ 0.4), 725 mg (72~ of theory) are obtained of a slightly yellowish oil.
Example 3 ; Preparation of a m i n o - P E G - a n g e 1 i c i n ( 5 ) Le A 29 093 - 12 -., '~'1` ' ' ' ,, , ' ' ' . . ' - . ' . ~ ~'''., ''`" ~' ' '' .
20995~2 Om~ S
H H
NH~
Il (C2~0)5" " "
4.87 g (20 mmol) of 4'-aminomethyl~4,5'-dimethylangelicin are dissolved in 25 ml of DME and reacted with 3.24 g (20 ml) of carbonyldiimidazo]Le at room temperature.
Complete reaction (according to TLC) was observed after 6 hours of stirring under nitrogen. The solution is slowly added dropwise to a solution of 16.85 g (60 mmol) of 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane in 40 ml of DMF at 80C and the mixture stirred at 70C for a further 12 hours. After cooling, the solution i5 concen-trated in vacuo and chromatographed on silica gel (eluent: chloroform/methanol/ammonia 90:10:1, Rf = 0.28).
7.1 g (65% of theory) are obtained of a slightly yellow oil.
,~
Le A 29 093 - 13 -,, ~.:
, : . , , . . .
20995~2 ExamPle 4~!
Preparation of Ln-S-Fn ester (6):
~ C02~Bu H H H~ ~-CO ~Bu r~N~ M~w~o-(cH2)n~-~ ,N 2 Il (C2~ )5~ ~ o ~ ~C02t3u \--C02t~Bu 6 n ~ 5 7 = 11 250 mg (0.37 mmol) of the compound 3 described in Example 2a) are dissolved in 3 ml of dry toluene. 65 mg (0.4 mmol) of carbonyldiimidazole are added. After 17 hours of stirring at 60C under N2, 3 is completely reacted (according to TLC, eluent: chlorofoxm/ethanol 15:1, Rr = 0.45) and a new product has formed (eluent:
see above, R~ = 0.63). 220 mg (0.4 mmol) of the compound 5 described in Example 3 are added and the reaction mixture is stirred at 90C for a further 24 hours. After cooling, the solution is concentrated in vacuo and the residue is chromatographed on silica gel (eluent:
toluene/ethanol 5 : 1, R~ = 0.36). 138 mg (30% of theory) -are obtained of a slightly yellow oil.
Le A 29 093 - 14 -~,~ . . . .
:' ' ' j' , ~ ' ' ~ ~, ,~ ' ' ,' , ' ' : ' ~`, . : "
.: : ' ' ' .
209~2 Example 4bl Preparation of Ln-S-Fn ester (7):
410 mg (0~54 mmol) of the compound 4 described in Example 2b) are activated with carbonyldiimidazole and subse quently reacted in analogy to Example 4a) with the amino-PEG-angelicin 5 described in E,xample 3). After chroma-tography on silica gel (elue:nt: toluene/ethanol 5:1, Rf = 0.31), 188 mg (26% of theory) are obtained of a yellowish oil.
Example 5a!
Preparation of Ln-S-Fn-tetracarboxylic acid (8):
~[~0 ~C02H
L~ C2 H 40 ) 5~ CH2 ) n~2 ~ n - 5 138 mg (0.11 mmol) of the tetraester 6 de cribed in Example 4a) are dissolved in 4 ml of dry benzene and 569 mg (5 mmol) of trifluoroacetic acid are added under N2. After 2 hours of stirring a~ 60C, the product Le A 29 093 - 15 -.
, .:
:, ~ , 2099~'12 separates out in benzene as an oil. According to TLC the reaction is complete. After cooling, the solution is concentrated in vacuo. The residue is dissolved in 5 ml of distilled water and extracted by shaking twice with 3 ml of diethyl ether. The aqueous phase is concentrated and chromatographed on RP 18 (eluent: methanol, R~ =
0.13). 70 mg (62% of theory) are obtained of a milky, viscous oil.
Example 5b !
Preparation of Ln-S-Fn tetraacid ~9):
45 mg (0.034 mmol) of the tetraester 9 described in Example 4b) are reacted with trifluoroacetic acid in analogy with Example 5a). 30 mg (81~ of theory) are obtained of a viscous oil.
Example 6 Preparation of N1-(angelicinamido)-N4,N9-dimethylspermine (10):
0~
H H
Le A 29 093 - 16 -.. . .
, ~ . ..
. ~ ~
, ~ . , . .
2~)99~2 4.87 g (20 mmol) of 4'-aminomethyl-4,5'-dimethylangelicin are activated with carbony].diirnidazole in analogy with Example 1. The resulting solution is added dropwise to a solution of 13.8 g (60 mmol) of N4,N9-dimethylspermine in 40 ml of DMF in anology with Example 1. After cooling, the solution is concentrated in vacuo and the residue is chromatographed on silica gel (eluent: chloroform/
methanol/ammonia 30:5:1, Rf = 0.11). 7.1 g t71% of theory) are obtained of a yellow oil.
Le A 29 093 - 17 -, .
: , .
: ~ ~
'.: :'~ .- ~ ' 20995~2 Il J
~o , h ~) ~0 ~0~ ;
Le A 29 093 - 18 -~, .~ : . , 2099~2 318 mg (0.5 mmol) of the compound 4 described in Example ~b are activated with carbonyldiimidazole and subse-quently reacted with the amino compound 10 described in Example 6 in analogy with Example 4. After chromatography on silica gel (eluent: chloroform/methanol/ammonia 70:45:1, Rf = 0.42), 132 mg (21% of theory) are obtained of a yellow oil.
Example 8 Preparation of Ln-S-Fn tetraacid (12): (EuPA) 30 mg (0.023 mmol) of the tetraester 11 described in Example 7 are reacted with trifluoroacetic acid in analogy with Example 5. 22 mg (92% of theory) are obtained of a yellow oil.
Example 9 Photoreaction of hairpin oligonucleotides with EuPA (12) 50 ~g of the hairpin oligonucleotide are taken up in 100 ~l of Tris-ECl buffer. The solution is left in the waterbath at 50C for 15 minutes. To permit 510w cooling to room temperature, the sample is taken out of the waterbath. Subsequently a further 400 ~l of water are added.
For the photoreaction, a 20-fold molar excess of EuPA is added to 15 ~g of the hybridised hairpin oligonucleotide.
:`
~ Le A 29 09~ - l9 -., .
~ ., . ~ . . : .
:~ ~ , , . , ~ :
.,: . , : . , 2099~42 The solution ls subsequently illuminated under a W lamp a~ 312 nm or 366 nm in an Eppendorf tube. The photo-reaction is followed using HPLC. Within 15 minutes, the pho toreaction was comple~e.
