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WO2004014941A1 - Procede de marquage selectif de peptides - Google Patents

Procede de marquage selectif de peptides Download PDF

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Publication number
WO2004014941A1
WO2004014941A1 PCT/DE2003/002534 DE0302534W WO2004014941A1 WO 2004014941 A1 WO2004014941 A1 WO 2004014941A1 DE 0302534 W DE0302534 W DE 0302534W WO 2004014941 A1 WO2004014941 A1 WO 2004014941A1
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Prior art keywords
groups
amino
peptide
labeled
group
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German (de)
English (en)
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WO2004014941A8 (fr
Inventor
Norman Koglin
Manja Lang
Annette Beck-Sickinger
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Universitaet Leipzig
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Universitaet Leipzig
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Priority to AU2003258472A priority Critical patent/AU2003258472A1/en
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Publication of WO2004014941A8 publication Critical patent/WO2004014941A8/fr
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • 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
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to a method for the selective labeling of peptides for use in biotechnological research, the pharmaceutical industry and medical diagnostics.
  • new and selectively labeled biomolecules are always required to carry out bioactivity studies. So one needs for the investigation of the interaction of peptides z. B. with peptide receptors in binding studies and for structure-activity relationships at certain positions radio-labeled and thus easily detectable peptides.
  • this label must not restrict biological activity. They have radioactively labeled peptides compared to e.g. B. fluorescence-labeled peptides have the advantage that they are highly sensitive detectable and change the biological and physicochemical properties of the biomolecules less. [1 ' 2 - 1
  • peptides are usually produced by chemical peptide synthesis.
  • a peptide is built up sequentially on a solid support material (resin).
  • the most commonly used isotopes in biochemistry as radioactive markers are 125 iodine ( I25 I), 35 sulfur ( 35 S) and tritium ( 3 H).
  • the methods for incorporating these radioactive markers into the peptide differentiate between direct and indirect labeling methods.
  • a direct marking is done e.g. B. by the use of radioactively labeled amino acids (z. B. 3: ⁇ S-methionine) for peptide synthesis.
  • the disadvantage here is that these labeled amino acids are very expensive and are also only available to a limited extent with protective groups suitable for peptide synthesis. It is also problematic that the use of radioactively labeled amino acids in the peptide synthesis has the consequence that the entire synthesis and a z. T. elaborate processing must take place in a radioactive laboratory.
  • Indirect labeling of peptides is preferably carried out by derivatization of primary amino groups (ie the N-terminus or ⁇ -amino group of lysine residues) by means of the Bolton-Hunter reaction. Radioactive acyl residues activated by N-hydroxysuccinimide (NHS) esters are transferred to amino groups.
  • NHS esters of radioactive acyl residues are commercially available, such as. B. the tritium-labeled [2,3- 3 H] propionyl NHS ester.
  • the reaction scheme for labeling a lysine residue with this NHS ester is shown in Formula 1.
  • Indirect labeling of peptides can either be carried out a.) With a peptide which is still attached to the support material after synthesis and is side-chain protected except for the site to be labeled, or b.) With a free peptide in solution.
  • radioactive labeling of a peptide bound to the carrier material is the possibility of selectively deprotecting and derivatizing amino acid side chains. This advantage is, however, after the synthesis by z. T. elaborate work steps lost under radioactive conditions.
  • the disadvantage here is that resin-bound peptides can only be used with approaches from the milligram range. Therefore, radioactive labeling on the resin requires considerable amounts of radioactivity and is very expensive.
  • the peptide is radioactively labeled according to the prior art after the peptide synthesis after the removal of all protective groups.
  • a selective marking is u. a. necessary if individual reactive groups are not allowed to be modified for the full biological activity of the peptide.
  • the object of the present invention is to provide a method for the selective labeling of peptides.
  • the step of selective radioactive labeling of the peptides in solution should be possible.
  • the peptides should also be able to have further amino groups.
