DK158948B - Indole derivatives and their use when preparing indole- carrier conjugates or affinity matrices - Google Patents

Description

in

DK 158948 B

The present invention relates to novel indole derivatives of the formula I as claimed in claim 1 and to their use.

Many indole compounds are biologically active. There can be either 5 naturally occurring substances or purely synthetic derivatives. In clinical and plant physiological contexts, it is important in biological systems in vivo and in vitro to be able to determine the concentrations of these compounds with a high degree of precision as well as to develop standardized assay-10 methods for determining these compounds.

Over the past few years, the number of published analytical methods based on immunochemical reactions has grown significantly. Among the main reasons for this, it can be mentioned that these 15 methods have a high sensitivity and specificity while the methods have a high capacity, which allows many analyzes per day. By using monoclonal instead of polyclonal antibodies, it is usually possible, by appropriate selection among the antibody-producing hydride cells, to increase antibody specificity and thereby minimize unwanted cross-reaction with related compounds. The use of monoclonal antibodies also has the great advantage that these assay methods can be standardized. This facilitates the ignition of analysis results from various laboratories.

Indole compounds are low molecular weight substances. In immunological contexts, these compounds act as haptenes and are thus not directly immunogenic. Therefore, in order to obtain an immune response 30, it is necessary to covalently link the hapten to a high molecular weight carrier, typically a protein. This will change the original hapten structure and reduce the similarity to the free hapten. This relationship will be expressed in the specificity of the antibodies produced against the free hapten and in the cross-reaction of the antibodies with related compounds. In general, the antibody specificity is at least towards the binding sites on the hapten molecule where the covalent binding is introduced.

2

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This problem can be illustrated by comparing two types of antibodies to the important p1 ante hormone, the auxin indole-3-acetic acid (IAA} of the formula:

_ _p. CH-COOH

Γ jTT

k in ndo1-3-acetic acid 10 In the literature, two methods are described for preparing immunogenic IAA protein conjugates. One method is based on a Mannich reaction between the indole nitrogen atom and a protein amino group. The method was used by N.S. Ranadive and A.H. Sehon, Canadian Journal of Biochemistry, 1967, vol. 45, pp. 1701-1710, in connection with the preparation of immunogenic 5-hydroxytrypta-min protein conjugates and later by W. Pengelly and F.J. Meinz, Planta, 1977, Vol. 136, pp. 173-180, for the preparation of immunogenic IAA-N-1 protein conjugates. The putative structure of this conjugate is shown by the formula: - - ch 2 -cooh 2 5 1 CH- i Δ

. NH

in

Protein IAA-N-1 protein.

30

The second method is based on the introduction of an amide bond between the carboxylic acid group and a protein amino group. N.S. Ranadive and A.H. Sehon, Canadian Journal of Biochemistry, 1967, vol. 45, pp. 1681-1688, has used such immunogenic indole-protein conjugates prepared by a symmetrical anhydride reaction of 5-hydroxyindole-3-acetic acid with a protein to produce antibodies. to the free hapten 5-hydroxyindole-3-acetic acid. E.W .. Weiler, Planta, 1981, Vol. 153, pp. 319-325, 3

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have used a mixed anhydride reaction between indole-3-acetic acid and a protein to introduce a corresponding amide bond between the ligand and a protein amino group. The structure of this conjugate is shown by the formula: 5.0 - [- CH2-c-HH protein γ

H

10 IAA-C-1 protein.

When comparing antibodies produced against the two types of IAA protein conjugates, a marked difference is observed with respect to the cross-reactivity of the antibodies with related compounds. Antibodies produced against IAA-N-1 protein conjugates are very specific to indole C-3 substitution and therefore only a small cross-reaction with related indole C-3 compounds is observed. In contrast, in ndolspec in the fitness, there are 20, and a considerable degree of cross-reaction is observed with other cyclic acetic acid derivatives, e.g. naphthalen-l-acetic acid. Antibodies produced against IAA-C-11 protein conjugates exhibit the opposite pattern of specificity. Thus, a significantly higher degree of cross-reaction is observed with the other C-3-substituted indoles than with antibodies produced against IAA-N-1 protein conjugates. In contrast, the indole specificity of these antibodies is significantly higher compared to antibodies produced against IAA-N-1 protein conjugates, and the degree of cross-reaction with e.g. naphthalene-1-acetic acid is therefore significantly lower.