; 5 Example 10 Photolabelling with EuPA (12) For the photolabelling, 50 ~1 of 1 M sodium tetraborate buffer pH 8.3 and 50 ~1 of EuPA (2 ~g/~l) were added to 2 to 5 ~g of DNA in 20 ~1 of TE buffer and the solution made up to 500 ~1 with double-distilled H20. The mixture was irradiated for 10 minutes at 312 nm using a W trans-illuminator, with the samples being kept on ice during this period.
The photolabelled DNA was subsequently precipitated with 1/10 volume of 3 M sodium acetate-pH 5.8 and 1 volume of isopropanol at room temperature and left to stand for 5 minutes. Subsequently, the DNA was centrifuged down at 10,000 rpm in an Eppendorf centrifuge, the supernatant decanted off and the DNA precipitate washed with 70%
; 20 strength ethanol. After the samples had been dried, the photolabelled DNA was taken up in T~. The photolabelling of the DNA with EuPA was subsequently monitored by agarose gel electrophoresis and microtitre tests.
. , .
~ i' ~ Le A 29 093 - 20 -.
: , - . - . .
2~9~2 Ex~mple 11 Detection of -the EuPA labelling in the microtitre test To detect the EUPA labelling of double-stranded DNA, the DNA was pipetted, after the labelling, into microtitre test plates in concentrations of 250 ng to 125 pg in 1:2 dilution steps. To attach the DNA to the polystyrene groups of the microtitre wells, the DNA was first diluted in the wells with PBSM buffer (10 mM Na phosphate pH 7.2 with 0.1 M MgCl2, 0.15 M NaCl, 3 M KCl) and incubated at room temperature overnight. Washing 2x with 200 ~1 of PBSM buffer subsequently took place, and the DNA was fixed to the wells by 10-minute irradiation with a W
transilluminator at 312 nm. The DNA fixed in this way was subsequently washed 4x with wash-concentrate buffer from Delfia/Pharmacia, in order to remove excess EuPA loaded with europium. As the negative control, unlabelled double-stranded DNA was treated in the same manner.
Following addition of 100 ~1 of enhancement solution from Wallac/Pharmacia, the tLme-resolved fluorescence of europium was measured, after 30 minutes at room tempera-ture, in a DELFIA 1232 fluorescence photometer from Wallac/ Pharmacia at 290 to 360 nm excitation/615 nm emission. Depending on the dilution of the DNA, fluore-scence signals of 212,000 to 1,700 were measured in the labelled DNA. The unlabelled DNA only gave low background - signals.
~.
Le A 29 093 - 21 -.
:
2~995~2 Example 12 Hybridisation of EuPA-labelled genomic DNA in the reversed phase test The preparation of EuPA-label:led DNA was carried out according to the method described in Example 10.
The hybridisation was carried out by conventional pro-cesses at an incubation temperature of 40 to 68C.
Different substances were added depending on the hybrid-isation temperature. With long gene probes, dextran sulphate or other polymers were employed in order to increase the speed and extent of the hybridisation.
Detergents and blocking reagents, such as dried milk, Denhardt's solution, heparin or SDS, were employed in order to suppress the non-specific binding of the DNA to the membrane. Denaturing agents, such as urea or form-amide, may be employed in order to reduce the melting temperature of the hybrid, so that lower hybridisation temperatures may be used. In addition, the non-specific binding of probes to non-homologous DNA on the blot may be reduced by the addition of heterologous DNA.
To prepare for the hybridisation, 100 ng of the unlabelled E.coli-specific gene probes (1.7 kb to 6 kb) were first denatured at 100C for 5 minutes, cooled to 0C, and khen transferred to pre-treated nitxocellulose or nylon membranes using a Minifold-II filtration apparatus from Schleicher and Schull and fixed at 80C
Le A 29 093 - 22 -.
- , -: ~ ;
;.~ ' ~ " ' ' ~
2099~42 for 2 hours.
The filters were hybridised in a sealed plastic film bag or plastic box with at least 20 ml of hybridisation solution per lO0 cm2 of filter at 68C for at least l hour.
The solution was replaced by 2.5 ml of hybridisation solution of lO0 cm2 of ilter 1;o which solution freshly denatured (100C, 5 minutes), EuPA-labelled, genomic DNA
from E.coli (l ~g) had been added. The filters were incubated at 68C for at least 6 hours with gentle shaking.
The filters were then washed 2 x 5 minutes at room tem-perature with at least 50 ml of 2xSSC, 0.1% SDS per lO0 cmZ of filter and 2 x 15 minutes at 68C with O.lxSSC, 0.1% SDS.
The filters were then directly employed for detecting the hybridised DNA. Depending on whether EuPA-DNA was used which was already loaded with europium, or which was subsequently loaded with europium, the following further i 20 steps were carried out in working up the filters for the fluorescence read-out. In the case of EuPA-labelled genomic DNA which was not loaded with europium, the filters were treated in lO0 ~M EuCl, 100 ~M EDTA and lxSSC pH 7.0 in a total volume of 2 ml at room tempera-ture for 2 hours. The filters were then washed six times with 2xSSC. Subsequently, the individual slots of the Le A 29 093 - 23 -., .
:~ .
... .
' ' ' ' ' -209~2 hybridisation blot were cut out and treated with 1 ml ofenhancement solution i.n 1.5 ml reaction tubes. After a 30-minute incuba-tion at room temperature, 200 ~1 of the samples from the individual slots were pipetted into microtitre plates and the samples were measured in a DELFIA 1232 fluorescence photo~eter from Wallac/Pharmacia at 290 to 360 nm excitation and 615 emission.
In the case of slo~ blots with EuPA-labelled DN~, which had been loaded with europium before the labelling, the indi~idual slots were cut out direc~ly after the hybrid-isation and 1 ml o enhancement solution wa~ added to them in 1.5 ml reaction tubes and then, as described above, the enhancement solution was added and measurement took place in a fluorescence photometer.
Solutions:
: ~0 x SSC: 3M NaCl, 0.3 M Na citrate pH 7.0 Hybridisation solution: 5xSSC; 0.1% N-lauroylsarcosine, Na salt, 0.02% SDS; 0.5% block-ing reagent (Boehringer), dissolve ~he solution at 50 to : 70C.
Example 13 Hybridisation with EuPA-labelled gene probe~
The preparation of Eu~A-labelled gene probes (1.7 to Le A 29 093 - 24 -2099!~2 6 kb) was car~ied out according to the method described in Ex~mple 10.
The EuPA-labelled gene probes may be employed in solid-phase or liquid hybridisations. Suitable solid phases S are, for example, nitrocellulose membranes, nylon membranes, polystyrene groups of microtitre plates or magnetic particles. The fluorescent hybridisation com-plexes of gene probes with complementary genomic DNA may be separated from free fluorescent gene probes using hydroxyapatite.