  • the object is achieved by a method for the selective labeling of amino groups in peptides, in which, in a first step, the peptide is produced in a chemical solid-phase synthesis in a manner known per se, wherein
  • the protective group at the position to be marked is selectively removed and the peptide is detached from the support material
  • a radioactive label or a label which can be detected by other methods is introduced into the selectively deprotected peptide thus obtained in solution via amino-reactive substances at the position to be labeled, and
  • the method according to the invention can advantageously be handled with just a few ⁇ g peptide and is not bound to method-related minimum quantities.
  • the method described below for labeling peptides with radioactive isotopes can also be used to introduce other markers that can be detected using other methods.
  • the amino group to be labeled selectively can advantageously be chosen freely, i. H. it is either an amino group in the side chain of an amino acid building block (e.g. an ⁇ group of a lysine) or the N-terminus of the peptide built up. Selective labeling on several amino groups is also possible.
  • Peptides in the sense of this invention are understood to mean organic compounds produced by chemical solid-phase peptide synthesis, which consist of two to approximately one hundred ⁇ -, ⁇ -, ⁇ -amino acids. These are connected to one another in any order, by means of amide or peptide bonds.
  • the peptide is acylated at the N-terminus or glycosylated, phosphorylated or otherwise derivatized at the N- or C-terminus or on side chains.
  • peptide synthesis starting from a free amino acid bound to a solid support (resin) with a terminal free ⁇ -amino group (N-terminus) sequentially built a peptide in which one amino acid building block with the C-terminus is added to the free N-terminus of the growing peptide chain per synthesis step.
  • the ⁇ -amino group of the added amino acid building block then forms the new N-terminus to which the next amino acid building block is added in the subsequent synthesis step.
  • amino acid building blocks are used for the synthesis in which, apart from the C-terminus, all chemically reactive groups of the amino acids, such as the ⁇ -amino group and other functional ones Groups in the side chains such as amino groups (lysine), or z.
  • B. free hydroxyl groups (tyrosine, serine) are provided with appropriate protective groups.
  • the protecting group on the ⁇ -amino group of the added amino acid building block is selectively removed during the synthesis in order to provide a new free N-terminus for the next synthesis step.
  • the remaining protective groups remain bound and are only removed at the end of the synthesis.
  • peptide synthesis follows either the Boc / Bzl strategy or the Fmoc / l Bu strategy. Depending on the synthesis strategy, different protective groups are used.
  • the Boc / Bzl strategy is characterized by graded acid instability.
  • the ⁇ -amino groups are protected by acid-labile tert-butoxycarbonyl groups (Boc groups).
  • the Boc group on the ⁇ -amino group is usually cleaved selectively by treatment with trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the remaining reactive groups of the amino acid side chains, such as. B. hydroxyl groups (Ser, Thr) and ⁇ -amino groups (Lys) are protected by benzyl (Bzl) groups or benzyloxycarbonyl (Z) - groups that z.
  • the ⁇ -amino groups are protected by the base-labile 9-fluoro-enylmethoxycarbonyl group (Fmoc group) and the remaining reactive groups of the amino acids are generally protected by acid-labile groups.
  • hydroxyl groups are mostly protected by tertiary butyl groups ( ⁇ Bu groups) and the ⁇ -amino groups of lysine by Boc groups.
  • the Fmoc group on the ⁇ -amino group is treated during the synthesis by treatment with a weak base, e.g. B. 20% piperidine in dimethylformamide (DMF) removed.
  • DMF dimethylformamide
  • an amino acid building block is inserted at at least one position to be marked later, which carries an amino group which is protected by a group which differs functionally from the protective groups on the amino groups which are not to be marked.
  • the protective group must differ functionally from the protective groups on the amino groups that form the peptide bond.
  • This functional difference makes it possible to selectively remove the protective group at the position to be marked in a further step of the method.
  • This functional difference is based on different chemical properties, such as resistance to certain chemical substances.
  • the amino group to be labeled later is initially protected during the synthesis by a group which can be split off by a method in which the amino protective groups on the amino groups which are not to be labeled are retained.