These results show that both the indole nitrogen atom and the carboxylic acid group are part of the hapten immunogenic determinant. The results clearly show that the antibody specificity is reduced against the functional group masked by the covalent linkage between the hapten and the carrier molecule. In connection with the preparation of an immunogenic hapten conjugate, it is important to obtain a high degree of substitution of the chosen one.

DK 158948 B

4 carrier molecules. Many indoles are labile. It is therefore important that all reactions take place under mild conditions. It is also important that the coupling between the hapten and the carrier molecule takes place in a solvent system compatible with the stability of the carrier molecule. This is especially important when a labile or functional protein is used as a carrier molecule, e.g. an enzyme. Coupling by means of activated esters in pure aqueous systems or systems containing water-miscible organic solvents is suitable in this 10 step.

The object of this invention is to provide novel indole carrier conjugates wherein any indole C-3 substituents appear in optimum analogue form with the respective free indole compound such that the substituents as defined below are retained in unaltered, active form.

Therefore, the present invention relates to novel indole derivatives which are characterized by the general formula I

2 0 D-A-0 ^ ----- R3 υζΧ 25 I. .

H '0

II

wherein R 3 represents hydrogen or -CH 2 C-OQ, where Q together with the acetic acid residue forms an ester, A represents a coupling residue selected from -C- and -C-,

II II

0 S

and D represents a nucleophilic group of formula Y-X-D 'wherein X represents a direct bond or a straight or branched, saturated or unsaturated hydrocarbon chain of 1-9 carbon atoms.

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more, D 'represents -ΝΉ-, -S- or -O-, and Y represents a carboxylic acid group, an ester thereof, -OH or tosylates or tresylates thereof.

Preferred indoles in levels according to the invention are those in which A derives from compounds selected from 1,1'-carbonyldiimidazole, 1,1'-carbonyldi (1,2,4-triazole), di (N-succinimidyl) - carbonate, 1,11-carbonylbis (2-methylimidazole) and 1,11-thiocarbonylimidazole.

10

Further preferred in ndo 1 which in levels of the invention are those wherein the nucleophilic group D is derived from compounds selected from amino-C 1-6 alkanoic acids and esters thereof, C tosylates or tresylates thereof.

Particularly preferred indole derivatives are compounds of the formula 0 0 20 11 11 H C 0 - (CH 2) n -NH-C-O-j 2 O-jpCH 2 -C-O-Q (III)

H

Wherein n is 0-9 and Q is as defined above, namely a protecting group as set forth in, for example: Theodora W. Greene: "Protective Groups in Organic Synthesis", John Wiley and Sons Inc, New York, 1981. Q can thus e.g. be -CH3, -CH2CH3 or -CH2C00CH3.

30

Further preferred indole derivatives are compounds of Form-1 and 35 6

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O O O

li II II

E-O-C- (CH2) n-NH-C-O - ^^ j__jCH2-C-O-Q (V) Ϊ

5 H

wherein n and Q have the above meanings, and -COOE is an ester group.

A particularly preferred indole derivative of the invention is the compound of formula 0

II II

H00C-CH2-CH2-NH-C-O> N2 CH2 "C-O-CH2-CH3 (VI)

H

Another particularly preferred indole derivative of the invention is the compound of formula 20 V 0 0 0 0

W II II II

[^ IJ-O-C-CH2-CH2-NH-C-O- ^ J ^ __ ^ CH2-C-O-CH2-CH3 (VII)

26 I

wherein V is H or -SO 3 H or a salt thereof, particularly preferably an alkali metal salt such as a L 1, Na or K salt.

The above derivatives of formulas VI and VII are particularly preferred compounds of the invention in that they enable the preparation of a ligand with great structural similarity to the important plant hormone, the auxin indole-3-acetic acid. 1

The present invention further relates to the use of the indole derivatives of the invention in the preparation of indole-carrier conjugates, particularly preferred conjugates in which the carrier is a protein. It is thereby possible to make a 7

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antigen or an enzyme assay or enzyme tracer with optimal resemblance to the free plant hormone, the auxin indole-3-acetic acid.