For example, a slot~blot hybridisation was carried out with EuPA-labelled, E.coli-specific gene probes (1.7 kb to 6 kb) and genomic DNA from E.coli.
For this purpose, the genomic E.coli DNA was denatured at 100C for 5 minutes and then cooled to 0C and then transferred to nitrocellulose or nylon membranes using a Minifold-II filtration apparatus from Schleicher and Schull in the concentrations 500 ng to 125 pg in 1:2 dilution steps. The prehybridisation and hybridisation were carried out as described in Example 12. 100 ng of EuPA-labelled E.coli gene probe were employed.
The read-out took place as described in Example 12 by individual measurement of the excised filter slots after treatment with enhancement solution in a DELFIA 1232 fluorimeter from Wallac/Pharmacia.
Le A 29 093 - 25 -,, .
~ ~ .
2099~i~2 Using the gene probes, 125 ng of genomic DNA from E.coli were Still readily detectable. This corresponds to a test sensitivi~y of 0.1 pg of DNA measured in the hybridisa-tion of pure pBR322 plasmid probe to pBR322 DNA.
Alternatively, microtitre hybridisation tests were carried out. For this purpose, the genomic E.coli DNA was denatured as described above, and then diluted samples of 10 ng to 45 pg were pipetted into microtitre wells and left to stand at room temperature overnight. Subse-quently, washing took place 2 x with 200 ~l of PBSMbuffer and the DNA was then fixed for 10 minutes at 312 nm using a W transilluminator. 200 ~l of hybridisa-tion solution (Example 12) witll 10 ng of EuPA-labelled gene probe were added and the hybridisation mixture was incubated at 68C for at least 6 hours. Subsequently, the microtitre wells were washed 2 x 5 minutes at room temperature with 2 x 200 ~l of 2 x SSC, 0.1 SDS and 2 x 15 minutes at 50C with 2 x 200 ~l of 0.1 x SSC, 0.1%
SDS.
The read-out took place as in Example 12 in a DELFIA 1232 fluorimeter from Wallac/Pharmacia after treatment of the wells with 100 ~l of enhancement solution.
Le A 29 093 - 26 -: ' ~ -. - : - ' - ' ; . ~ . ~ , . . .
Gene probe diagnostics makes possible the detection of infectious diseases and genetic defects. Prerequisites for the broad application of gene probe diagnostics are adequate sensitivity of detection, simplicity in perform-ance and the avoidance of radioactivity.
One variant of gene probe diagnostics proceeds by way of the direct photochemical labelling of the DNA/RNA to be detected; subsequently hybridisation occurs to gene probes with complementary nucleic acid sequences [N. Dattagupta, P.M.M. Rae, E.D. Huguenel, E. Carlson, A. Lyga, J.S. Shapiro, J.P. Albarella, Analytical Bio-chemistry 177, 85 (1989); J.P. Albarella, R.L. Minegar/
W.L. Patterson, N. Dattagupta, E. Carlson, Nucleic Acids Research 17, 4293 (1989)].
Furocoumarins which are linked to biotin by way of suitable spacer molecules have been shown to be very suitable for the photobiotinylation of nucleic acids.
After hybridisation to a gene probe with a complementary Le A 29 093 - 1 -, .. .
:
' ~
~" ~
, 2~99~L2 nucleic acid sequence, and a separation step, detection -takes place, for example by addition of a complex of antibiotin-antibody or avidin or streptavidin with alkaline phosphatase. For the cletection, a colour reac-tion, which is elicited by alkaline phosphatase, iscarried out in an additional step [J.J. I.eary, D.J. Brigati, V.C. Ward, Proc. Natl. Acad. Sci. USA 80, 40~5-~059 (1983)].
A disadvantage of the detection system using biotin is the wide distribution of biotin in biological systems.
A possible alternative would be direct photolaballing of the DNA/RNA to be de~ected, for example using a fluore-scent dye. However, this has been found not to be prac-ticable under the conditions of the photoreaction, because of preferential energy wasting. In addition, a suitable label would have to be photoinert.
Surprisingly, lanthanide chelates which are linked to suitable furocoumarins by means of a spacer have been found to be suitable.
Lanthanide chelates, in particular europium chelates, are already being routinely employed in immunodiagnostics [P. Degan, A. Abbondandolo, G. Montangnoli, J. of Bio-luminescence and Chemiluminescence 5, 207 (1990)]. A
particular advantage of using them is the possibility of time-resolved measurement of fluorescent light. Their application in gene probe diagnostics has now also been Le A 29 09-3 - 2 -:~, ' ~ :
-.
2~99~2 described for the first time [A. Oser, W.K. Roth,G. Valet, Nucleic Acids Res., 3, 1181 (1988)], though in this work labelling with europium chelate reagents takes place using a costly procedure. In addition, the use of S europium chelate primers for the PCR reaction has been described [P. Dahlen, A. Iitia, V.-M. Mukkala, P. Hurs-kainen, M. Kwiatkowski, Molecular and Cellular Probes 5, 143-149 (1991)].
According to the invention, a labell.ing reagent of the general formula Ln-S-Fu is synthesised, where:
Ln = a lanthanide ion-chelating structure, S = a spacer molecule and 5 Fu = a furocoumarin derivative as a photochemically linkable structure.
The lanthanide ion-chelating structure (Ln) is a pyridine derivative of the formula /~ --C02R
~-X-~ ,N
~, ~co2R
- N
\~02R
where Le A 29 093 - 3 -~ j, 1 . ~ . . ~ . .
.
.
2~9~9L2 X represents C5- to C14-arylene, optionally containing ~ hetero atom grouping, or C1- to Cz4-alkylene containing hetero atom groupings [N, O, S ~lx, more than once)], Y and optionally X ~ Y represents N-oxysuccinimido, N-maleimido, NH2~ OH, COCH2-halogen, halogen, NCO, NCS, CHO, COOH, SH, CO-halogen, COOCOR1, CH=CHC02R1, ~ , - S- S ~ CH2--CH2 o ~ 503H
-N J -C- N
o where R1 represents hydrogen,- a saturated or unsatu-rated C1- to C20-alkyl radical, optionally substitu-ted by a phenyl gxoup, or a phenyl group, R represents, in each case independently of the others, hydrogen, ammonium or an equivalent of an alkali metal or a 1/2 equivalent of an alkaline earth metal.
The synthesis of the pyridine derivative Ln takes place according to methods which are known per se [see, e.g., F. Vogtle and C. Ohm, Chem. Ber. 117, 849 to 854 (1984);
.:
Le A 29 09~ _ 4 _ ,.
!
' ;' . . . ' ` ` ' .