  • all amino groups which are not to be labeled are preferably protected by photolabile protective groups (for example Nvoc), and only the amino group at the point to be labeled is protected with a chemically labile protective group (SG) such as.
  • SG chemically labile protective group
  • this is achieved in that, in the peptide synthesis, photolabile protective groups, for. B. nitroveratryloxycarbonyl (Nvoc).
  • photolabile protective groups for. B. nitroveratryloxycarbonyl (Nvoc).
  • Nvoc nitroveratryloxycarbonyl
  • Lysines that are protected on the side chain with Nvoc are e.g. B. after reacting N ⁇ -protected lysines (eg N ⁇ -Boc-Lys or N -Fmoc-Lys) with Nvoc chloride 00] .
  • the N-terminal amino group is to be selectively labeled, the N-terminal amino group, which is still protected after the last coupling step of the synthesis, is deprotected and the peptide is cleaved from the support material, while all other amino groups not to be labeled in the side chains are removed with one photolabile protecting group are derivatized.
  • an N ⁇ -photolabile-protected amino acid is preferably used in the last coupling step of the peptide synthesis.
  • This N ⁇ -photolably protected amino acid is synthesized analogously to the N ⁇ -photolably protected lysine (Vossmeyer: Journal of applied physics 1998).
  • the N-terminal amino group is deprotected after the peptide synthesis and is protected in a subsequent synthesis step by reacting the free N-terminal amino group with the acid chloride of the photolabile protective group (e.g. Nvoc chloride).
  • the photolabile protective groups used in the process according to the invention are preferably groups of the ort / zo-nitrobenzyl type, particularly preferably nitroveratryloxycarbonyl (Nvoc).
  • Nvoc chloride Its acid chloride (Nvoc chloride) has the following formula (Formula 2):
  • Such photolabile protective groups can be easily removed again by UV radiation at a wavelength of 200-600 nm.
  • 1 - ⁇ & compounds of the ortho-nitrobenzyl type undergo photoenolization under the influence of light and there is a fragmentation to ortho-nitrosobenzaldehydes.
  • the lamp strength can vary within the range of 10 W -1000 W.
  • the cleavage is preferably carried out at temperatures between 0 ° C. and 30 ° C. and within 1 minute to 5 hours.
  • the chemically labile protective group on the amino group to be labeled is selectively removed after the synthesis by hydrofluoric acid (HF) (in the case of the Boc / Bzl strategy) or trifluoroacetic acid (TFA) (in the case of the Fmoc Bu strategy).
  • HF hydrofluoric acid
  • TFA trifluoroacetic acid
  • the photolabile protective groups on the remaining amino groups that are not to be labeled are not removed in the process.
  • the peptide in solution now only contains a free amino group at the position to be labeled, which is unprotected and can be labeled selectively. This selective labeling takes place, as explained in more detail later, with a radioactive amino-reactive reagent, preferably an N-hydroxysuccinimide (NHS) ester.
  • a radioactive amino-reactive reagent preferably an N-hydroxysuccinimide (NHS) ester.
  • the photolabile protective groups on the remaining amino groups are removed by UV light after the labeling. This is shown schematically in Fig. 3_.
  • the photolabile groups are not introduced directly, but previously groups (e.g. Dde) that differ in their reactivity to a chemical agent, such as. B. hydrazine, distinguish from the remaining protective groups (SG).
  • SG protective groups
  • all amino groups which are not to be labeled with a hydrazine-labile protective group particularly preferably with 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) or 1, are preferred during the peptide synthesis - (4, 4-dimethyl-2,6-dioxocyclohex-l-ylidene) -3-methylbutyl- (ivDde-) (Novabiochem, Laufelfingen, Switzerland).
  • Such amino acids such as. B. N ⁇ -Fmoc-N ⁇ -Dde-lysine are commercially available (Novabiochem, Laufelfingen, Switzerland).
  • an amino group in a side chain e.g. B. an ⁇ -amino group of the lysine or the N-terminus
  • an amino group in a side chain e.g. B. an ⁇ -amino group of the lysine or the N-terminus
  • an amino group in a side chain e.g. B. an ⁇ -amino group of the lysine or the N-terminus
  • the amino group is provided with a base-labile protective group (e.g. Fmoc).
