OLIGONUCLEOTIDE HYBRIDIZATION PROBES AND MEANS FOR THE SYNTHESIS OF THE MOST PREFERRED PROBES

Abstract: Oligonucleotide probe labeled at one of its teminal positions with a non-linear branched polymer carrying a plurality of negatively charged groups, preferably phosphodiester groups, and optionally also analytically indicatable groups covalently linked to terminal ends of the branches of said polymer. The preferred indicatable groups are lanthanide chelates. A compound that can be used for the synthesis of the probe is also disclosed. The compound has formula (I), where R1 and R2 are lower alkyl; R3 is lower alkyl (C1-C7), or aryl each of which optionally being provided with electron-withdrawing substituents; and A1, A2 and A3 are hydrogen, A'-O-B, A''-O-B or A'''-O-B, at most one of A1-3 being hydrogen and A', A'' and A''' being a hydrocarbon chain providing a distance of 5-9 atoms between the phosphorous atom and the oxygen directly bound to B, and B being a protecting group. In the preferred compound at least two of A1-3 are identical while the other is hydrogen.

Description: Oligonucleotide hybridization probes and means for the synthesis of the most preferred probes Field of the Invention Oligonucleotides are now widely used as probes for the detection of specific genes. Various substances have been used as labels for DNA probes. Attention has been mostly focused on alternatives of radioisotopic labels becuase of th associated problems of safety, stability and waste disposal. Recently pub¬ lished reports on the use of lanthanide chelates as markers of DNA probes ( O-A-88/02784, Nucl. Acid. Research ^, 1181-1196, Nucl. Acid. Research _, 1017-1028) showed that the fluorescence markers indeed offer a powerful alternative to the radioisotopes. We have presented the idea of using polymeric material as carrier of a multi¬ plicity of europium or terbium chelates and bound to the oligomeric probe (WO-A-88/02784). Here we present a new and fully automated method for the synthesis of oligo- nucleotide-based hybridization probes of the type mentioned earlier. Moreover while the polymeric linker which is formed is strongly acidic, it drastically decreases the background obtained in hybridization tests, and the specific spatial nature of the probe is presumably responsible for the high hybridiza¬ tion efficiency. Description of Prior Art and strategy for making the invention There is a strong tendency to employ short oligonucleotides instead of longer poly-DNA fragments as hybridization probes since these a) offer better specificity b) are characterized by much higher hybridization kinetics c) hybridize in at relatively low temperatures d) are easily accessible in large quantities e) offer wider possibilities for derivatization. Today, however, oligonucleotide probes are generally less sensitive in the detection when compared to the poly-DNA probes. The obvious explanation of this fact is that oligomers could not be derivatized to such a high extent with the labels as it was possible to do with polymeric DNA. In (EP-A- 212,951) base modified mono eric nucleotides were suggested for introducing a derivatizable linear chain subsequently labelled with the appropriate chelate. This idea has been investigated by us (Nucleosides and Nucleotides, in press) and we encountered considerable difficulties in introducing the large amount of modified nucleotides as aimed. In our hands the modified tail consisted maximally of 40 to 60 nucleotides when the complementary hybridization part which we attempted to achieve was a 50-mer. Additional complications, i.e. only 50-60 % of the total functional group could be derivatized with the europium chelate, together with increased assay background caused by the remaining aminogroups made this approach difficult. In (WO-A-88/02784) we described a method in which the step by step procedure can be avoided by synthetic coupling of the 5'-thiol modified oligo-DNA to previously activated linear polymers labelled with large amounts of chelates. Despite the promising sensitivity the method was rather laborious and complicated as several separations on different synthetic stages were necessary. We seeked to find a method for the synthesis of oligo-DNA based hybridizatio probes which would involve a minimal number of separation stages, utilize a neutral, or preferably acidic polymer, and which would result in the incorporation of large amounts (hundred or more) of lanthanide chelates to a single oligo-DNA chain. We concluded that in order to achieve reproducible results, as much of synthesis as possible should be performed automatically under controlled conditions - preferably as a continuation of the oligo-DNA synthesis. Theoretically, this could be realized in several wa s, but we preferred a method which would not involve the use of aqueous solutions. Among several alternatives which were tested we focus our attention on a process which cou be described as the hydroxyl amplification procedure. The Invention The invention comprises three major aspects: 1) novel oligonucleotide probes 2) novel compounds that can be used for the synthesis of the most preferred probes of the invention, and 3) a method for the synthesis of the most preferred probes. Embodiments 1 and 2 in generalized forms are defined in th claims that form an integral part of the descriptive part of this specification. Derivatives having the following general structure have been synthesized (n- 3 and 6) DMT-0-(CH 2 ) n -CH-(CH 2 ) n -0-DMT I 0 I P 'N' 0-CH,-CH 2 -CN To some extent these compounds resemble the mononucleotide blocks used in th phosphite triester approach as all comprise a phosphoroamidite function whic can readily be activated by tetrazole, and a dimethoxytrityl group which can easily be removed by mild acids. However, contrary to the mononucleotide blocks which have only one DMTr grou at the 5' position of the deoxyribose ring, the compounds of this invention contain two, three or more of such groups. Our intention was to use the above compounds for coupling to the free 5' hydroxyl of the accomplished oligonucleotide sequence built on the commercia Gen Assembler (Pharmacia, Sweden). Oxidation of the existing P ~ to P ~ with iodine followed by removal of DMTr groups will result in an oligomer having two reactive hydroxyl groups starting from only one on the original oligonucleotide. The process of coupling, oxidation and detritylation could repeated thus generating increased amounts (theoretically for the case illustrated in the experimental part: OH = 2 n , where n is the number of coupling reactions) of hydroxyls bound to the DNA via a synthesized and high branched polyphosphate diester polymer. The reagents employed in this amplification process should obviously fulfil two criteria: 1. They should be as small as possible to keep the size of the polymeric par on the minimum mass level. 