~ ' ' . ' ' ' ' ~';'" ' ' ' ' ' ' ' ' ' 2099~42 R. Singh and G. Just, J. Org. Chem. 54, 4453 (1989)].
The spacer is a polyalkylamine, a polyethylene glycol or a combination of these.
Polyalkylamines have the follo~ing general formula:
~ R
-N--L - t CH2 ) X N--]~f where R represents H, Cl-C7-alkyl, aryl (such as, e.g., phenyl, naphthyl or anthracyl ), hydroxyl or C~-C7-alkoxy;
x represents a number between 2 and 7;
y represents a number between 3 and 10.
R can occur differently in the possible variants mentioned above, i.e. it must not be identical for each R
repetition of the -(CH2)~-N- unit in the spacer. The same J iS also the case for x, i.e. x must not be identical for each repetition of the (CH2)X- unit in the spacer.
.
Preferably the Rs, independently of each other, = H, Cl-C4-alkyl (e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, tert-butyl); x = 2, 3, 4 or 5; and Le A 29 0~3 - 5 -: .. : , . ... .
~ , - -~, , . .. . : ~ . .
'' : ' . :
2~99~2 y = 3, 4, 5 or 6.
Particularly preferred are N-4,N-9-dimethylspermine derivative~ of the formula H ¦ 1 3 -N-~CH2)3-N-(C~)4-N-~cH2)3 N
Polyethylene glycols have the fc)llowing general formula -o-[-(cH2)x-~)-]
where x is = 2, 3, 4 or 5 and y is = 3, 4, 5 or 6.
Preferred are polyethylene glycols with x = 2, 3, 4 or 5;
y = 3, 4, 5 or 6. Particularly preferred are polyethylene glycols with x = 2 and y = 4, 5 or 6.
Spacer molecules with combined amine/glycol structures have the following general formula:
-Zl ~ (CH2)X-~2 3y-1 ~CH2)X-?.3-where Zl, Z2 and Z3, independently of each other, represent O
or NR, Le A 29 093 - 6 -'~
2099~2 R represents H, Cl-C7-alkyl, aryl (such as, e.g., phenyl, naphthyl or anthracyl), hydroxyl or Cl-C7-alkoxy;
x represents a number between 2 and 7;
y represents a number between 3 and 10.
Preferred are spacer structures with ZZ = O and zl, Z2 =
NR where the Rs = H, Cl-C4-alkyl (e.g. mel:hyl, ethyl, n-propyl, n-butyl, i-butyl, tert-butyl); x = 2, 3, 4 or 5; and y = 3, 4, 5 or 6.
Particularly preferred are structures with ZZ = O, zl z3 = NR, R = H, methyl, ethyl, x = 2, y = 6-Suitable photochemically linkable structures are inparticular furocoumarins, such as, for example, angelicin (isopsoralen) or psoralens as well as derivatives of these which react photochemically with nucleic acid.
Le A 29 093 - 7 -.. . .
` `
:
' .
~: :
2099~i~2 Angelicin deri.vatives hava the followi.ng general formula:
R2 Rl O ~r R~
where Rl, R2 and R3, independently of each other, represent H or Cl-C7-alkyl, and R4 represents H, Cl-C7-alkyl or a lower alkyl with hydroxyl, Cl-C7-alkoxy, amino, halo or N-phthalimido substituents.
Particularly preferred are angelicin derivatives which contain the following Rl-R4 groupings:
Rl R2 R3 R4 __ ___________________________________________ H H H H
~H3 H CH3 CH20CH3 H H CH3 CH2Cl H H C~I3 C~2 -O
Le A 29 093 - 8 -,.. .,.: ~ .
, . - . . .
- . ., :
, : ~ - , ~ ,: . :
- :
. :;:- . . ..
2099~2 Other compounds with different R's may also be synthes-ised by processes known from the literature.
Suitable psoralens have the following general formula:
~2 R
1 '~
R~:~R6 R5~o where Rl, R3 and R~, independently o:E each other, represent H
or Cl-C7-alkyl, R4 represents H, Cl-C7-alkyl or Cl-C7-alkyl with hydroxyl, Cl-C7-alkoxy, amino, halo or N-phthalimido substituents, R2 and R5, independently of each other, represent H, hydroxyll carboxyl, carbo-Cl-C7-alkoxy or Cl-C7-alkoxy.
-Angelicin deri~ati~es are ad~antageous in comparison with psoralens because of the monoadduct formation.
The sequence of the binding of the lanthanide ion chela-ting agent, the spacer and the furocoumarin is arbitrary.
It is thus possible, inter alia, first to link the chelating agent Ln wi~h the spacer S and subsequently to ~, . .
. .
Le A 29 093 - 9 - ~
::
~: . , , ' ':
:
:~ - : :
20995~2 react the product with the furocoumarin Fu. Conversely, Fu-S may first be constructed and then reacted with Ln.
The linking of the moieties is effected in a manner known per se.
Examples Example la !
Preparation of 2,6-biR[N,N-bis(t-butoxycarbonylmethyl)-; amino-methyl]-4-(5-hydroxypent-1-inyl)pyridine (1):
--C02t Bu --N 1n = 3 ~02t~u HO----~CH2~n~ ~----C02tBu 2= 9 ~2 Bu 6 g (10 mmol) of 2,6-bis[N,N-bis(t-butoxycarbonylmethyl)-amiino-methyl3-4-bromopyridine (prepared as described by H. Tokalo, P. Pasanen and J. Kaukare in Acta Chem. Scand.
Ser. B 42, (1988) 373) are dissolved in a mixture of freshly distilled tetrahydrofuran, 15 ml, and 15 ml of trie~hylamine. The solution is degassed and 1 g (12 mmol) of 5-hydrox~pent-1-ine is introduced. The catalyst, consisting of a mixture of 280 mg (0.4 mmol) of bis(tri-phenylpho~phine)palladium(II) chloride, 840 mg (3.2 mmol) of ~riphenylphosphine and 117 mg (0.61 mmol) of Cu(I) iodide, is added at room temperature and with stirring.
The reaction is complete according to TLC after 7 hours Le A 29 093 - 10 -,~ ~
.: .
, of refluxing. After cooling to room temperature and subsequent filtration, the solution is concentrated in vacuo and chromatographed over silica gel (eluent: ethyl acetate, R~ = 0.61).
4.6 g (68% of theory) are obtained of a slightly yellow-ish solid with a melting point of 90C.
Example lb) Preparation of 2,6-bis[N,N-bis(t-butoxycarbonylmethyl)-amino-methyl-4-(11-hydroxyundec-1-inyl)pyridine (2):
6 g (10 mmol) of 2,6-bis[N,N-bis(t-butoxycarbonyl-methyl)amino-methyl]-4-bromopyridine are reacted with 1.93 g (12 mmol) of l-undecin-10-ol under the action of Pd catalysis in analogy with Example la)~ After chromato-graphy on silica gel (eluent: ethyl acetate, R~ = 0.52), 7 g (77% of theory) are obtained of a yellow solid with a melting point of 57 to 59C.