  • an amino acid component which contains an amino group protected with an acid-labile (hydrazine-stable) protective group in the side chain is incorporated in the site to be labeled, z.
  • a lysine whose ⁇ -amino group (N ⁇ ) is Boc-protected e.g. N ⁇ -Fmoc-N ⁇ -Boc-lysine).
  • the amino acid component to be selectively labeled accordingly contains an amino group protected with a base-labile protective group (for example Fmoc).
  • a base-labile protective group for example Fmoc
  • an amino acid building block is used in the synthesis in the last coupling step in the Fmoc Bu strategy, the ⁇ -amino group (N ⁇ ) of which contains an acid-labile (hydrazine-stable) protective group (e.g. . Boc) is protected.
  • an amino acid module is used in the last coupling step, the ⁇ -amino group of which is protected with a base-labile protective group (e.g. Fmoc).
  • the protective group which it carries during the last coupling step is removed after the synthesis. removed.
  • this is a base-labile protective group (e.g. Fmoc), which is characterized by a weak base, such as. B. 20% piperidine is removed.
  • the protective group at the N-terminus which should not be labeled is an acid-labile protective group (e.g. Boc), which is protected by an acid, such as. B. TFA is removed.
  • hyrazine-labile protective groups such as. B. Dde
  • hydrazine for. B removed with 2% hydrazine in DMF [9] .
  • protective groups which can be removed under weakly acidic conditions such as preferably 4-methyltrityl- (Mtt) or 4-methoxytrityl (Mmt), are used.
  • These protective groups are selectively split off with a dilute acid within 4 times 2 minutes instead of with hydrazine, e.g. B. with 1% TFA in dichloromethane (DCM).
  • a scavenger preferably 1% to 5% TIS (triisopropylsilane) is added.
  • the protective group on the amino acid to be labeled preferably a Boc group, which can only be removed under strongly acidic conditions (> 50% TFA), is not removed.
  • an activated derivative of the photolabile protecting group e.g. Nvoc chloride
  • N-hydroxybenzotriazole HOBt, Novabiochem
  • DIPEA diisopropylethyl min
  • DMF N, N'-dimethylformamide
  • the molarity of Nvoc chloride in the reaction solution should be as high as possible (0.1-0.5 M), but the volume should also be sufficient to completely cover the resin.
  • the volume of the respective reaction solution depends on the size of the peptide and is approx. 0.5 ml to 1 ml.
  • the reagents which are also pre-swollen in DMF, are added to the reagents, which are also dissolved in DMF, and left for at least 3 hours at room temperature or overnight react with shaking. After the reaction has ended, the KAISER test is used to check whether all the free amino groups have reacted. u * If this is not the case, the reaction is carried out again.
  • Nvoc group at all relevant positions on the polymeric support is better and easier to carry out than is the case with the direct use of N ⁇ - Nvoc-protected Lys derivatives in the synthesis. ° 0 - 'This variant also advantageously does not require the use of special Nvoc-derivatized amino acids which are not commercially available.
  • the acid-labile protecting group e.g. Boc
  • an acid such as. B. TFA removed.
  • the base-labile protective group e.g. Fmoc
  • a weak base such as. B. 20% piperidine in DMF
  • the peptide now in solution now contains only a free amino group at the position to be labeled, which is unprotected and can be labeled selectively.
  • the photolabile protective groups on the remaining amino groups can be easily removed by UV light after the marking.
  • this embodiment of the invention is preferred since N ⁇ -Dde-protected lysines, in contrast to N ⁇ -Nvoc-protected lysines, are commercially available.
  • this embodiment is only suitable for building up short peptides using the Boc / Bzl strategy.
  • the amino groups which should not be labeled are protected with base-labile protective groups (e.g. Fmoc).
  • base-labile protective groups e.g. Fmoc
  • amino acid units provided with base-labile protective groups on their side chains are used for synthesis (e.g. N ⁇ -Boc-N ⁇ -Fmoc-lysine).