2. They should be big enough so that the distance between the formed phosphotriester group and the liberated hydroxyl will be sufficiently lon to prevent any side reactions. In addition to criterion 1, the maximal size of the reagent (number of methylene groups n) is also regulated by the fact that large aliphatic chai possess increased hydrophobic properties which finally result in nonspecifi binding of the probe to the commonly used polystyrene hybridization strip t unnecessarily increasing the background signal. The side reactions mentioned in 2 are known from ribonucleotide chemistry. has been found that the free 2' hydroxyl group in synthetic oligoribo- nucleotides attacks intramolecularly the adjusted 3' phosphotriester group the process which results in degradation of the molecule. Indeed, when the first derivative (n=l) was used we could not achieve any amplifiation of th originally existing amounts of hydroxyls. This process was monitored spectr photometrically following the absorbance of released DMTr cation. The usual increase of absorbance after the first coupling assumed immediately a const value already from the second and subsequent couplings. The above effect co be explained assuming intramolecular attack at the OH group on the protoniz phosphotriester group and formation of cyclic phosphate. OH OH I I tμ-ONA-OH — — The derivative where π=o w s shown to b li ive as theore ically expected, allowing amplifica ion with tin. awr ue factor l. c) -L.fι. The maximal amplification factor 2 in a sin .. L cυupli ιv,; v. Iu a.-. v.rv difficult to achieve even with a l r.^u .-xcess of r_.u;ι j nts ,mά r l ng., coupling time. is worthwhile to note that amplification value for compound n=6 approaches 1 already after 9 coupling . Most probably the size of the pore on the solid support is responsible for this decreased yield and further iπprov.i.icp.ts may be possible by using materials having larger pores but no systematic studios have been performe us. Compound (n=3) was found to be an optinal substrate. It could be obtained inexpensive starting materials in high yield. This derivative couples to f hydroxy group in an expected manner with no detectable tendency to side reactions as was also shown on the model compounds synthesized in solution Further, it is possible to go as far as to 15 coupling reactions without diminishing the amplification factor below 1.5. The average coupling yields using this reagent were also generally higher than those obtained with compound (n=6). The oligo-DNA probe, still bound to the solid phase support and having a phosphotriester polymer with free hydroxyl groups at its highly branched arms, was finally labelled with the appropriate label. The choice of label that can be used is governed by several factors: a) the labels should be sufficiently small to ensure optimal employment of existing derivatizable hydroxyls b) the bond between the label on probe and the label itself should be resistant to the deprotection conditions normally used for oligo-DNA deprotection (high pH, concentrated ammonia) c) the label should not be a hapten or other group the visualization of which is based on their high affinity to other biological materials (e.g. biotin-avidin), since the presented cumulation of the label In one limited space would not pay enough with the increased sensitivity of the probe. The protected form of strongly chelating ligands offers the best alternative for such a label. We employed ligands previously described by us in SE 8702824-7, and used the in form of activable phosphoroamidites. Thus the whole synthesis of the prob was very similar at all stages and could be easily automated. The accomplish synthesis was finished by deprotection of the synthesized probe, incorporati of lanthanide ion into the probe and separation of the polymeric material fr the low molecular components using fast FPLC gel filtration. Detailed description of the Invention The oligonucleotidic hybridization probes have the common structure given in formula II. This formula represents an ideal rather than the actual probe in view of the fact that the couplings of amplificating reagent do not always go with quantitative yield. In the real probe some of the arms of synthetic polymer may be shorter than others, nevertheless we believe that even these hydroxyl that are present "inside" the polymer can react with ligand due to the much less bulky properties of the last reagent. 5 where H ( denotes a oligonucleotidic hybridization probe that ca be a sequence complementary to the gene fragment to be analyzed and has a minimum length of 16 and maximum of 200-400 nucleotides. Sequences which comprise 35 to 100 nucleotides are preferred. L denotes a label which can be chosen among several labels known to those skilled in the art. In the preferred embodiment the group L is connencted t the rest of the probe through an ordinary phosphodiester bond but the invention also includes probes where the label is bound to the probe using another type of stable bond (see for instance Protective Groups in Organic Synthesis; Greene TN, John Wiley & Sons Inc., 1981) formed in the last reaction. The sign m denotes the amount of repeated amplification cycles using reagen with the general formula III and the m preferred by us is 11 which theoretically should give over 200 fold OH amplification. Where R j _ and R2 may be the same or different and consist of straight or branched alkyl groups. R, and R„ may be a part of a homocyclic or heterocy ring system containing one or more heteroatoms. These groups in the preferred embodiment are R^=R = isopropyl in which ca the phosphoroamidite formed is characterized by very good storage stabilit R 3 denotes alkyl, alk laryl, aryl or any group having additional function groups (mostly electron-withdrawing) introduced for instance to achieve ea and faster deprotection. These groups and the rules that govern their choi are known to those skilled in the art. The preferred OH amplificating reagent forms the phosphate type polymer in process called phosphite triester chemistry. This process is characterized today as the most effective (high yield) method for phosphotriester bond formation. However, this invention is not limited to the exemplified phosphi triester method but would also include phosphodiester, phosphotriester and also H-phosphonate chemistry. Especially the H-phosphonates present an interesting alternative to the phosphite triester chemistry as they need onl minor changes in the synthetic procedure. All the above mentioned methods ar well known to those who are skilled in the art (Gait, M.J. Oligonucleotide synthesis - a practical approach, IRL Press, 1984). A represents a straight or branched aliphatic carbon chain in which the distance between the oxygen connected to B and the phosphorus atom in the central phosphoroamidite group is at least 6 atoms or more. If A denotes a simple aliphatic carbon chain of type -(CH. )_- it means that the lowest number of carbon atoms that can be present is 3. This situation, advocated previously, was preferred. Finally, B denotes a protecting group, preferably an acid labile (such as dimethoxytrityl, pixyl, monomethoxytrityl, etc.), but another type of protecting group may theoretically also be employed provided proper changes the other existing groups in the synthesized molecule. Rules that govern the chemistry of protecting groups are published and commonly known.The importan matter is that it shall be possible to remove B selectively from a growing polymer chain without concomitant removal of R 3 . The reagents exemplified here are based on the triols, however the method is not anyhow limited to the triols. Compounds possessing more than three hydroxyls can be used for the same purpose as well as cyclic pol ' y-hydroxy compounds. The only limiting factor to be kept in mind in view of mentioned side reactions is that the distance between any of the hydroxyl oxygens shou be four atoms or longer. The following examples are presented to illustrate the present invention. In all examples the commercial chemicals used were of the highest quality and the remaining reagents were prepared using conventional procedures. Support for flash chromatography Kieselger G60, and TLC Plates Kieselgel 60F-254 were obtained from Merck. NMR spectra were recorded with a Jeol JNM GX400 using tetramethylsilan ( ) or 80%, H 3 P0 H ( P) as standards. The oligonucleotide hybridization probes were synthesized with Gen Assembler (Pharmacia, Sweden) using standard reagents and solvents recommended by the manufacturer. Example 1 1,4,7-Heptanetriol (1) Lithium aluminium hydride (11.25 g, 0.296 mol) was suspended in 200 ml dry dietylether in a one-litre three-necked round-bottomed flask equipped with a dropping funnel, reflux condenser, and a magnetic stirrer. 