Example 2a) Preparation of 2,6-bislN,N-bis(t-butoxycarbonylmethyl)-amino-methyl]-4-(5-hydroxypentyl)pyridine (3):
/--C02~BU
~--N 3 n = 5 ~C ~02tBu HO--(CH2)n ~ ~--co2~Bu 4 = 11 ~--C2 Bu Le A 29 093 - 11 -- :: :~ . ~ :
.
::
~099~42 472 mg (0.7 mmol) of the compound 1 described in Example 1 are dissolved in 20 ml of abs. ethano:L and 2~ mg of 10%
Pd/C are added. The solution is ~igorously stirred at 45 to 50C under positive hydrogen pressure. The reaction is complete within 1 hour (accordin~ to TLC). After cooling and removal of the catalyst, the solution is concentrated in vacuo and the residue is chromatographed on silica gel (eluent: ethyl acetate, RE = 0.52). 242 mg (51% of theory) are obtained of a slightly yellowish oil.
An improvement of the yield (56% of theory) i9 achieved if PtO2 is used as the catalyst under the same reaction conditions.
Example 2b) Preparation of 2,6-bis~N,N-bis(t-butoxycarbonylmethyl)-amino-methyl-4-(11-hydroxyundecyl)pyridine (4):
1.0 g (1.32 mmol) of the compound 2 described in Example lb) is hydrogenated with PtO2 catalysis (100 mg) in analogy with Example 2a). After chromatography on silica gel (eluent: chloroform/ethanol 15 : 1, R~ ~ 0.4), 725 mg (72~ of theory) are obtained of a slightly yellowish oil.
Example 3 ; Preparation of a m i n o - P E G - a n g e 1 i c i n ( 5 ) Le A 29 093 - 12 -., '~'1` ' ' ' ,, , ' ' ' . . ' - . ' . ~ ~'''., ''`" ~' ' '' .
20995~2 Om~ S
H H
NH~
Il (C2~0)5" " "
4.87 g (20 mmol) of 4'-aminomethyl~4,5'-dimethylangelicin are dissolved in 25 ml of DME and reacted with 3.24 g (20 ml) of carbonyldiimidazo]Le at room temperature.
Complete reaction (according to TLC) was observed after 6 hours of stirring under nitrogen. The solution is slowly added dropwise to a solution of 16.85 g (60 mmol) of 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane in 40 ml of DMF at 80C and the mixture stirred at 70C for a further 12 hours. After cooling, the solution i5 concen-trated in vacuo and chromatographed on silica gel (eluent: chloroform/methanol/ammonia 90:10:1, Rf = 0.28).
7.1 g (65% of theory) are obtained of a slightly yellow oil.
,~
Le A 29 093 - 13 -,, ~.:
, : . , , . . .
20995~2 ExamPle 4~!
Preparation of Ln-S-Fn ester (6):
~ C02~Bu H H H~ ~-CO ~Bu r~N~ M~w~o-(cH2)n~-~ ,N 2 Il (C2~ )5~ ~ o ~ ~C02t3u \--C02t~Bu 6 n ~ 5 7 = 11 250 mg (0.37 mmol) of the compound 3 described in Example 2a) are dissolved in 3 ml of dry toluene. 65 mg (0.4 mmol) of carbonyldiimidazole are added. After 17 hours of stirring at 60C under N2, 3 is completely reacted (according to TLC, eluent: chlorofoxm/ethanol 15:1, Rr = 0.45) and a new product has formed (eluent:
see above, R~ = 0.63). 220 mg (0.4 mmol) of the compound 5 described in Example 3 are added and the reaction mixture is stirred at 90C for a further 24 hours. After cooling, the solution is concentrated in vacuo and the residue is chromatographed on silica gel (eluent:
toluene/ethanol 5 : 1, R~ = 0.36). 138 mg (30% of theory) -are obtained of a slightly yellow oil.
Le A 29 093 - 14 -~,~ . . . .
:' ' ' j' , ~ ' ' ~ ~, ,~ ' ' ,' , ' ' : ' ~`, . : "
.: : ' ' ' .
209~2 Example 4bl Preparation of Ln-S-Fn ester (7):
410 mg (0~54 mmol) of the compound 4 described in Example 2b) are activated with carbonyldiimidazole and subse quently reacted in analogy to Example 4a) with the amino-PEG-angelicin 5 described in E,xample 3). After chroma-tography on silica gel (elue:nt: toluene/ethanol 5:1, Rf = 0.31), 188 mg (26% of theory) are obtained of a yellowish oil.
Example 5a!
Preparation of Ln-S-Fn-tetracarboxylic acid (8):
~[~0 ~C02H
L~ C2 H 40 ) 5~ CH2 ) n~2 ~ n - 5 138 mg (0.11 mmol) of the tetraester 6 de cribed in Example 4a) are dissolved in 4 ml of dry benzene and 569 mg (5 mmol) of trifluoroacetic acid are added under N2. After 2 hours of stirring a~ 60C, the product Le A 29 093 - 15 -.
, .:
:, ~ , 2099~'12 separates out in benzene as an oil. According to TLC the reaction is complete. After cooling, the solution is concentrated in vacuo. The residue is dissolved in 5 ml of distilled water and extracted by shaking twice with 3 ml of diethyl ether. The aqueous phase is concentrated and chromatographed on RP 18 (eluent: methanol, R~ =
0.13). 70 mg (62% of theory) are obtained of a milky, viscous oil.
Example 5b !
Preparation of Ln-S-Fn tetraacid ~9):
45 mg (0.034 mmol) of the tetraester 9 described in Example 4b) are reacted with trifluoroacetic acid in analogy with Example 5a). 30 mg (81~ of theory) are obtained of a viscous oil.
Example 6 Preparation of N1-(angelicinamido)-N4,N9-dimethylspermine (10):
0~
H H
Le A 29 093 - 16 -.. . .
, ~ . ..
. ~ ~
, ~ . , . .
2~)99~2 4.87 g (20 mmol) of 4'-aminomethyl-4,5'-dimethylangelicin are activated with carbony].diirnidazole in analogy with Example 1. The resulting solution is added dropwise to a solution of 13.8 g (60 mmol) of N4,N9-dimethylspermine in 40 ml of DMF in anology with Example 1. After cooling, the solution is concentrated in vacuo and the residue is chromatographed on silica gel (eluent: chloroform/
methanol/ammonia 30:5:1, Rf = 0.11). 7.1 g t71% of theory) are obtained of a yellow oil.