The invention further relates to the use of an indole in the waters of Formula I as previously defined, particularly preferably the indole derivatives of Formula VI or VII in the preparation of an affinity matrix. Hereby an affinity nine matrix can be obtained for purification of the antibodies corresponding to the above antigens or for purification of biologically important hormone receptors.

10

Thus, the invention will apply to the preparation of immunogenic indole-carrier conjugates for antibody production as well as to the preparation of enzyme assays or enzyme tracers which will be used in analytical contexts.

Furthermore, in Level 1, these will be useful in the preparation of affinity gels for the purification of indole-specific antibodies and biologically important indole receptors.

20 25 30 35

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8

Example 1

Synthesis of 5- (N- (3-sulfosuccinimidoxycarbonylethyl) carbamoyl) oxy-indolyl-3-acetic acid ethyl ester, Na salt of formula (4).

5

HAY

-rrCH2-COOH ->

10 L YY

t U) * ν - = ^ o 0 ch2-co-ch2-ch3 (21 -> o 9 _r-Cao-C-O-CH7 ~ CHq (3) ~> HOOC “CH 2" CH 2 ** NH- - 2 2

Na03S ^^ O Q ^ O

^-O-C-CH₂-CH₂-NH-C-O₂ ^--j₂-CH₂-α-O-CH₂-CH3 (4), 30

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5-hydroxy-indoly-3-acetic acid ethyl ester of formula (1)

ISLAND

CH2-C-O-CH2-CH3 5 mmol of 5-hydroxy-indolyl-3-acetic acid is dissolved in 100 ml of absolute ethanol and 0.5 ml of concentrated 37% HCl is added.

The reaction mixture is closed under Ar and left for 24 hours at room temperature with stirring. The volume is then reduced to approx. 50 ml with a rotary evaporator at 40 ° C and H2 O is added until the reaction mixture is milked. After freezing to -20 ° C and thawing to room temperature, (1) 15 is isolated in crystalline form.

H-NMR. (90 MHz) δ 7.2 (dd, 1H), 7.04 (d, 1H), 6.9 (dd, 1H), 6.68 (dd, 1H), 4.07 (q, J = 7 , 3 Hz, 2H), 3.63 (d (allylic), 2H), 2.89 (OH), 2.85 (NH), 1.20 (t, J = 7.3 Hz, 3H).

20

Mass spectrum m / e 146 (base peak), 219 (N- (carboxyethyl) carbamoyl) -oxy-indoly-3-acetic acid ethyl ester of formula (3)

ISLAND

5 mmol (1) is dissolved in 10 ml of dry dioxane and 10 mmol of carbonyl diimidazole is added. The reaction mixture is closed under Ar and stirred for 1 hour. The solvent is then removed in a rotary evaporator at 40 ° C. The evaporation residue containing 35 (2) is washed with cold 0.1 M sodium phosphate buffer, pH 7.0, and immediately reacted with 7.5 mmol of 3-aminopropanoic acid, dissolved in 10 ml of 50% H2 O dioxane, pH 8.0. The reaction mixture is closed under Ar and stirred for 24 hours. The volume is then reduced to about 2 ml

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10 on a rotary evaporator at 40 ° C and 10 ml of ethanol is added. After cooling, the formed precipitates are filtered off. The filtrate is purified by ion exchange chromatography on QAE-Sephadex® G-25 (Pharmacia Fine Chemicals) in 50% H 2 O ethanol, pH 7.0. Elution is performed with NaCl saturated 50% H 2 O ethanol, adjusting the pH to 4.0 by adding 1 N hydrochloric acid. The ethanol is removed from the eluate by treatment in a rotary evaporator at 40 ° C and (3) is taken up in ethyl acetate. After drying over magnesium sulfate, the fraction is evaporated to an oil.

10 H-nmr. (90 MHz) δ 7.61 (NH, 1H), 7.34 (d, 1H), 7.26 (s, 1H), 7.14 (d, 1H), 6.78 (dd, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.67 (s, 2H), 3.30 (m, 2H), 2.45 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). Mass spectrum m / e 146 (base peak), 219, 261, 334.