  • the base-labile group e.g. Fmoc
  • a weakly basic solution e.g. 20% piperidine in DMF
  • a photolabile protective group e.g. Nvoc
  • Peptide now only contains a free amino group at the position to be marked, which is unprotected and can be selectively marked.
  • the photolabile protecting groups on the The remaining amino groups can be easily removed by UV light after the marking.
  • the peptide in which the amino group to be labeled is selectively deprotected according to one of the previously described variants of the method according to the invention, is labeled in solution with an amino-reactive substance as follows.
  • N-hydroxysuccinimide (NHS) esters are preferably used as amino-reactive substances. As described schematically in Formula 1, these NHS esters transfer an acyl group which contains the corresponding marker from the NHS ester to the amino group.
  • the acyl group preferably contains radioactive isotopes, such as. B. 1 death ( 123 I), 35 sulfur ( 35 S) and tritium ( 3 H).
  • markers are bound to the acyl group, such as the biotin detectable via the affinity for streptavidin / avidin, or immunologically detectable haptens, such as, for. B. digoxygenin or fluorescein.
  • Dyes which can be detected by fluorescence can also be used as markers, with the use of photolabile protective groups in the process according to the invention followed by the splitting off of the protective group at a wavelength at which the dyes are not bleached.
  • the labeling reaction can be carried out with just a few micrograms of peptide. Since these small amounts can only be weighed out with difficulty, a stock solution of the peptide purified with preparative HPLC and protected in a photolabile manner was prepared in bidistilled water, the desired amount (e.g. 2 nmol, approx. 10 ⁇ g depending on the molecular weight of the peptide) removed from the stock solution and lyophilized.
  • the desired amount e.g. 2 nmol, approx. 10 ⁇ g depending on the molecular weight of the peptide
  • the labeling reaction can be carried out either in an organic solvent such as DMF or in an aqueous buffer at neutral or slightly basic pH values. leads.
  • the photolabile protected peptide is dissolved in a small amount of 0.1% DIPEA / DMF or in an aqueous and slightly basic buffer.
  • a slightly acidic buffer e.g.
  • pH 6.5 pH 6.5
  • pH 6.5 pH 6.5
  • the pH value there is a compromise between the reactivity of the amino group (stronger at higher pH values) and the stability of the labeling reagent in an aqueous medium (lower at higher pH values).
  • the peptide is the cheaper component and the nucleophile in the labeling reaction and should be used in a slight excess to increase the yield.
  • Radioactive NHS esters or OSu activatable fluorescent dyes are commercially available (e.g. from Amersham). If the labeling reagent is dissolved in toluene, it is removed in a stream of N 2 before the reaction. The reaction is preferably carried out in a silanized vessel (for example an Eppendorf tube) in order to keep adsorption of ester and peptide on the vessel wall low. If there is a double excess of peptide (eg 2 nmol), 1 nmol of the labeling substance is used. The peptide dissolved in buffer or DMF is added to the labeling reagent and mixed well. The reaction takes about 1-2 hours at room temperature.
  • the photolabile protective groups on the amino groups not to be labeled are separated off under the conditions described by means of UV light in an aqueous medium.
  • the photolabile protective group is removed almost quantitatively from the peptide.
  • the cleavage is carried out in an acidic environment.
  • the labeling batch is mixed with a 0.1% aqueous TFA / ACN solution.
  • the ACN content can already be adjusted to the subsequent starting conditions of the HPLC gradient.
  • the peptide is then purified in a known manner. This is preferably done by means of HPLC (High Performance Liquid Chromatography). Since the labeling never runs 100% and, in addition, the Nvoc peptide has been used in excess of radioactive ester, the derivatized peptide must be separated from the non-derivatized peptide. Since the two peptides usually differ only slightly with regard to their elution behavior, depending on the group transferred, a gradient elution with two elution agents was expediently chosen for separation. The gradient may depend on the peptide sequence and the quality of the column used and cannot therefore be generalized for all sequences. The eluents used are: 0.08% TFA in acetonitrile and 0.1% TFA in water.