Le A 29 093 - 17 -, .
: , .
: ~ ~
'.: :'~ .- ~ ' 20995~2 Il J
~o , h ~) ~0 ~0~ ;
Le A 29 093 - 18 -~, .~ : . , 2099~2 318 mg (0.5 mmol) of the compound 4 described in Example ~b are activated with carbonyldiimidazole and subse-quently reacted with the amino compound 10 described in Example 6 in analogy with Example 4. After chromatography on silica gel (eluent: chloroform/methanol/ammonia 70:45:1, Rf = 0.42), 132 mg (21% of theory) are obtained of a yellow oil.
Example 8 Preparation of Ln-S-Fn tetraacid (12): (EuPA) 30 mg (0.023 mmol) of the tetraester 11 described in Example 7 are reacted with trifluoroacetic acid in analogy with Example 5. 22 mg (92% of theory) are obtained of a yellow oil.
Example 9 Photoreaction of hairpin oligonucleotides with EuPA (12) 50 ~g of the hairpin oligonucleotide are taken up in 100 ~l of Tris-ECl buffer. The solution is left in the waterbath at 50C for 15 minutes. To permit 510w cooling to room temperature, the sample is taken out of the waterbath. Subsequently a further 400 ~l of water are added.
For the photoreaction, a 20-fold molar excess of EuPA is added to 15 ~g of the hybridised hairpin oligonucleotide.
:`
~ Le A 29 09~ - l9 -., .
~ ., . ~ . . : .
:~ ~ , , . , ~ :
.,: . , : . , 2099~42 The solution ls subsequently illuminated under a W lamp a~ 312 nm or 366 nm in an Eppendorf tube. The photo-reaction is followed using HPLC. Within 15 minutes, the pho toreaction was comple~e.
; 5 Example 10 Photolabelling with EuPA (12) For the photolabelling, 50 ~1 of 1 M sodium tetraborate buffer pH 8.3 and 50 ~1 of EuPA (2 ~g/~l) were added to 2 to 5 ~g of DNA in 20 ~1 of TE buffer and the solution made up to 500 ~1 with double-distilled H20. The mixture was irradiated for 10 minutes at 312 nm using a W trans-illuminator, with the samples being kept on ice during this period.
The photolabelled DNA was subsequently precipitated with 1/10 volume of 3 M sodium acetate-pH 5.8 and 1 volume of isopropanol at room temperature and left to stand for 5 minutes. Subsequently, the DNA was centrifuged down at 10,000 rpm in an Eppendorf centrifuge, the supernatant decanted off and the DNA precipitate washed with 70%
; 20 strength ethanol. After the samples had been dried, the photolabelled DNA was taken up in T~. The photolabelling of the DNA with EuPA was subsequently monitored by agarose gel electrophoresis and microtitre tests.
. , .
~ i' ~ Le A 29 093 - 20 -.
: , - . - . .
2~9~2 Ex~mple 11 Detection of -the EuPA labelling in the microtitre test To detect the EUPA labelling of double-stranded DNA, the DNA was pipetted, after the labelling, into microtitre test plates in concentrations of 250 ng to 125 pg in 1:2 dilution steps. To attach the DNA to the polystyrene groups of the microtitre wells, the DNA was first diluted in the wells with PBSM buffer (10 mM Na phosphate pH 7.2 with 0.1 M MgCl2, 0.15 M NaCl, 3 M KCl) and incubated at room temperature overnight. Washing 2x with 200 ~1 of PBSM buffer subsequently took place, and the DNA was fixed to the wells by 10-minute irradiation with a W
transilluminator at 312 nm. The DNA fixed in this way was subsequently washed 4x with wash-concentrate buffer from Delfia/Pharmacia, in order to remove excess EuPA loaded with europium. As the negative control, unlabelled double-stranded DNA was treated in the same manner.
Following addition of 100 ~1 of enhancement solution from Wallac/Pharmacia, the tLme-resolved fluorescence of europium was measured, after 30 minutes at room tempera-ture, in a DELFIA 1232 fluorescence photometer from Wallac/ Pharmacia at 290 to 360 nm excitation/615 nm emission. Depending on the dilution of the DNA, fluore-scence signals of 212,000 to 1,700 were measured in the labelled DNA. The unlabelled DNA only gave low background - signals.
~.
Le A 29 093 - 21 -.
:
2~995~2 Example 12 Hybridisation of EuPA-labelled genomic DNA in the reversed phase test The preparation of EuPA-label:led DNA was carried out according to the method described in Example 10.
The hybridisation was carried out by conventional pro-cesses at an incubation temperature of 40 to 68C.
Different substances were added depending on the hybrid-isation temperature. With long gene probes, dextran sulphate or other polymers were employed in order to increase the speed and extent of the hybridisation.
Detergents and blocking reagents, such as dried milk, Denhardt's solution, heparin or SDS, were employed in order to suppress the non-specific binding of the DNA to the membrane. Denaturing agents, such as urea or form-amide, may be employed in order to reduce the melting temperature of the hybrid, so that lower hybridisation temperatures may be used. In addition, the non-specific binding of probes to non-homologous DNA on the blot may be reduced by the addition of heterologous DNA.
To prepare for the hybridisation, 100 ng of the unlabelled E.coli-specific gene probes (1.7 kb to 6 kb) were first denatured at 100C for 5 minutes, cooled to 0C, and khen transferred to pre-treated nitxocellulose or nylon membranes using a Minifold-II filtration apparatus from Schleicher and Schull and fixed at 80C
Le A 29 093 - 22 -.
- , -: ~ ;
;.~ ' ~ " ' ' ~
2099~42 for 2 hours.
The filters were hybridised in a sealed plastic film bag or plastic box with at least 20 ml of hybridisation solution per lO0 cm2 of filter at 68C for at least l hour.
The solution was replaced by 2.5 ml of hybridisation solution of lO0 cm2 of ilter 1;o which solution freshly denatured (100C, 5 minutes), EuPA-labelled, genomic DNA
from E.coli (l ~g) had been added. The filters were incubated at 68C for at least 6 hours with gentle shaking.
The filters were then washed 2 x 5 minutes at room tem-perature with at least 50 ml of 2xSSC, 0.1% SDS per lO0 cmZ of filter and 2 x 15 minutes at 68C with O.lxSSC, 0.1% SDS.
The filters were then directly employed for detecting the hybridised DNA. Depending on whether EuPA-DNA was used which was already loaded with europium, or which was subsequently loaded with europium, the following further i 20 steps were carried out in working up the filters for the fluorescence read-out. In the case of EuPA-labelled genomic DNA which was not loaded with europium, the filters were treated in lO0 ~M EuCl, 100 ~M EDTA and lxSSC pH 7.0 in a total volume of 2 ml at room tempera-ture for 2 hours. The filters were then washed six times with 2xSSC. Subsequently, the individual slots of the Le A 29 093 - 23 -., .