4—Ketopimelic acid dimethyl ester (25 g, 0.124 mol) was dissolved in dry dietylether (50 ml) and added dropwise to the suspension maintaining a steady reflux. After the exothermic reaction had ceased the reaction mixture was refluxed for additional 30 in. Then the reaction mixtur was transferred to a one-litre separating funnel and shaken with water. The alkaline water phase was poured into a beaker and made acidic with dilute sulfuric acid. The precipitate was filtered off and the filtrate was made slightly alkaline with concentrated ammonia. The precipitate was filtered off and the filtrate was evaporated to dryness and the residue coevaporated with ethanol. The inorganic salts were separated from the organic oil by resuspension in ethanol and filtration. The residual brown oil was purified b vacuum distillation. The yield was 11 g (60%); b.p. 195-205°C/0.5 mmHg. ^NMR, (400 MHz, CDC1 3 ): 1.56 (4H, m); 1.95 - 2.10 (4H, m); 3.54 (1 H, broad s); 3.60 (4H, t); 3.80 (3 H, s). Example 2 1,7,13-Tridecanetriol (2) Magnesium sponge (1.2 g, 50.1 mmol) was added to 100 ml of dry THF in a 500 m round-bottomed flask, and the flask was kept in an ultrasonic bath for 20 min. The catalytic amounts of iodine and 1 ,2-dibromoethane were added, followed by addition of 6-chloro-l-0-tetrahydropyranylhexane (11.05 g, 50.1 mmol) dissolved in dry THF (20 ml). The mixture was refluxed under nitrogen until almost all magnesium had dissolved. The reaction flask was put into an ice-bath and ethylformiate (1.85 g, 25.05 mmol) was added slowly, maintaining a temperature around 35°C. After stirring for 15 min at room temperature, water (30 ml) and saturated aqueous ammonium chloride (30 ml) ware added, and the contents were transferred to a separating funnel and extracted with diethylether (3 x 100 ml). The combined organic extract was dried with sodium sulfate and evaporated. The residual oil (containing 1 ,13-di-0-tetrahydropyranyl-7-hydroxytridecane) was dissolved in 100 ml of acetic acid/tetrahydrofuran/water (4:2:1) and stirred for 12 h at 50°C. After this the solvent was removed on the vacuum evaporator and coevaporated once with ethanol and once with toluene. The residual oil was crystallized from chloroform. The yield was 1.16 g (20%). ^N R, (400 MHz, CDC1 3 ): 1.30-1.609 (16H, m); 1.90-2.10 (4H, πi); 3.57 (1H, broad s); 3.61 (4H, t); 4.00 (3 H, broad s). Example 3. 1,7-Di-0-(4,4'-dimethoxytrityl)-4-hydroxy heptane (3) (1) (1.35 g, 9.12 mmol) was dissolved in dry pyridine (80 ml) followed by addition of 4,4'-dimethoxytritylchloride (7.43 g, 21.96 mmol). The solution was stirred for 12 h and was poured onto saturated aqueous sodium bicarbonate (250 ml) and extracted with chloroform (3 x 150 ml). The combined organic extract was concentrated, coevaporated with toluene and flash chromatographe on silica gel CH^Cl^: petroliumether, 1:1, as eluent). Evaporation of the solvent gave the pure product as oil. The yield was 5.08 g (74%). R f (MeOH/CHCl 3 , 5:95) = 0.66. 1 HNMR, (400 MHz, CDC1 3 ): 1.40-1.80 (84, m); 3.09 (4H, t); 3.55 (1H, s); 3.70 (1H, broad s); 3.75 (12H, s); 6.77-7.45 (26H, m) Example 4. l,13-Di-0-(4,4'-di ethoxytrityl) 7-hydroxytridecane (4) Compound (4) was prepared starting from (2) in a reaction analogical to Example 3, and was obtained as oil in % yield. R f (Me0H/CHCl3 5:95) = 0.8 1 HNMR, (400 MHz, CDCI 3 ): 1.30-1.50 (16H, ra); 1.80-2.05 (4H, m); 3.10 (4H, t) 3.41 (1H, s); 3.57 (lri, ra); 3.76 (12H, s); 6.75-7.46 (26H, m) . Example 5. 1,7-Di-0-(4,4'-dimethox tri yl)heptane 4—N,N-diisopropylcyanoethylphosphor- amidite (5) (N,N-Diisopropylamino)cyanoethylphosphonamidic chloride (1.73 ml, 8.83 mmol was added at once to a stirred solution of (3) (5.11 g, 6.79 mmol) and N,N-diisopropylethylarnine (2.48 ml, 14.26 mmol), and the mixture was stirre for 15 min. The mixture was evaporated to dryness and the residue suspended a small amount of ethylacetate/petroliu ether/triethylamine (15:75:10) and loaded onto a short silica column made in the same solvent. The appropriate fractions were pooled together and evaporated to dryness. The yield was 4.4 (65%);R f (EtAc/pet. et/Et 3 15:75:10) = 0.29. 1 HNMR, (400 MHz, CDC1 3 ) : 1.15 (6H, d); 1.18 (6H, d) 1.56-1.76 (8H, m) ; 2.45-2.58 (4H, m); 3.52-3.63 (1H, m); 3.67-3.75 (1H, ra); 3.77 (12H, s); 6.75-7.46 (26H, ) . 31 P-NMR, (161 MHz, CDCI 3 /8O 7. H 3 P0u ext.): 147.2 ppm. Example 6. 1,13-Di-0-(4,4'-dimethoxytrityl)tridecane 7-N,N-diisopropylcyanoethylphosphor- a idite (6) Compound (6) was prepared starting from (4) similarly to the synthesis of (5) from Example 5, except for the eluent, which in this case was ethylacetate/petroliumether/triethylamine (10:80:10). The yield was 65%. R f (EtAc/pet. et./Et 3 N 10:80:10) = 0.21. ^NMR, (400 MHz, CDC1 3 ) : 1.16 (12H, d); 1.30-1.54 (16H, m) ; 1.82-2.10 (4H, m) ; 3.50-3.60 (1H, m); 3.65-3.73 (1H S m); 3.77 (12H, s); 6.74-7.40 (26H, m) . 31 P-NMR, (161 MHz, CDCI 3 /8O % H 3 P0_> ext.): 147.3 ppm. Example 7 Construction of the probe A 50-mer oligodeoxynucleotide having a sequence complementary to λ-phage DNA was synthesized on Gen-Assembler (Pharmacia) using standard building blocks and reagents. The use of silica-based support is essential for the performance and isolation of final products since another, chemically inert e.g. polymer-based support gave very pure results probably by trapping the product in its pores. The released dimethoxytrityl cation from the deprotection of accomplished 50-mer was collected, its concentration was measured spectrophotometrically and used as reference during the continuation of the synthesis. Couplings of the OH amplifying reagent were made under standard DNA-synthesis conditions with the exception that the amounts of reagent were gradually increased as the polymerase chain was growing. At the end of this coupling cycle, several coupling cycles had to be performed without the usual detritylation to achieve a high amplification factor. Post-coupling oxidations and detritylations were made using standard reagents under standard conditions. The amounts of dimethoxytrityl cation from each condensation were collected and the coupling yields were measured spectro¬ photometrically. The final hydroxy-amplified oligo-polymer was labelled with a phosphoroa ido form of the protected ligand by repeating the coupling procedure five times followed by oxidation of phosphorus and washing* The high efficiency of ligand coupling was routinely checked by attempts for incorporation of T-block in the final stage and lack of any DMT-cation in its acidic deprotection. The cassette containing synthesis support was then subjected for 0.3 M MaOH (0.5 ml) for 4 h at RT followed by 35% aq. ammonia (1.0 ml) at 60°C for 16 h. To ensure that the formed probe will contain europium and not other metal ions present in the added reagents, europium citrate (0.5 M, 100 μl) was addd to the deprotection mixture already before the addition of NaOH. The deprotected material was separated from the support by centrifugation and the reaction mixture was separated using a FPLC gel filtration system, using sodium bicarbonate 0.5 M/Et 3 N 2% as the eluting