15

Preparation of 5- (N- (3-sulfonsuccinimidoxycarbonylethyl) carbamoyl) -oxy-indiolyl-3-acetic acid ethyl ester, Na salt of formula (4).

20 O

Na0-j | -CH2 -? - 0-CH2-CH3 25 Dissolve 1 mmol (3) in ΙΟΟμΙ of dimethylformamide and 900 μΐ of water are added together with 1 mmol of N-hydroxysulfosuccinimide Na salt.

To this mixture is added, with stirring, 1.2 mmol of N- (3-dimethylaminopropyl) -N1-ethyl carbodiimide hydrochloride. The product is stirred for another 15 minutes at room temperature. It is used without further purification.

35 11

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Example 2

Preparation of a hapten carrier conjugate with indole-3-acetic acid ethyl ester or indole-3-acetic acid as a ligand and a protein as carrier.

Bovine serum albumin (Cohn fraction V) is dissolved in 0.2 M potassium phosphate buffer, pH 7.4, concentration 100 mg / ml. To 100 μΐ of this solution are added two portions of 8 mg of 5- (N- (3-sulfosuc-10 c in n in doxy-carbonylethyl) carbamoyl) -oxy-indolyl-3-acetic acid ethyl ester, Na salt at 2 hour intervals. The reaction mixture is stirred for 16 hours at room temperature. Thereafter, the reaction mixture is dialyzed for 5 days at 4 ° C with stirring against 5x2 1 10 mM potassium porosate, 0.15 M NaCl, pH 7, ft, with daily change of the outer liquid.

The final indole-3-acetic acid ethyl ester conjugate is analyzed by UV spectroscopy.

The prepared indo-β-acetic acid ethyl ester conjugate can be converted to the corresponding indole-3-acetic acid conjugate by eventually removing the ethyl group from the otherwise finished conjugate. This can be achieved by treating the indole-3-acetic acid ethyl ester conjugate with an ethyl esterase. In the spectrum, cf.

25a, an indole shoulder with reversing key is observed at 294-295 nm.

FIG. Ib shows a UV spectrum of bovine serum albumin (.....) and indole-3-acetic acid conjugate (-). FIG. 1a shows a diffuse purification spectrum of conjugate and carrier protein.

The concentrations are adjusted at Xmax such that A (278) = 0.45.

35

Example 3 12

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Synthesis of 5- (N-succinimidoxycarbonylethyl) carbamoyl) -oxy-indolyl-3-acetoxyacetate methyl ester of formula (4).

5 HCl) -CH2-C00H -►

10 H

0 o

in! II

HO .. CH2-C-O-CH <? - C-O-CH3 (1) - y · CX / «

H

9Π ν = Λ 0 0 0

20 \ 11 II II

N-C-O, CH2-C-O-CH2-C-O-CH3 (2) - £ υςί

L H -J

25 .... ... · o 0 0 II II 11 hooc-ch2-ch2-nh-c-o ^ CH2-C-O-CH2-C-O-CH3 (3) _ £

30 I

H

CO O o 0 0 <t ir 11 li N-0-C-CH 2 -CH 2 -NH-C-0 ^^ CH 2 -C-0-CH 2 -C-O-CH 3 (4) * ¢ 0

H

13

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5-Hydroxy-indoleyl-3-acetoxyacetate methyl ester of formula (1) OO η η HO CH2-C-O-CH2-C-O-CH3 (1)

H

Dissolve 5 mmol of 5 ~ hydroxyindolyl-3-acetic acid in 12 ml of dry dimethylsuoxide (residual water content <0.03%). and 1 ml of triethylamine is added and the reaction vessel is closed under Ar. With vigorous stirring at room temperature, 6 mmol of bromoacetic acid methyl ester is added over 10 min. When the addition is complete, stir for another 30 minutes at room temperature. Then, the reaction mixture is diluted with 25 ml of ethyl acetate and washed with 4 x 50 ml of 0.1 M NaHCO3 followed by washing with 50 ml of saturated aqueous NaCl and finally with 50 ml of water. The organic phase is dried over anhydrous MgSO 4 and the desiccant is filtered off. Ethyl acetate is removed in a rotary evaporator at 40 ° C, whereby the product crystallizes. The crystalline product is washed with cold ether.

iHnmr (90 MHz): 7.30-6.62 (m, 4H), 4.65 (s, 2H), 3.77 (d, J = 0.6Hz, 2H), 3.70 .2.5 (s, 3H),

Mass spectrum m / e: 263 M + (15.7), 173 (9.8), 146 (100), 117 (5.3).