:~ .
... .
' ' ' ' ' -209~2 hybridisation blot were cut out and treated with 1 ml ofenhancement solution i.n 1.5 ml reaction tubes. After a 30-minute incuba-tion at room temperature, 200 ~1 of the samples from the individual slots were pipetted into microtitre plates and the samples were measured in a DELFIA 1232 fluorescence photo~eter from Wallac/Pharmacia at 290 to 360 nm excitation and 615 emission.
In the case of slo~ blots with EuPA-labelled DN~, which had been loaded with europium before the labelling, the indi~idual slots were cut out direc~ly after the hybrid-isation and 1 ml o enhancement solution wa~ added to them in 1.5 ml reaction tubes and then, as described above, the enhancement solution was added and measurement took place in a fluorescence photometer.
Solutions:
: ~0 x SSC: 3M NaCl, 0.3 M Na citrate pH 7.0 Hybridisation solution: 5xSSC; 0.1% N-lauroylsarcosine, Na salt, 0.02% SDS; 0.5% block-ing reagent (Boehringer), dissolve ~he solution at 50 to : 70C.
Example 13 Hybridisation with EuPA-labelled gene probe~
The preparation of Eu~A-labelled gene probes (1.7 to Le A 29 093 - 24 -2099!~2 6 kb) was car~ied out according to the method described in Ex~mple 10.
The EuPA-labelled gene probes may be employed in solid-phase or liquid hybridisations. Suitable solid phases S are, for example, nitrocellulose membranes, nylon membranes, polystyrene groups of microtitre plates or magnetic particles. The fluorescent hybridisation com-plexes of gene probes with complementary genomic DNA may be separated from free fluorescent gene probes using hydroxyapatite.
For example, a slot~blot hybridisation was carried out with EuPA-labelled, E.coli-specific gene probes (1.7 kb to 6 kb) and genomic DNA from E.coli.
For this purpose, the genomic E.coli DNA was denatured at 100C for 5 minutes and then cooled to 0C and then transferred to nitrocellulose or nylon membranes using a Minifold-II filtration apparatus from Schleicher and Schull in the concentrations 500 ng to 125 pg in 1:2 dilution steps. The prehybridisation and hybridisation were carried out as described in Example 12. 100 ng of EuPA-labelled E.coli gene probe were employed.
The read-out took place as described in Example 12 by individual measurement of the excised filter slots after treatment with enhancement solution in a DELFIA 1232 fluorimeter from Wallac/Pharmacia.
Le A 29 093 - 25 -,, .
~ ~ .
2099~i~2 Using the gene probes, 125 ng of genomic DNA from E.coli were Still readily detectable. This corresponds to a test sensitivi~y of 0.1 pg of DNA measured in the hybridisa-tion of pure pBR322 plasmid probe to pBR322 DNA.
Alternatively, microtitre hybridisation tests were carried out. For this purpose, the genomic E.coli DNA was denatured as described above, and then diluted samples of 10 ng to 45 pg were pipetted into microtitre wells and left to stand at room temperature overnight. Subse-quently, washing took place 2 x with 200 ~l of PBSMbuffer and the DNA was then fixed for 10 minutes at 312 nm using a W transilluminator. 200 ~l of hybridisa-tion solution (Example 12) witll 10 ng of EuPA-labelled gene probe were added and the hybridisation mixture was incubated at 68C for at least 6 hours. Subsequently, the microtitre wells were washed 2 x 5 minutes at room temperature with 2 x 200 ~l of 2 x SSC, 0.1 SDS and 2 x 15 minutes at 50C with 2 x 200 ~l of 0.1 x SSC, 0.1%
SDS.
The read-out took place as in Example 12 in a DELFIA 1232 fluorimeter from Wallac/Pharmacia after treatment of the wells with 100 ~l of enhancement solution.
Le A 29 093 - 26 -: ' ~ -. - : - ' - ' ; . ~ . ~ , . . .
Claims (5)
1. Labelling reagent of the general formula Ln-S-Fu where Ln is = a lanthanide ion-chelating structure, S is = a spacer molecule and Fu is = a furocoumarin derivative.
2. Labelling reagent according to Claim 1, where the lanthanide ion-chelating structure (Ln) is a pyri-dine derivative of the formula where X represents C5- to C14-arylene, optionally containing a hetero atom grouping, or C1- to C24-alkylene containing hetero atom groupings [N, O, S (1x, more than once)], Y and optionally X + Y represents N-oxysuccin-imido, N-maleimido, NH2, OH, COCH2-halogen, Le A 29 093 - 27 -halogen, NCO, NCS, CHO, COOH, SH, CO-halogen, COOCOR1, CH=CHCO2R1, , , , , where R1 represents hydrogen, a saturated or unsaturated C1- to C20-alkyl radical, optionally substituted by a phenyl group, or a phenyl group, R represents, in each case independently of the others, hydrogen, ammonium or an equivalent of an alkali metal or a 1/2 equivalent of an alkaline earth metal.
3. Labelling reagent according to Claim 1, where the spacer is a polyalkylamine, polyethylene glycol or a combination of these.
4. Labelling reagents according to Claim 1, where Fu is an angelicin derivative of the following general formula:
Le A 29 093 - 28 - where R1, R2 and R3, independently of each other, represent H or C1-C7-alkyl, and R4 represents H, C1-C7-alkyl or a lower alkyl with hydroxyl, C1-C7-alkoxy, amino, halo or N-phthalimido substituents.
Le A 29 093 - 28 - where R1, R2 and R3, independently of each other, represent H or C1-C7-alkyl, and R4 represents H, C1-C7-alkyl or a lower alkyl with hydroxyl, C1-C7-alkoxy, amino, halo or N-phthalimido substituents.