buffer (pll 11.5). The polymeric probe came in the void easily separating from shorter fragments. Example 8 The ability of the λ-phage specific Eu-labelled 50-mer probe (from Example 7) to hybridize with λ-phage DNA was checked in a direct hybridization assay. Different amounts of heat-denaturated λ-phage calf thymus DNA or herpes simplex virus type IF DNA (5 ng/well) were coated onto raicrotiter wells in PBSM-solution (= 8m Na 2 HP0 £+ , 1.5 M H^O^, pH 7.2, 140 mM NaCl, 3 mM KC1, 0.1 M MgCl„). After overnight incubation the wells were aspirated and UV-irradiated (254 nm, 250 μW/cm 2 ) for 5 min. To block nonspecific binding of the probe the microtiter wells were prehybridized in 0.9 M NaCl, 20 mM Tris-HCl pH 7.5, 0.1 % sodium dodecyl sulfate, 0.5 % polyvinylpyrolidone 100 μg/ml sodium polyacrylate, 50 μM EDTA at 50°C for 30 min. The hybridization was performed in the same solution having 100 ng/ml Eu-labelled 50-mer as a probe at 50°C for 3 h. Microtiter wells were washed in 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1 % sodium dodecyl sulfate, 50 μM EDTA at * The protected ligand employed was compound 35 of Swedish patent application 8702824-7, having the group £ C1C-H.0P0 " replaced with the group ((CH )„CH) NP0(CH„)„CN (compare also compound 53). This ligand is novel. We have therefore included the experimental part of 8702824-7 in this specification. 37°C for 3 x 10 min. After washing the amount of europium in each well was measured by adding 200 μl of enhancement solution (0.1 M acetate buffer adjusted to pH 3.2 with potassium hydrogen phthalate, containing 15 μM 2-naphtoyltrifluoroacetone, 50 μM tri-n-octylphosphine oxide and 0.1 % Triton X-100) and measuring in a time-resolved fluorometer (Arcus 1230, Wallac Oy) after 15 min incubation at room temperature. Typically microtiter wells containing only 5 ng calf thymus DNA or herpes simplex virus DNA gave 1000-1500 cps. 50 ng of λ-phage DNA gave a signal of 112000 cps. The detection limit was 400 pg of λ-phage DNA. EXPERIMENTAL PART OF SWEDISH PATENT APPLICATION 8702824-7. Example 1. 2,6-Dimethyl-4-(2-phenylethyl)ρyridine (2) Liquid ammonia (150 ml) was introduced to a 250 ml three-necked round bottome flask equipped with a mechanical stirrer, dropping funnel and outlet tube an immersed in a dry ice/ethanol bath. Sodium amide was generated by addition o 20 mg of iron nitrate (Fe 3+ ) followed by metallic sodium (2.09 g, 0.09 mol) The deep blue solution was stirred for one hour and the solution of collidin (1) (10.06 g, 0.083 mol) in 20 ml of dry diethylether was introduced Into th reaction — addition time 15 min. The formed yellow suspension was stirred fo an additional 45 min followed by the addition of benzylchloride (6.33 g, 0.0 mol) dissolved in 10 mi of dry diethylether. The reaction mixture was stirre for 45 min and the excess of sodium amide was neutralized by addition of ammo nium chloride (4.82 g, 0.09 mol) dissolved in 20 ml of H 2 0. Ammonia was evapo rated and the residue was partitioned between water and diethylether. Th collected etheral phase was dried over sodium sulphate and evaporated. Th brown residual oil was fractionated collecting a fraction distilling a 130°C/0.1 mmHg Yield = 6.17 g (55%), oil. Rf = 0.57 (System A) E l mm. (60 MHz, CDC1 3 ): 7.30-7.05 (m, 7H) , 2.90-2.70 (m, 4H) , 2.46 (s, 6H) Example 2 2 > 6-Dimethyl-4-(2-(4-nitrophenyl)ethyl) pyridine (3) Compound (2) (20 g, 88.9 mmols) was dissolved in THF (150 ml), and nitric ac (6.7 ml, 60% aq. solution, 1 eq) was added. Diethylether was added to t clear solution until it remained dim y, and the mixture was left in the fre zer for crystallization. The white crystals of nitrate (quantit. yield) we added in small portions to well-chilled sulphuric acid (150 ml) never allowi the temperature to reach 10°C, whereafter the mixture was warmed at 50°C for 10 min. The resulting brown solution was poured onto ice and neutralized with solid sodium hydrogen carbonate. The organic material was extracted with chloroform (3x200 ml), and after drying over sodium sulphate, the chloroform extract was flash chromatographed using 4% ethanol/chloroform as a solvent. The appropriate fractions were collected and evaporated yielding a pure yellow solid. Yield: 22.82 g (95%) Rf = 0.62 (System A) H X NMR (400 MHz, CDC1 3 ): 8.14 (d, 2H, J = 8.7 Hz), 7.30 (d, 2H, J=8.7 Hz), 6.75 (s, 2H), 3.05-2.98 (m, 2H) , 2.90-2.84 (m, 2H) , 2.48 (s, 6H) Example 3. 2,6-Dimethyl-4-(2-(4 nitrophenyl)ethyl) pyridine N-oxide (4) Compound (3) (22.82 g, 84.5 mmol) was dissolved in chloroform (100 ml), and 16 g (94 mmol) of m-chloroperbenzoic acid (mCPBA) was added in small portions at RT over a period of 30 min. The mixture was stirred for an additional 2 h and after a negative TLC test for the starting material it was worked up by parti¬ tioning between sat. sodium hydrogen carbonate and chloroform. The combined chloroform extracts (3x200 ml) were evaporated yielding a light yellow solid material that was TLC pure. Yield: 24.55 g (100%) Rf = 0.40 (System A) H X NMR (400 MHz, CDC1 3 ): 8.14 (d, 2H, J=8.7 Hz), 7.29 (d, 2H, J=8.7 Hz) 6.93 (s, 2H), 3.06-3.00 (m, 2H), 2.92-2.86 (m, 2H) , 2.50 (s, 6H) Example 4. 2-Acetoxymethyl-4-(2-(4-nitrophenyl)ethyl)-6-methylpyridine (5) Compound (4) (24.0 g) was suspended in 100 ml of acetic anhydride. The mixture was refluxed for 20 min which resulted in a homogenous dark solution. Acetic anhydride was evaporated on a Rotavapor and the oily residue was neutralized with saturated sodium hydrogen carbonate, followed by extraction with chloro¬ form (3x200 ml). The chloroform phase was evaporated and the crude material was flash chromatographed using 2% ethanol/chloroform as a solvent. The pure fractions were evaporated yielding oil that was TLC and NMR pure. Yield: 19.55 g (69%) Rf - 0.71 (System A) E^NMR (400 MHz, CDC1 3 ): 8.14 (d, 2H, J=8.7 Hz), 7.31 (d, 2H, J=8.7 Hz) 6.95 (s, 1H), 6.91 (s, 1H) , 5.14 (s, 2H) , 3.08-3.92 (m, 2H), 2.97-2.91 (m, 2H) , 2.53 (s, 3H), 2.14 (s, 3H) Example 5 2-Acetoxymethyl-4-(2-(4-nitrophenyl)ethylj-6-methylpyridine N-oxide (6) Compound (5) (19 g, 60.5 mmol), was oxidized as described in Example 3. The crude, single spot on the TLC product was isolated after standard work up. Yield: 18.97 g (95%), oil. Rf = 0.45 (System A) ^NMR (400 MHz, CDCI 3 ): 8.17 (d, 2H, J=8.8 Hz), 7.32 (d, 2H, J=8.8 Hz) 7.03 (s, 1H), 7.01 (s, 1H) , 5.38 (s, 2H), 3.10-3.03 (m, 2H), 3.00-2.93 ( , 2H) , 2.52 (s, 3H), 2.20 (s, 3H) Examnle 6. 2,6-Bisacetoxymethyl-4-(2-(4-nitrophenyl)ethyl) pyridine (7) Compound (6) (18.5 g, 56 mmol), was converted to product (7) in a synthesis analogous to the synthesis in Example 4. The neutralized, end-extracted pro¬ duct was evaporated and flash chromatographed using 2% ethanol/chloroform as a solvent. The pure fractions containing the product were combined and evapo¬ rated. Yield: 12.04 g (61%), oil Rf = 0.72 (System A) B^ MR (400 MHz, CDC1 3 ): 8.14 (d, 2H, J=8.8 Hz), 7.31 (d, 2H, J=8.8 Hz), 7.07 (s, 2H), 5.18 (s, 4H) , 3.10-2.80 (m, 4H) , 2.15 (s, 6H) Example 7. 2,6-Bishydroxymethyl-4-(2-(4-nitrophenyl)ethyl) pyridine (8) Diacetate (7) (12.0 g, 34.1 mmol), was dissolved in 50 ml of ethanol. To this solution stirred at RT, sodium hydroxide 5 M, 20 ml was added at once. After 10 min, when the TLC test for the substrate was negative, the mixture was neutralized with citric acid, and partitioned between sat. sodium hydrogen carbonate and ethanol/chloroform 1:1. The extraction was repeated three times using 100 ml of organic solvent for each extraction. The combined extracts were evaporated and the residual mixture was flash chromatographed using finally 8% ethanol/ chloroform as a solvent. The appropriate pure fractions were collected and evaporated. Yield: 4.75 g (52%), yellow solid Rf = 0.35 (System B) H MR (400 MHz, DMS0-d b ); 8.15 (d, 211, J=8.5 Hz), 7.55 (d, 2H, J=8.5 Hz), 7.22 (s, 2H), 5.33 (t, 2H, exchangeable, J=5.5 Hz), 4.48 (d, 4H, J=5.5 Hz) 3.10-3.02 (m, 2H) , 3.00-2.94 (m, 2H) Example 8. 