5- (N- (carboxyethyl) carbamoyl) oxyindolin-3-acetoxyacetate methyl ester of formula (3).

0 0 0

II II II

hooc-ch2-ch2-nh-c-o ch2-c-o ~ ch9-c-o-ch3

H

Mole ratios and reaction conditions for the preparation of this compound are identical to that of Example 1.

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14 H-nmr (90 MHz): 7.45-6.72 (m), 4.66 (s), 3.85 (d, J = 0.61), 3.69 (s), 2.66 (q).

Quarter at approx. 3.4 cannot be seen for water peak.

5

Mass spectrum m / e: 378 M + (0.08), 277 (1.8), 263 (22.3), 173 (13.7), 146 (100), 117 (6.6).

5- (N- (suedin midoxycarbonylethyl) carbamoyl) -oxyindolyl-3-acetoxy-acetate methyl ester of formula (4).

CO 0 0 00

<β Η II II

N-O-C-CH 2 -CH 2 -NH-C-0n _ ^^ CH 2 -C-O-CH 2 -C-O-CH 3

H

Dissolve 1 mmol (3) together with 1 mmol N-hydroxysuccinimide in 2.5 20 ml of dry, peroxide-free dioxane. Then 1.2 mmol of N, N'-dicyclohexylcarbodiimide is added. The reaction mixture is closed under Ar and stirred for 12 hours at room temperature. The N, N'-dicyclohexylurea formed is filtered off and the dioxane is removed in a rotary evaporator at 40 ° C. The resulting oil is taken up in 1.5 ml of acetone and the last residue of Ν, Ν'-dicyclohexylurea is removed by filtration. Acetone is removed in a rotary evaporator at 40 ° C. The evaporation residue containing (4) is used without further purification.

30 35 15

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Example 4

Preparation of a hapten carrier conjugate with ndol-3-acetic acid as a ligand and a protein as carrier 5β-s-s-f) N-1 10.

V

2.

"h!" # o o o o tf "K (in il V4 ^ / - NH-C-CH2-CH2-NH-C-0 CH2-C-O-CH9-C-O-CHo W '

ψ H

3.

30 ϋ? / I

35 H

4th

16

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N / hs - 5 w Æl o o o iML 11 11 W / _NH-C-CH 2 -CH 2 -NH-C-0 / v ^^ CH 2 -C-0 “, Na +

Ogr 10 li 1st step. Preparation of BSA Gel 0.5 g of bovine serum albumin (Cohn fraction V) (BSA) is dissolved in 0.1 M NaHCO 3 and 1 mM Na 2 EDTA. The protein solution reacts overnight with 5 g of Thiopropyl Sepharose® (Pharmacia Fine Chemicals) previously washed with 0.1 M NaHCO 3 and 1 mM Na 2 CO 3. The reaction takes place under gentle mechanical stirring at room temperature. Then the gel is washed with 0.1 M NaHCO 3 and 1 mM 20 Na 2 EDTA until the wash water no longer shows absorption at 280 nm. Wash with another 5 volumes of H 2 O. Unreacted gluthathione-2-pyridyl disulfide groups are reduced by treating the washed gel with 20 ml of 50 mM mercaptoethanol in 0.1 M acetic acid, pH 4.5. The gel is then washed with 0.1 M acetic acid of pH 4.5 followed by washing with 0.1 M carbonate buffer and 1 mM Na 2 EDTA, pH 8.0. In both cases, a 10 times surplus is used.