5. Labelling reagent according to Claim 1, where Fu is a psoralen with the following general formula:
where R1, R3 and R5, independently of each other, repres-ent H or C1-C7-alkyl, R4 represents H, C1-C7-alkyl or C1-C7-alkyl with hydroxyl, C1-C7-alkoxy, amino, halo or N-phthalimido substituents, Le A 29 093 - 29 -R2 and R5, independently of each other, repres-ent H, hydroxyl, carboxyl, carbo-C1-C7-alkoxy or C1-C7-alkoxy.
where R1, R3 and R5, independently of each other, repres-ent H or C1-C7-alkyl, R4 represents H, C1-C7-alkyl or C1-C7-alkyl with hydroxyl, C1-C7-alkoxy, amino, halo or N-phthalimido substituents, Le A 29 093 - 29 -R2 and R5, independently of each other, repres-ent H, hydroxyl, carboxyl, carbo-C1-C7-alkoxy or C1-C7-alkoxy.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/273,759 US5528920A (en) | 1993-07-02 | 1994-07-12 | Process for laminating a thin film of lihium by controlled detachment |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4222255.9 | 1992-07-07 | ||
| DE19924222255 DE4222255A1 (en) | 1992-07-07 | 1992-07-07 | Photochemical labeling of nucleic acids with europium chelate reagents and their use in gene probe test systems |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2099542A1 true CA2099542A1 (en) | 1994-01-08 |
Family
ID=6462656
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2099542 Abandoned CA2099542A1 (en) | 1992-07-07 | 1993-07-02 | Photochemical labelling of nucleic acids with europium chelate reagents and their use in gene probe test systems |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0578067A1 (en) |
| JP (1) | JPH0694720A (en) |
| CA (1) | CA2099542A1 (en) |
| DE (1) | DE4222255A1 (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6297018B1 (en) | 1998-04-17 | 2001-10-02 | Ljl Biosystems, Inc. | Methods and apparatus for detecting nucleic acid polymorphisms |
| US6406297B1 (en) | 1999-02-18 | 2002-06-18 | The Regents Of The University Of California | Salicylamide-lanthanide complexes for use as luminescent markers |
| US6515113B2 (en) | 1999-02-18 | 2003-02-04 | The Regents Of The University Of California | Phthalamide lanthanide complexes for use as luminescent markers |
| US6949639B1 (en) | 2000-05-05 | 2005-09-27 | Wallac Oy | Oligonucleotide labeling reactants and their use |
| US7745142B2 (en) | 1997-09-15 | 2010-06-29 | Molecular Devices Corporation | Molecular modification assays |
| US8173800B2 (en) | 2006-08-15 | 2012-05-08 | The Regents Of The University Of California | Luminescent macrocyclic lanthanide complexes |
| US8507199B2 (en) | 2007-01-25 | 2013-08-13 | Lumiphore, Inc. | Multi-color time resolved fluorophores based on macrocyclic lanthanide complexes |
| US8551453B2 (en) | 2003-12-30 | 2013-10-08 | The Regents Of The University Of California | Aromatic triamide-lanthanide complexes |
| US9273059B2 (en) | 2009-08-24 | 2016-03-01 | Lumiphore, Inc. | Macrocyclic HOPO chelators |
| US9556122B2 (en) | 2006-07-10 | 2017-01-31 | The Regents Of The University Of California | Luminescent 1-hydroxy-2-pyridinone chelates of lanthanides |
| US11453652B2 (en) | 2013-03-15 | 2022-09-27 | Lumiphore, Inc. | Di-macrocycles |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2837132B2 (en) * | 1996-03-29 | 1998-12-14 | 株式会社ダイケン | Mounting structure of lid for ceiling inspection port |
| DE102011001368B4 (en) | 2011-03-17 | 2013-01-31 | Bundesanstalt für Materialforschung und -Prüfung (BAM) | Lanthanoid chelates containing particles, their preparation and their use in bioanalysis |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0187332B1 (en) * | 1985-01-10 | 1989-02-01 | Molecular Diagnostics, Inc. | Photochemical method of labelling nucleic acids for detection in hybridization assays |
| SE8502573D0 (en) * | 1985-05-23 | 1985-05-23 | Jouko Kanakre | FLUORESCENT LANTHANIDE CHELATES USEFUL AS LABELS OF PHYSIOLOGICALLY ACTIVE MATERIALS |
-
1992
- 1992-07-07 DE DE19924222255 patent/DE4222255A1/en not_active Withdrawn
-
1993
- 1993-06-24 EP EP93110109A patent/EP0578067A1/en not_active Withdrawn
- 1993-06-28 JP JP17848693A patent/JPH0694720A/en active Pending
- 1993-07-02 CA CA 2099542 patent/CA2099542A1/en not_active Abandoned
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7745142B2 (en) | 1997-09-15 | 2010-06-29 | Molecular Devices Corporation | Molecular modification assays |
| US6297018B1 (en) | 1998-04-17 | 2001-10-02 | Ljl Biosystems, Inc. | Methods and apparatus for detecting nucleic acid polymorphisms |
| US6406297B1 (en) | 1999-02-18 | 2002-06-18 | The Regents Of The University Of California | Salicylamide-lanthanide complexes for use as luminescent markers |
| US6515113B2 (en) | 1999-02-18 | 2003-02-04 | The Regents Of The University Of California | Phthalamide lanthanide complexes for use as luminescent markers |
| US6864103B2 (en) | 1999-02-18 | 2005-03-08 | The Regents Of The University Of California | Phthalamide-lanthanide complexes for use as luminescent markers |
| US7018850B2 (en) | 1999-02-18 | 2006-03-28 | The Regents Of The University Of California | Salicylamide-lanthanide complexes for use as luminescent markers |
| US7404912B2 (en) | 1999-02-18 | 2008-07-29 | The Regents Of The University Of California | Salicylamide-lanthanide complexes for use as luminescent markers |
| US7442558B2 (en) | 1999-02-18 | 2008-10-28 | The Regents Of The University Of California | Phthalamide-lanthanide complexes for use as luminescent markers |
| US6949639B1 (en) | 2000-05-05 | 2005-09-27 | Wallac Oy | Oligonucleotide labeling reactants and their use |
| US8551453B2 (en) | 2003-12-30 | 2013-10-08 | The Regents Of The University Of California | Aromatic triamide-lanthanide complexes |
| US9556122B2 (en) | 2006-07-10 | 2017-01-31 | The Regents Of The University Of California | Luminescent 1-hydroxy-2-pyridinone chelates of lanthanides |
| US8173800B2 (en) | 2006-08-15 | 2012-05-08 | The Regents Of The University Of California | Luminescent macrocyclic lanthanide complexes |
| US8729258B2 (en) | 2006-08-15 | 2014-05-20 | The Regents Of The University Of California | Luminescent macrocyclic lanthanide complexes |
| US8507199B2 (en) | 2007-01-25 | 2013-08-13 | Lumiphore, Inc. | Multi-color time resolved fluorophores based on macrocyclic lanthanide complexes |
| US9273059B2 (en) | 2009-08-24 | 2016-03-01 | Lumiphore, Inc. | Macrocyclic HOPO chelators |
| US10352938B2 (en) | 2009-08-24 | 2019-07-16 | Lumiphore, Inc. | Macrocyclic HOPO chelators |
| US11453652B2 (en) | 2013-03-15 | 2022-09-27 | Lumiphore, Inc. | Di-macrocycles |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0694720A (en) | 1994-04-08 |
| DE4222255A1 (en) | 1994-01-13 |
| EP0578067A1 (en) | 1994-01-12 |
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