2,6-Bisbromomethyl-4-(2-(4-nitrophenyi)ethyl ) pyridine (9) To the dihydroxy compound (8) (2.7 g, 9.44 mmol), in 35 ml of dry dichloro- methane, phosphorus tribromide (3.63 g, 1.26 ml, 13.41 mmol) was added and the mixture was refluxed for 15 min. The reaction mixture was neutralized with saturated sodium hydrogen carbonate and extracted with chloroform (3x50 ml). The combined extracts were concentrated and crystallized from ethyl acetate. Yield: 3.91 g (84%) - white crystals Rf=0.73 (System C) H MR (400 MHz, CDC1 3 ); 8.15 (d, 2H, J=8.5 Hz), 7.28 (d, 2H, J=8.5 Hz) 7.14 (s, 2H), 4.49 (s, 4H) , 3.05-3.02 (m, 2H) , 3.01-2.37 (ra, 2H) Example 9. 2,6-Bis {Ή,N-bis(ethoxycarbonylmethyl)aminomethyl)-4-(2- (4-nitrophenyl)ethyl) pyridine (10) Compound (9) (3.27 g, 7.9 mmol) and iminodiacetic acid diethylester (5.78 g, 30.5 mmol), were coevaporated together with toluene and redissolved in dry acetonitrile (50 ml). Solid sodium carbonate (10 g) was added and the mixture was refluxed for 2 h, whereafter the salts were filtered out and the filtrate was evaporated. The residue was flash chromatographed and the fractions con¬ taining the product evaporated to dryness. To achieve material free from any co-chromatographed iminodiacetic acid diethylester, the oily product was tri¬ turated with petrolether (3x20 ml) which yielded material free from any conta¬ minations. Yield: 5.09 g (80%), oil Rf = 0.27 (System C) H^MR (400 MHz, CDC1 3 ); 8.08 (d, 2H, J=8.8 Hz) 7.29 (s, 2H) , 7.27 (d, 2H, J=8.8 Hz) 4.13 (q, 8H) , 3.95 (s, 4H) , 3.53 (s, 8H), 3.04-2.90 (m, 4H) , 1.23 (t, 12H) Example 10. 2,6-Bis , -bis(ethoxycarbonylmethyl)aminomethyl )-4-(2-(4-aminophenyl)ethyl) pyridine (11) To the solution of compound (10) (4.8 g, 7.5 mmol) in 50 ml of ethanol, 10% palladium on carbon (100 mg) was added followed by sodium borohydride (378 g, 10 mmol). The reaction mixture was stirred at RT for 5 min and partitioned between sat. sodium hydrogen carbonate and chloroform. The chloroform extracts (3x50 ml) were concentrated and flash chromatographed to give compound (11) as oil after evaporation. Yield: 3.89 g (85%) Rf = 0.37 (System A) H 1 NMR (400 MHz, CDC1 3 ); 7.28 (s, 2H) , 6.93 (d, 2H, J=7.3 Hz), 6.60 (d, 2H, J=7.3 Hz), 4.17 (q, 8H) , 4.00 (s, 4H) , 3.59 (s, 8H) , 2.87-2.79 (m, 4H) , 1.27 (t, 12H) Example 11. 2,6-Bis(N,N-bis(carboxymethyl)aminomethyl-4-(2-(4-aminophenyl) hyl) pyridine and its europium chelate (12) Compound (11) (250 mg) in 20 ml of ethanol, was treated with 1 M sodium hydr¬ oxide (10 ml) at RT for 3 h. The pure on TLC product (solvent system aceto- nitrile/water 4:1) was neutralized with 1 M hydrochloric acid and concentra¬ ted. To the residue dissolved in water (25 ml), europium chloride hexahydrate (60 mg) dissolved in 5 ml of water was added and the mixture was stirred for 30 min. The excess of europium salt was removed by raising the pH to 8.5 with saturated sodium carbonate solution and filtration of the precipitate. The clear solution was evaporated almost to dryness and (12) was precipitated by addition of 100 ml of acetone. The product was washed on the filter with ace¬ tone and dried. Example 12. Europium chelate of 2,6-Bis(N,N-bis(carboxymethyl)aminomethyl)-4-(2-(4-iso- thiocyanatophenyl)ethyl) pyridine (13) To the amino chelate (12) (100 mg) dissolved in 5 ml of water and vigorously stirred, tiophosgene (80 μl) dissolved in 3 ml of chloroform was added at once and the mixture was stirred at RT for 1 h. The water phase was separated, extracted with chloroform (3 x 3 ml) and con¬ centrated to a volume of 0.5 ml. Addition of ethanol (10 ml) precipitated (13) quantitatively as white solid. The TLC (System Acetonitrile/H 2 04:1) and fluo¬ rescence developing with acetonyl acetone/EtOH (1:20) showed only a single product being negative to a fluorescamine test for free amines. IR (in KBr); 2100 cm "1 Example 13. 4-(7-Bromoheptyl)-2,6-dimethyl pyridine (14) Sodium amide was formed from sodium (1.0 g, 43.5 mmol) in liquid ammonia (100 ml) according to Example 1. Collidine (5.0 g, 41.3 mmol) dissolved in tetrahydrofurane (5 ml) was dropped in and after 45 min a well cooled solution of dibromohexane (51.2 g, 210 mmol) in THF (100 ml) was quickly added — addition time 5 min. The reaction mixture was stirred for 1 h at -40°C and then left stirring overnight during which time ammonia evaporated. The residual THF solution was evaporated and the residue was partitioned bet¬ ween water and diethylether. The etheral phase was treated with hydrochloric acid solution (2.0 M, 200 ml), -and the pyridinium salts extracted to the aqueous phase were liberated on addition of sodium hydroxide (5 M) to get a slightly alkaline solution. The oily products formed were reextracted and separated by silica gel column chromatography. Yield: 3.54 g (30%) oil Rf=0.48 (System C) H X NMR (60 MHz, CDCl 3 ) ; 6.73 (s, 2H) , 3.37 (t, 211, J=8.5 Hz), 2.44 (s, 6H) , 1.70 (t, 2H), 1.30-1.60 (m, 10H). As a byproduct 1,7-(Bis-4-(2,6-dimethylpyτidyl)heptane (15) was isolated from the same reaction mixture in a 35% yield. H L NMR (400 MHz, CDC1 3 ); 6.77 (s, 4H) , 2.48 (s, 12H), 1.58 (t, 4H, J=7.0 Hz), 1.30 (s, 12H). Example 14. 4-(7-Phthalimidoheptyl)-2,6-dimethylpyridine (16) A mixture of compound (14) (3.54 g, 12.5 mmol), potassium phthalimide (2.54 g, 13.7 mmol) and dimethylformamide (25 ml) was heated at 125°C for 6 h. DMF was evaporated and the residue coevaporated twice with n-butanol and twice with toluene. The dry crude product was purified by flash chromatography. Yield: 3.54 g (81%) viscous oil Rf=0.46 (System A) H 1 NMR (60 MHz, CDC1 3 ); 7.60-7.95 (m, 4H) , 6.76 (s, 2H) , 3.68 (t, 2H, J=7 Hz), 2.48 (s, 6H), 2.36-2.60 (m, 2H) , 1.23-1.77 (m, 10H) Examples 15-21. From 4-(7-Phthalimidoheptyl)-2,6-dimethylpyridine N-oxide (17) to 4-(7- phthalimidoheptyl)-2,6-bis(N,N-bis(ethoxycarbonylmethyl)aminomethyl) pyridine (23). Compounds 17-23 in Scheme 4 These compounds were prepared following the conditions from Examples 3, 4, 5, 6, 7, 8 and 9 respectively. Therefore, only final results will be presented here. Example 15. 4-(7-Phthalimidoheptyl)-2,6-dimethylpyridine-N-oxide (17) Yield: 95% oil Rf=0.38 (System A) H X NMR (60 MHz, CDC1 3 ); 7.60-7.95 (m, 4H) , 6.97 (s, 2H) , 3.68 (t, 2H, J=7 Hz), 2.54 (s, 6H), 2.36-2.60 (m, 2H), 1.23-1.77 (m, 10H) . Example 16. 2-Acetoxymethyl-4-(7-phthalimidoheptyl)-6-methylpyridine (18) Yield: 89% oil Rf=0.70 (System A) H X NMR (60 MHz, CDC1 3 ); 7.60, 7.95 (m, 4H) , 6.96 (s, 1H) , 6.92 (s, 1H) , 5.14 (s, 2H), 3.69 (t, 2H, J=7 Hz), 2.51 (s, 3H) , 2.40-2.65 (m, 2H), 2.14 (s, 3H) , 1.23-1.83 (m, 10H). Example 17. 2-Acetoxymethyl-4-(7-phthalimidoheptyl)-6-methylpyridine-N-oxide (19) Yield: 96% oil Rf=0.57 (System A) H 1 NMR (60 MHz, CDC1 3 ); 7.61-7.96 (m, 4H) , 7.06 (s, 2H) , 5.39 (s, 2H) , 3.67 (t, 2H, J=7 Hz), 2.51 (s, 3H) , 2.42-2.67 (m, 2H) , 2.20 (s, 3H), 1.23-1.83 (m, 10H). Example 18. 2,6-Bisacetoxymethyl-4-(7-phthalimidoheptyl)pyridine (20) Yield: 80% oil Rf=0.71 (System A) H X NMR (60 MHz, CDC1 3 ); 7.61-7.96 (m, 4H), 7.10 (s, 2H) , 5.18 (s, 4H) , 3.67 (t, 2H, J=7 Hz), 2.45-2.67 (m, 2H) , 2.14 (s, 6H) , 1.23-1.77 (m, 10H). Example 19 . 