Step 2. Coupling of ligand to BSA gel 30 5 g of BSA gel are suspended in 50 ml of 0.1 M carbonate buffer and 1 mM EDTA, pH 8.0. 0.1 mmol of 5- (N- (succinimidoxycarbonylethyl) carbamoyl) -oxyindoly-3-acetoxyacetate methyl ester is dissolved in 1 ml of dimethylformamide. Every half hour, 0.2 ml of this solution is added to the gel suspension with mechanical stirring at room temperature. Half an hour after the last addition of ligand, the gel is first washed with equilibration buffer and then with 50 mM borate buffer and 1 mM Na 2 EDTA, pH 8.6. For washing, 100 ml of each buffer is used.

17

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Step 3. Removal of the ligand ester group by esterase treatment

The gel (2) is suspended in 50 ml of 50 mM borate buffer and 1 mM 5 Na 2 EDTA and 1300 U esterase (carboxylic acid ester hydrolase EC 3.1.1.1.) Dissolved in 2 ml of the above buffer is added. The reaction mixture is shaken for 48 hours.

Step 4. Reduction of disulfide bond between gel and hapten-10 conjugate

The esterase-treated gel is transferred to a column and washed with 0.1 M sodium phosphate buffer and 1 mM Na 2 EDTA, pH 8.2 until the wash water no longer shows absorption at 280 nm.

15 Then wash with another 5 column volumes of the same buffer. The column is eluted with 50 mM mercaptoethanol and 0.1 M sodium phosphate buffer pH 8.2 until the eluate no longer shows absorption at 280 nm. The eluate containing the indole-3-acetic acid BSA conjugate is dialyzed for 2 hours against running tap water followed by dialysis against 2 times 2 1 50 mM sodium phosphate buffer, pH 7.0.

In FIG. 2 is a UV spectrum of this conjugate (A) and of BSA (B). The protein concentration in both cases is 0.23 mg BSA per ml 0.1 M calcium phosphate buffer, pH 7.0 (protein determination by Lowry's method).

1%

Using E28O nm = 10 as the extinction coefficient of 30 BSA and 6280 = 5200 M_1 as the don molar extinction coefficient for the ligand, a degree of substitution of 34.4 moles of ligand per mole of carrier BSA can be calculated. Particular attention is paid to the shoulder in the region of 290-295 nm, which shows a reversal key as an indication that the extra UV absorption originates from an indole structure, cf. la.

18

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Preparation of an IIA-C5 acarose cell.

10 g of Sepharose 4B (Pharmacia Fine Chemicals, Sweden) is transferred to a BUchner funnel and washed as follows: 5 1) 100 ml 0.9% NaCl.

2) 100 ml of 0.4 M NaOH.

Excess liquid is removed and the gel is transferred to a container where it is suspended in 12 ml of 0.1 M NaOH. With mechanical stirring at 40 ° C, 1 ml of epichlorohydrin (1-chloro-2,3-epoxypropane) is added. Stirring is continued at 200 rpm for another 2 hours.

The epoxide activating gel is washed on a BUchner funnel according to the following procedure: 1) 100 ml 0.9% NaCl.

2) 100 ml water / acetone 80/20.

3) 100 ml water / acetone 50/50.

4) 100 ml water / acetone 20/80.

5) 100 ml of acetone.

6) 100 ml water / acetone 20/80.

7) 100 ml water / acetone 50/50.

8) 100 ml water / acetone 80/20.

9) 100 ml 0.9% NaCl.

25

Two 0.5 g samples are taken and analyzed for epoxide content according to Sundberg, L. and J. Porath, J. Chromatogr., 1974 90 87. Result: 37 pmol epoxide per g wet gel. This structure is shown as (1) below.

30

With mechanical stirring, the epoxide activating gel is reacted with 20 ml of 10% ammonia water. After stirring for 8 hours, the gel is transferred to a BUchner funnel and washed by the following procedure: 1) 100 ml of 0.1 M HCl.

2) 100 ml of water.

3) 100 ml of 0.1 M NaOH.

4) 100 ml of water.

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19

Two samples are taken to determine the amino structure by titration with 0.025 M HCl. Result: 35 μιηοΐ amino structure per g wet gel. This structure is shown as (2) below.