2,6-Bishydroxymethyl-4-(7-phthalimidoheptyl)pyridine (21) The alkaline hydrolysis of phthalimido diester (20) gave as expected both the product with hydrolyzed ester functions only (21) as well as the biproduct with an open phthalimido ring system. Both products were collected after short column purification since the latter is cyclizing back under conditions applied in the next step. Total yield: 55% white crystals Rf=0.31 (System B) H^MR (60 MHz, CDC1 3 ) for pure (21); 7.61-7.96 (m, 4H) , 7.20 (s, 2H) , 5.35 (s, broad, 2H) , 4.50 (s, 4H) , 3.67 (t, 2H, J=7 Hz), 2.45-2.67 (m, 2H) , 2.14 (s, 6H) , 1.23-1.77 (m, 10H). Example 20. 2,6-Bisbromomethy1-4-(7-phthalimidoheptyl)pyridine (22) Yield: 78% white crystals Rf=0.73 (System A) H X NMR (60 MHz, CDC1 3 ); 7.61-7.96 (m, 4H) , 7.28 (s, 2H) , 4.52 (s, 4H) , 3.67 (t, 2H, J= 7Hz), 2.45-2.67 (m, 2H), 2.14 (s, 6H), 1.23-1.77 (m, 10H). Example 21. 4-(7-Phthalimidoheptyl)-2,6-bis N,N-bis(ethoxycarbonylmethyl)-aminomethyl) pyridine (23) Yield: 93% oil Rf=0.62 (System A) H 1 NMR (400 MHz, CDC1 3 ); 8.06-8.08 (m, 2H) , 7.93-7.95 (m, 2H), 7.51 (s, 2H) , 4.40 (q, 8H, J=7.1 Hz), 4.23 (s, 4H), 3.90 (t, 2H, J=7.0 Hz), 3.83 (s, 8H), 2.81 (t, J=7.6 Hz), 1.95-1.55 (m, 10H), 1.49 (t, 12H, J=7.1 Hz). Example 22. 4-(7-Aminoheptyl)-2,6-bis(N,N-bis ethoxycarbonylmethyl)aminomethyl)pyridine (24) Compound (23) (400 mg, 0.55 mmol) was dissolved in dioxane (5 ml) and sodium hydroxide (2 M, 5 ml) was added. The mixture was stirred at RT for 1.5 h, neutralized with cone , hydrochloric acid and evaporated almost to dryness. The residue was treated with hydrazine hydrate (2.0 ml) dissolved in ethanol (10 ml) and refluxed for 6 h. Volatile matter was evaporated and coevaporated with dry acetonitrile. The obtained residue was suspended in dry ethanol (50 ml) previously treated with thionyl chloride (4 ml). The mixture was refluxed for 2 h, filtered, evaporated and partitioned between saturated sodium hydrogen carbonate and chloroform. Combined chloroform extracts were dried over sodium sulphate, concentrated and finally purified using short column chromato¬ graphy. Yield: 232 mg (71%) oil Rf=0.15 (System B) H X NMR (400 MHz, CDC1 3 +CD 3 0D); 7.29 (s, 2H) , 4.17 (q, 8H, J=7 Hz), 4.00 (s, 4H), 3.60 (s, 8H), 2.72 (t, 2H, 7.0 Hz), 2.58 (t, 2H, J=7.3 Hz), 1.33-1.61 (m, 10H) , 1.26 (t, 12H, J=7.0 Hz). Example 23. Hydrolysis of compound (24) and synthesis of aminochelate (25) Hydrolysis of compound (24) was performed analogously to Example 11. The obtained product was precipitated using standard acetone precipitation. Example 24. 4-(3-Benzyloxypropyl)-2,6-dimethyl-pyridine (26) Sodium amide solution in liquid ammonia was prepared from sodium (1.03 g,- 44.6 mmol) in 100 ml of liquid ammonia according to Example 1. Collidine (5 g, 41.3 mmol) in dry tetrahydrofurane was dropped in and the mix¬ ture was stirred for 60 min at -40°C. To this stirred mixture l-benzoxy-2- bromoethane (Tetrahedron Lett. _28_, 2639-42, 1979) (8.24 g, 39.2 mmol) dissol¬ ved in dry tetrahydrofurane (20 ml) was added dropwise during 30 min and the reaction mixture was stirred overnight. After evaporation of all volatile matter the residue was partitioned between sodium hydrogen carbonate and chloroform. Evaporation of chloroform phase gave an oily residue which was distilled at reduced pressure — Bp. 145-150/12 mmHg. H i MR (60 MHz, CDC1 3 ); 7.30 (s, 5H) , 6.73 (s, 2H) , 4.46 (s, 2H) , 3.43 (t, 2H) , 2.60 (t, 2H), 2.42 (s, 6H), 1.84 (m, 2H). Examples 25-31. From 4-(3-Benzyloxypropyl)-2,6-dimethylpyridine-N-oxide (27) to 4-(3-Benzyl- oxypropyl)-2,6-bis(N,N-bis(ethoxycarbonylmethyl)aminomethyl)pyridin e (33). Compounds 27-33 in Scheme 5. These compounds were prepared following the conditions from Examples 3, 4, 5, 6, 7, 8 and 9 respectively. Therefore, only the final results will be presen¬ ted here. Example 25 4-(3-Benzyloxypropyl)-2,6-dimethylpyridine-N-oxide (27) Yield: 90% oil Rf=0.42 (System A) B^NMR (60 MHz, CDC1 3 ); 7.30 (s, 5H) , 6.91 (s, 2H) , 4.46 (s, 2H),3.43 (t, 2H) , 2.60 (t, 2H), 2.44 (s, 6H) , 1.84 (m, 2H) . Example 26. 2-Acetoxymethyl-4-(3-beuzyloxypropyl)-6-methylpyridine (28) Yield: 70% oil Rf=0.64 (System A) H X NMR (60 MHz, CDC1 3 ); 7.30 (s, 5H) , 6.95 (s, 1H) , 6.90 (s, 1H) , 5.11 (s, 2H) , 4.46 (s, 2H), 3.46 (t, 2H), 2.66 (t, 2H) , 2.46 (s, 3H) , 2.09 (s, 3H), 1.86 (m, 2H) . Example 27. 2-Acetoxymethyl-4-(3-benzyloxypropyl)-6-methylpyridine-N-oxide (29) Yield: 97% oil Rf=0.5 (System A) H L NMR (60 MHz, CDC1 3 ) ; 7.30 (s, 5H) , 7.03 (s, 2H), 5.34 (s, 2H) , 4.46 (s, 2H) , 3.45 (t, 2H), 2.68 (t, 2H), 2.46 (s, 3H), 2.13 (s, 3H) , 1.86 ( , 2H), Example 28. 2,6-Bisacetoxymethyl-4-(3-benzyloxypropyl)pyridine (30) Yield: 85% oil Rf=0.69 (System A) E l ΗMR (60 MHz, CDC1 3 ); 7.30 (s, 5H) , 7.08 (s, 2H) , 5.14 (s, 4H) , 4.46 (s, 2H) , 3.45 (t, 2H), 2.70 (t, 2H), 2.09 (s, 6H) , 1.86 (m, 2H). Example 29. 2,6-Bishydroxymethyl-4-(3-benzyloxyρropyl)pyridine (31) Yield: 57% white crystals Rf=0.45 (System B) H L NMR (60 MHz, CDC1 3 ); 7.31 (s, 5H) , 6.99 (s, 211), 4.68 (s, 4H), 4.49 (s, 2H) , 3.69 (s, 2H, exchangeable), 3.49 (t, 2H) , 2.74 (t, 2H) , 1.89 (m, 2H). Example 30. 2,6-Bisbromomethyl-4-(3-benzyloxyproρyl)pyridine (32) Yield: 54% oil Rf=0.70 (System C) H L NMR (60 MHz, CDC1 3 ); 7.32 (s, 5H) , 7.14 (s, 2H) , 4.51 (s, 2H) , 4.48 (s, 4H) , 3.45 (t, 2H), 2.71 (t, 2H) , 1.90 (m, 2H). Example 31. 4-{3-Benzyloxypropyl)-2,6-bis(N,N-bis(ethoxycarbonylmethyl)-aminomethyl) pyridine (33) Yield: 97% oil Rf=0.49 (System A) H X NMR (60 MHz, CDC1 3 ); 7.30 (s, 5H), 7.08 (s, 2H) , 4.46 (s, 2H) , 4.14 (q, 8H) , 3.95 (s, 4H), 3.60 (s, 8H) , 3.43 (t, 211), 2.60 (t, 2H) , 1.90 (m, 2H), 1.30 (t, 9H). Example 32. 4-(3-Hydroxypropyl)-2,6-bis^N,N-bis(ethoxycarbonylmethyl)aminomethyl) pyridine (34) Compound (33) (520 mg, 0.83 mmol) and hydrobromic acid (5 ml, 47%) were refluxed together for 3 h. The homogenic mixture was cooled down and most of the acid was evaporated under reduced pressure. The residue was dissolved in 20 ml of dry ethanol and refluxed for 2 h. This reestrified mixture was evaporated, dissolved in chloroform and the residual acid was extracted by sat. sodium hydrogen carbonate. The organic phase was evaporated and the title compound was purified by short column chromato¬ graphy. Yield: 376 mg (84%) oil Rf=0.32 (System A) H i NMR (60 MHz, CDC1 3 ); 7.12 (s, 2H), 4.14 (q, 8H) , 3.97 (s, 4H) , 3.45-3.60 (m, 10H), 2.68 (t, 2H), 1.86 (m, 2H), 1.21 (t, 12H). Example 33. Synthesis of phosphodiester (35) derivative of compound (34) Compound (34) (350 mg, 0.65 mmol) was coevaporated twice with dry pyridine, dissolved in 10 ml of dry pyridine and o-chlorophenylphosphoro-bistriazolide solution in dry acetonitrile (0.25 M, 6.0 ml, 1.5 mmol) was added. The mixture was stirred at RT for 60 min. Triethylammonium bicarbonate (10 ml, 1.3 M, pH 7.3) was added and the mixture was stirred for 5 min whereafter it was parti¬ tioned between sat. sodium hydrogen carbonate and chloroform. The combined chloroform extracts (3x30 ml) were evaporated and the phosphodi¬ ester (35) purified by column chromatography using 20% MeOH/chloroform as eluent. Yield: 460 mg (92%) oil Rf=0.51 (System D) H L NMR (400 MHz, CDCI 3 ); 6.90-7.70 (m, 611), 4.15 (q, 8H) , 4.04 (t, 2H) , 3.98 (s, 4H), 3.58 (s, 8H), 3.06 (q, 6H), 2.66 (t, 2H) , 1.92 ( , 2H), 1.31 (t, 9H) , 1.24 (t, 12H) Example 34. L-Lysine ethyl ester (36) in Scheme 6. Thionyl chloride (5.0 ml, 8.06 g, 68 mmol) was dropped into 500 ml of ice- cooled dry ethanol. The stirred mixture was kept for 20 min at this tempera¬ ture and L-lysine hydrochloride (20 g, 109 mmol) was added. The mixture was then refluxed for 3 h and concentrated to a volume of about 200 ml. 200 ml of diethylether was added and the crystallized product filtered off. Yield: 29 g (97%)-dihydrochloride. Rf=0.20 (System F) Example 35. ω-N-(4-Nitrobenzoyl)-L-lysine ethyl ester (37) L-lysine HC1 (5 g, 27.4 mmol) dissolved in 50 ml of water was titrated with 5 M NaOH to pH 10.5. 4-Nitrobenzoyl chloride (6.6 g, 36 mmol) in dioxane (50 ml) and 5 M NaOH were slowly added keeping the vigorously stirred reaction mixture at pH 10.5. After complete addition and disappearance of the pink colour the reaction mixture was acidified with cone. HC1 to pH 2 and extracted four times with diethylether. The aqueous phase was concentrated to dryness, coevaporated twice with 200 ml of dry ethanol and suspended in 250 ml of dry ethanol pre¬ viously treated with 10 ml of thionyl chloride. The mixture was refluxed for 3 h, filtered and evaporated. The residual material was partitioned between saturated sodium bicarbonate and chloroform/ethanol 1:1 and the organic phase was dried over magnesium sulphate yielding a crude product which was purified by flash chromatography using 5% EtOH/chloroform as eluent. Yield: 1.08 g (12%) oil-crystallizing on standing Rf=0.23 (System A) H X NMR (60 MHz, CDC1 3 ): 8.25 (d, 2H, J=9 Hz), 7.93 (d, 2H, J=9 Hz),6.87 (s, broad, 1H) , 3.99-4.34 (q, 2H), 3.30-3.60 (m, 3H), 1.40-1.75 (m, 8H) , 1.11-1.37 (t, 3H) Example 36. α-N-(Methoxycarbonylmethyl)-ω-N-(4-nitrobenzoyl)-L-lysine ethyl ester (38) Compound (37) (0.54 g, 1.7 mmol) was coevaporated with toluene, dissolved in dry acetonitrile (10 ml) and bromacetic acid methylester (0.265 g, 1.7 mmol) was added followed by pulverized dry sodium carbonate (2.0 g). The mixture was refluxed for 3 h. Filtration of the inorganic salts and evaporation of the acetonitrile gave an oily crude product which was purified by flash chromatography. Yield= 0.45 g (68%) oil Rf=0.26 (System A) H X NMR (60 MHz, CDCl 3 ): 8.25 (d, 2H, J=9 Hz), 7.93 (d, 2H, J=9 Hz), 6.63 (s, broad, 1H) , 3.95-4.30 (q, 2H) , 3.68 (s, 3H) , 3.30-3.60 (m, SH), 1.40-1.75 (m, 7H) , 1.11-1.37 (t, 3H) . Example 37. ω-N-Monomethoxytrityl-L-lysine ethyl ester (39) Dry triethylamine (1.8 ml, 18 mmol) was added to a suspension of (36) (1.5 g, 6 mmol) in 20 ml of dry pyridine. To this mixture stirred at RT, solid mono- methoxytrityl chloride (1.96 g, 6 mmol) (MMTrCl) was added in small portions during a period of 1 h whereafter the mixture was stirred for additional 2 h. A standard sodium bicarbonate work-up followed by extraction with chloroform yielded a crude product contaminated by α-MMTr isomer. The pure title product was easily isolated by flash column chromatography due to the large Rf difference between the isomers. Yield: 1.35 g (48%) oil Rf=0.43 (System A) H X NMR (400 MHz, CDC1 3 ): 7.5-6.75 (m, 14H), ' 4.18-4.13 (q, 2H) , 3.78 (s, 3H) , 3.45-3.37 (m, 1H) , 2.14-2.10 (t, 2H, J=7 Hz), 1.75-1.35 (m, 9H), 1.26 (t, 3H) Example 38. g-N-(Methoxycarbonylmethyl)-ω-N-monomethoxytrityl-L-lysine ethyl ester (40) A partially protected L-lysine derivative (39) (1.0 g, 2.13 mmol) was conver¬ ted to product (40) using the method described in Example 36. Yield: 0.81 g (70%) oil Rf=0.73 (System A) ^ M (400 MHz, CDC1 3 ); 7.46-6.77 (m, 14H), 4.19-4.14 (q, 2H) , 3.77 (s, 3H) , 3.70 (s, 3H), 3.31-3.45 (q, 2H) , 3.22-3.25 (t, 1H) , 2.09-2.12 (t, 2H), 1.35-1.70 (m, 6H) , 1.23-1.27 (t, 3H) Example 39. ω-N-Trifluoroacetyl-L-lysine ethyl ester (41) Compound (36) (2.0 g, 8.1 mmol), dissolved in 10 ml of dry ethanol was treated with dry triethylamine (4.09 g, 40.4 mmol). Ethyl trifluoroacetate (1.5 g, 10.5 mmol) was added to the stirred suspension formed, and the mixture was refluxed for 6 h. All volatile matters were then evaporated and the residue partitioned between saturated sodium hydrogen carbonate and chloroform/ethanol 1:1. The combined organic phase (5x60 ml) was evaporated, coevaporated with toluene and flash chromatographed to get the title product in form of colourless oil. Yield: 1.9 g (87%) Rf- 0.72 (System D) H 1 NMR (400 MHz, CDC1 3 ); 7.10 (t, 1H, exchangeable), 4.21-4.16 (q, 2H) , 3.45-3.40 (m, 1H), 3.38-3.31 (m, 2H) , 1.84 (s, 2H, exchangeable), 1.82-1.40 (m, 6H), 1.28 (t, 3H) . Example 40. g-N-(Methoxycarbonylmethyl)-oj-N-trifluoroacetyl-L-lysine ethyl ester (42) L-lysine derivative (41) (1.0 g, 3.7 mmol) was converted to the product (42) in a method analogous to Example 36. Yield: 1.05 g (83%) oil Rf= 0.48 (System A) H L NMR (60 MHz, CDC1 3 +CD 3 0D); 4.4-4.0 (q, 2H) , 3.68 (s, 3H) , 3.5-3.1 (ra, 5H) , 1.8-1.4 (m, 6H), 1.23 (t, 3H) . Example 41. ω-N-(4-Hydroxybutyryl)-L-lysine ethyl ester (43) L-lysine ethyl ester x 2 HCl (36) (2 g, 8.1 mmol) in 30 ml of dry ethanol was treated with dry triethylamine (5.63 ml, 40.5 mmol) and γ-butyrolactone (0.7 g, 8.1 mmol) and the obtained suspension was refluxed for 3 h. Evaporation of volatile matters and coevaporation with toluene yielded a crude product which was purified by flash chromatography using 20% methanol/chloro- form as solvent. Yield: 1.54 g (73%) oil Rf=0.28 (System D) 1 H X NMR (400 MHz, CDC1 3 +CD 3 0D) : 4.30-4.22 (q, 2H) , 3.72-3.77 (m, 1H) , 3.58-3.65 (t, 2H), 3.18-3.28 (m, 2H), 2.30-2.36 (t, 2H), 1.40-2.00 (m, 8H) , 1.28-1.34 (t, 3H). Example 42. g-N-(Methoxycarbonylmethyl)-_r-N-(4-hydroxybutyryl)-L-lysine ethyl ester (44) Compound (43) (1.22 g, 4.68 mmol) in 20 ml of dry acetonitrile was converted to product (44) in a reaction analogous to that in Example 36. Yield: 1.04 g (64%) oil Rf=0.18 (System A) H X NMR (400 MHz, CDCl 3 ); 6.25 (s, broad, 1H) , 4.16-4.21 (q, 2H), 3.73 (s, 3H) , 3.67-3.69 (t, 2H), 3.33-3.49 (m, 2H) , 3.20-3.30 (m, 3H), 2.34-2.37 (t, 2H) , 1.40-1.90 (m, 8H) , 1.26-1.30 (t, 3H). Example 43. 2-(N,N-Bis(ethoxycarbonylmethy )aminomethyl-6-(N-methoxycarbonylmethyl-N-(5-N- (4-nitrobenzoyl)-l-ethoxycarbonylam_nopentyl))aminomethyl pyridine (47) Step A: 2,6-Bisbromomethyl pyridine (241 mg, 0.91 mmol) in dry acetonitrile (10 ml) was reacted with compound (38) (360 mg, 0.91 mmol) in the presence of 2 g powderized dry sodium carbonate at RT with vigorous stirring. The resulting mixture composing of unreacted pyridine derivative, monobromodiester (45) (Rf=0.72 in System A) and tetraester (46) (Rf=0.47 in System A) was evapora¬ ted, coevaporated with toluene and flash chromatographed to obtain pure (45) in a 52% yield - oil. H MR (400 MHz, CDC1 3 ); 8.23 (d, 2H, J=8.5 Hz), 8.00 (d, 2H, J=8.5 Hz), 7.25-7.55 (m, 3H) , 6.83 (s, 1H, broad), 4.49 (s, 2H) , 4.15-4.23 (q, 4H) 3.91-4.06 (dd, 2H, J=5 Hz), 3.54-3.69 (dd, 2H, J-8 Hz), 3.63 (s, 3H) , 3.42-3.52 (m, 3H), 1.50-1.95 (m, 6H) , 1.30 (t, 3H). Continuation of this chromatographic purification resulted in isolation o symmetrical tetraester (46) in a 18% yield - oil. ^NMR (400 MHz, CDCl 3 ); 8.24 (d, 4H, J=9 Hz), 8.01 (d, 4H, J=9 Hz), 7.37 (s, 3H), 6.94 (t, 2H, broad), 4.16 (q, 4H, J=6.1 Hz), 3.9 (dd, 4H, J=15 Hz), 3.58 (d, d, 4H, J=17.7 Hz), 3.63 (s, 6H), 3.38-3.50 (m, 6H), 1.50-1.80 (m, 12H), 1.28 (t, 6H) Step B : Compound (45) (100 mg, 0.17 mmol) was coevaporated with dry acetonitrile, dissolved in 3 ml of acetonitrile, and 1 g of dry powderized sodium carbonate was added followed by iminoacetic acid diethyl ester (36 mg, 0.19 mmol). The mixture was refluxed overnight, filtered and evaporated. The residue was chromatographed yielding pure title compound (47). Yield: 92% oil Rf=0.57 (System A) H X NMR (400 MHz, CDC1 3 ); 8.25 (d, 2H, J=7 Hz), 8.02 (d, 2H, J=7 Hz), 7.40-7.55 (m, 3H), 6.91 (t, 1H, broad), 4.12-4.22 (m, 6H) , 3.89-4.04 (dd, 2H, J=15 Hz), 4.00 (s, 2H) , 3.52-3.68 (dd, 2H, J=17.5 Hz), 3.63 (s, 311), 3.59 (s, 4H), 3.40-3.50 (m, 3H) , 1.50-1.80 (m, 6H) , 1.20-1.30 (m, 9H). Example 44. 