5 g of aminogel are suspended in 10 ml of 0.1 M NaHCO3 and 1.75 x 10 “4 mol of 5- (N- (succinimidoxy-carbonylethyl) carbamoyl) -oxyindolyl-3-acetoxyacetate methyl ester dissolved in 10 ml of dioxane is added over 1 hour. After an additional 1 hour with mechanical stirring, the gel is transferred to a BUchner funnel and washed as follows: 1) 100 ml of 0.1 M NaHCO 3.

2) 100 ml of water.

3) 100 ml water / acetone 80/20.

4) 100 ml water / acetone 50/50.

5) 100 ml water / acetone 20/80.

6) 100 ml of acetone.

7) 100 ml water / acetone 20/80.

8) 100 ml water / acetone 50/50.

9) 100 ml water / acetone 80/20.

10) 100 ml 50 mM borax, 1 mM Na 2 EDTA.

The gel (3) is suspended in 10 ml of 50 mM borax, 1 mM Na2EDTA and treated with 1500 U esterase (carboxylester hydrolase EC 3.1.1.1.) For 48 hours with shaking. The gel is transferred to a 25 BUchner funnel and washed with 200 ml of 50 mM borax. The structure obtained is shown as (4) below. Using an e280 = 5200 M "1 for the ligand, it is calculated that the gel contains 28 μπιοί ligand per cell. g wet gel.

30 35

Claims (10)

20 DK 158948 B | -OH | "0-ch2-ch -ch2 ΟΗΟΗ-O-CH2-CH-CH2" nh2 (2) O -CH2 "nh" c "ch2" CH2-NH- vo HO ίΟΗ 0 0 «I Η II ^ -CH2" C "OH -O-CHa-CH -CH2" NH-C-CH2-CH2-NH- C- Ov / ^ w ^ (03 <*> H Patent Claims 1. Indole derivatives characterized by the general formula I, DA-0 fVxR3 (i '· H 0' 11 where R3 represents hydrogen or -CHaC-OQ, with Q together with the acetic acid residue forms an ester, A denotes a coupling residue selected from 21 DK-158948 B -C- and -C-, II II 0 S and D represent a nucleophilic group of the formula YX-D ', where X 5 represents a direct bond or a straight or branched, saturated or unsaturated hydrocarbon chain of 1-9 carbon atoms, D' represents -NH-, -S- or -O-, and Y represents a carboxylic acid group, an ester thereof, -OH or tosylates or tresylates thereof. 10 Indole derivatives according to claim 1, characterized in that the coupling residue A is derived from compounds selected from Ι, Ι'-carbonyldiimidazole, 1,1'carbonyldi- (1,2,4-triazole), di - '(N-succinimidyl) - carbonate, 1,1,1-carbonyl-bis (2-methylimidazole) and 1,1'-thiocarbonylimidazole. 3. Indole derivatives according to claim 1 or 2, which are suitable in that the nucleophilic group D is derived from compounds selected from am in no-C1-6 alkanoic acids and esters thereof. Cjj6-αkancanthiol is and tosylates or tresylates thereof and Cj.g alk alkanols and tosylates or tresylates thereof. Indole derivatives according to claims 1-3, characterized in that they have the formula III 25 0 0 II II H00C- (CH2) n-NH-c-0 ~ ^^ Jj - - j | CH2 - c "O" Q (111) f H where n is 0-9 and Q has the meaning given above. H wherein n and Q are as defined above and -C00E is an ester group. "Λ Indole derivative according to claim 4, characterized in that it has the formula VI 0 0 II II H00C-CH 2 -CH 2 -NH-C-O 2 n 2 _ ^ CH 2 -C-O-CH 2 -CH 3 H Indole derivatives according to Claim 1, characterized in that they have the formula V 0 0 0 II II II E-O-C- (CH 2) n "NH'c_0 ^^ Jj --- ^ CH 2 -c -0 "Q (V) Indole derivative according to claim 5, characterized in that it has the formula VII 20 V 0 0 0 0 \ A »11 11] N-O-C-CH 2 -CH 2 -NH-C-O ' -C-O-CH 2 -CH 3 (VII) In which V is H or -SO 3 H or a salt thereof. Use of a compound according to claim 1 in the preparation of indole-carrier conjugates or affinity matrices. Use according to claim 8, wherein the carrier is a protein. Use of a compound according to claim 6 or 7 in the preparation of affinity matrices.