2-(N,N-Bis(ethoxycarbonylmethyl))aminomethyl-6-(N-methoxycarbonylmethyl-N-(5- N-(4-aminobenzoyl)-l-ethoxycarbonylaminopentyl) )aminomethyl pyridine (48) Solid sodium borohydride (11.3 mg, 0.3 mmol) was added to a mixture of com¬ pound (47) (100 mg, 0.15 mmol) and 2 g of palladium on carbon (10%) in 5 ml of dry ethanol. The mixture was stirred at RT for 10 min and partitioned between saturated sodium hydrogen carbonate and chloroform. Evaporated organic extracts were flash chromatographed yielding (48) in form of oil. Yield: 89% Rf= 0.37 (System A) H X NMR (400 MHz, CDC1 3 ); 7.63 (d, 2H, J=8.7 Hz), 7.38-7.55 (m, 3H) , 6.64 (d, 2H, J=8.7 Hz), 6.28 (t, 1H, broad), 4.12-4.20 (m, 6H) , 3.89-4.05 (dd, 2H, J-15.2 Hz), 4.01 (s, 2H), 3.70 (s, 3H), 3.64 (s, 4H), 3.50-3.67 (dd, 2H, J=17.7 Hz), 1.45-1.80 (m, 6H) , 1.20-1.30 (m, 6H9. Example 45. Hydrolysis of compound (48), formation of europium chelate and its conversion to the isothiocyanate (49) xnis stanαarα cycie or reactions nas Deen maαe ro iowmg tne general prescrip¬ tions from Example 12. The product was characterized on the basis of a) IR spectroscopy - the presence of isothiocyanate vibration at 2070 cm -1 b) Thin layer chromatography using acetonitrile/water (4:1) as the solvent - single spot with Rf=0.28 showing positive (UV-360 nm) test for europium 3 " after spraying with ethanolic solution of 2-naphthoyltrifluoroacetone (2-NTA) and negative test for free amino group - spraying with fluoram. c) HPLC chromatography - using ion exchanging Ultrapac Column - TSK DEAE-5PW (LKB). Running conditions: Triethylammonium bicarbonate buffer, pH 7.3, linear gradient 0.01—>0.60 M in 30 min. Flow 1.2 ml/min and detection at 278 nm. Example 46. 2-(N,N-Bis(ethoxycarboπylmethyi>)aminomethyl-6-(N-methoxycarbonylmethyl-N-(5- N- trifluoroacetyl-1-ethoxycarbonylaminopentyl))aminomethyl pyridine (50) This product has been synthesized following the two-stage procedure from Example 43, and using compound (42) as a substrate in step A. The final chro¬ matographic separation yielded the desired product as a colourless oil in a 45% overall yield (based on starting 2,6-dlbromomethyl pyridine). Rf=0.48 (System A) H 1 NMR (400 MHz, CDC1 3 ); 7.64 (t, 1H, J=7.7 Hz), 7.45 (d, 1H, J=7.7 Hz), 7.42 (d, 1H, J=7.7 Hz), 7.15 (s, 1H, broad), 4.13-4.22 (m, 6H), 4.03 (s, 2H), 3.88-4.04 (dd, 2H, J=15 Hz), 3.66 (s, 3H), 3.61 (s, 4H), 3.48-3.68 (dd, 2H, J=17.8 Hz), 3.33-3.45 ( , 3H), 1.50-1.80 (m, 6H) , 1.20-1.32 (m, 9H). Example 47. Hydrolysis of compound (50), formation of europium chelate and its conversion to the bromoacetamido derivative (51) Hydrolysis of compound (50) (0.75 g, 1.14 mmol), and Its chelate formation has been performed according to the general descriptions from Example 12. To the water solution of this europium chelate N-ethyl-N,N-diisopropylamine (0.29 g, 2.24 mmol) was added, followed by bromoacetylchloride (0.54 g, 3.43 mmol) dissolved in 10 ml chloroform. The solution was stirred for 15 min. Chloroform phase was separated and the water phase was neutralized with sodium bicarbonate. The desired product (51) was obtained in form of white powder after concentration of the water phase and precipitation with acetone. Example 48. 2-(N,N-Bis(ethoxycarbonylmethy]))aminomethyl-6-(N-methoxycarbonylmethyl-N-(5- N-(4-hydroxybutyryl)-l-ethoxycarbonylaminopentyl) )-aminomethyl pyridine (52) This product has been synthesized following the two-stage procedure from Example 43 and using compound (44) as a substrate in step A. Yield: 56 % oil Rf=0.25 (System A) H^MR (400 MHz, CDC1 3 +CD 3 0D); 7.66 (t, 1H, J=7.6 Hz), 7.47 (d, 2H, J=7.6 Hz), 6.28 (s, broad, 1H), 4.17 (q, 6H, J=7.0 Hz), 4.03 (s, 2H), 3.96 (dd, 2H, J=15.1 Hz), 3.70 (t, 2H, J=7.0 Hz), 3.67 (s, 3H) , 3.61 (s, 4H) , 3.59 (dd, 211, J=17.3 Hz), 3.43 (t, 1H, J=5.6 Hz), 3.23 (m, 2H), 2.36 (t, 2H, J=6.8 Hz), 1.86 (m, 211), 1.72 (m, 2H) , 1.38-1.60 (m, 4H) , 1.29 (t, 3H), 1.27 (t, 6H). Example 49. Synthesis of (53)-phosphoramidate derivative of compound (52) To compound (52) (0.52 g, 1 mmol) dissolved in anhydrous dichlormethane (10 ml) was added diisopropylethylamine (0.78 ml, 4.6 mmol) followed by 2-cyano- ethyl-N,N-diisopropylaminophosphochloridate (0.47 g, 2 mmol). After 15 min stirring at RT the mixture was washed with cold sodium bicarbo¬ nate, extracted with dichloromethane, evaporated and purified by silica gel column chromatography using petroleum ether/ riethylamine 9:1 as eluating solvent. Yield: 85% (colourless oil Rf=0.42 (System A) P31NMR (400 MHz, CDC1 3 ); 148.5 ppm. singlet (reference 85% phosphoric acid) Scheme 2. Rj = protected side chain of any multifunctional —aminoacid Scheme . , ysine (A) R-X (B) R-χ= monomethoxyiπtyl chloride = CF 3 C0 2 Et = 0CH 2 CH 2 CH =0 ."•chi'iiic (i. Scheme 8.

Claims: Claims 1. An oligonucleotide probe comprising a sequence with 16-200, preferably 35-100, nucleotides and being labeled at one of its terminal positions with a non¬ linear branched polymer carrying a plurality of negatively charged groups, preferably phosphodiester groups, and optionally also a plurality of analytically indicatable groups, preferably luminogenic or luminescent e.g. lanthanide chelate, covalently linked to terminal ends of the branches of said polymer. 2. The probe of claim, c h a r a c t e r i z e d in that said terminal position is the 5 '-terminal end. 3. The probe of any of claims 1-2, c h a r a c t e r i z e d in that the polymer has the structure: 0 i -0- (P u -O-CHs (-A-0-) r)mLn (i) 0 0 where (ii) represents a repetitive unit having none, one, two or three terminal ends (A-0-) ; A is a straight, branched or cyclic aliphatic carbon chain providing a distance of at least 6 and at most 10 atoms between two neighbouring phosphorous atoms; L is bound to said terminal ends and represents (a) hydrogen or (b) derivatized forms of terminal ends carrying hydrogen (OH) or (c) the analytically indicatable group bound to one of said terminal ends or to a derivatized form thereof allowing such binding; s is an integer 0 or 1 and r an integer 2 or 3 and s + n = 3; m is an integer 2-50, preferably 5-15, that equals the times the unit (ii) is repetitive in the longest chain of the non-linear polymer starting from the its link to the oligonucleotide sequence; and n is the number of analytically indicatable groups, the relation between r, m and n being k =n where k is an amplification factor greater than 1.00 but smaller or equal to r. 4. The probe of claim 3, characterized in that r=2 and s=l. 5. Organic compound having the structure where R, and R~ are identical or different and consist of lower alkyl (C--C-) selected from linear, branched and cyclic alkyl groups, with preference for R-=R 2 =isopropyl; R., is alkyl (C,-C 7 ) , alkylaryl, arylalkyl or aryl each of which optionally being provided with electron- withdrawing substituents such as cyano, the preferred R-. being cyanoethyl; and A,, A- and A., are selected from the group consisting of hydrogen, A'-O-B, A' '-0-B and A' ' '-O-B, at most one of A, 3 being hydrogen and A' , A - ■ and A 1 ' ' being a straight, branched or cyclic aliphatic hydrocarbon chain providing a distance of at least 5 and at most 9 atoms between the phosphorous atom and the oxygen directly bound to B, and B being a protecting group such as dimethoxytrityl, pixyl and monomethoxytrityl; preferably at least two of A. , are identical while the other is hydrogen.

Inventors: HURSKAINEN PERTTI, KWIATKOWSKI MAREK, SUND CHRISTIAN, HURSKAINEN, PERTTI, KWIATKOWSKI, MAREK, SUND, CHRISTIAN
Applicants: PHARMACIA AB, WALLAC OY
Application year: 1989
Main classification: C12Q1
IPC codes: C07F9, C07H21, C12Q1