  • Another object of the invention is the use of peptides in which all amino groups not to be labeled are protected by protective groups, preferably photolabile protective groups, and at least one amino group to be labeled is unprotected for the production of a selectively radioactively labeled peptide.
  • protective groups preferably photolabile protective groups
  • at least one amino group to be labeled is unprotected for the production of a selectively radioactively labeled peptide.
  • the peptide is first synthesized using solid phase peptide synthesis using the orthogonal Fmoc / tert-butyl protecting group strategy on a milligram scale (13.5 ⁇ mol).
  • a commercially available Lys derivative with 1 - (4,4-dimethyl-2,6- dioxocyclohex-l-ylidene) ethyl (Dde) protected amino function (Novabiochem) used in the side chain.
  • This Dde protective group of the Lys side chain (s) is selectively removed directly on the resin with 2% hydrazine solution in DMF after the synthesis.
  • the later to be modified amino group, a certain Lys-side chain or the N-terminus is then still protected with acid labile Boc protecting group. If the N-terminus is marked, an N ⁇ -Boc-protected amino acid must be used for this in the last coupling step of the peptide synthesis. If, on the other hand, an N ⁇ -Fmoc-protected amino acid was used in the last coupling step, the N-terminus is already freely available on the resin after synthesis. After removal of the Dde protective group ⁇ ) all free amino groups were then provided with the photolabile Nvoc protective group simultaneously.
  • the photolabile Nvoc group was installed on the resin at the positions that should be free again after the radioactive labeling. Based on the number of free amino functions present, 5 equivalents of 6-nitroveratryloxycarbonyl chloride (Nvoc-Cl, Sigma-Aldrich), 5 equivalents of N-hydroxybenzotriazole (HOBt, Novabiochem) and 10 equivalents of diisopropylethylamine (DIPEA, Fluura) were used. 0.5-1 ml of N, N'-dimethylformamide (DMF, Biosolve) served as solvent. The reagents, which are also dissolved in DMF, are added to the resin which has been swollen in DMF and allowed to react for at least 3 h at room temperature or overnight with shaking. After the reaction is over, check with the KAISER test whether all free amino groups have reacted. 1 - 1 ⁇ If this is not the case, the reaction is carried out again.
  • Nvoc-Cl 6-nitroveratryloxycarbonyl chloride
  • the resin is washed thoroughly with DMF, methanol, dichloromethane and diethyl ether and dried in vacuo.
  • the peptide is then cleaved from the resin with 1 ml of trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the acid-labile side chain protective groups are removed at the same time.
  • a mixture of thioanisole (Fluka) and thiocresol (Fluka) in a ratio of 1: 1 (v / v) is used as the scavenger for the removal of the reactive intermediates that occur.
  • a mixture of ethanedithiol (Fluka) and thiocresol (3: 7 (v / v)) is used as the scavenger.
  • the ratio of TFA to scavenger is always 9: 1.
  • the peptides dissolved in TFA are separated from the polymeric support by filtration and precipitated by ice-cooled diethyl ether.
  • the scavenger and protective group fragments are separated by centrifugation and repeated washing with cooled diethyl ether.
  • the peptides are then dried in vacuo, dissolved in a tert-butanol-water mixture and lyophilized.
  • the peptide is synthesized by means of solid phase peptide synthesis with the orthogonal Fmoc / tert-butyl protective group strategy on a milligram scale (13.5 ⁇ mol) .'- 5 - 1
  • lysine derivatives with photolabile protected amino functions are used in the synthesis. These lysine derivatives are not commercially available and are therefore shown separately before the start of the synthesis.
  • the implementation of the synthesis of an N ⁇ -Nvoc-protected lysine derivative is described in the literature (Rusiecki, Bioorganic & Medicinal Chemistry Letters 1993). After synthesis, such peptides only contain a free amino group in the form of the N-terminus, which can then be selectively labeled.
  • the peptide is synthesized as described in Example 2, but the labeling takes place on a Lys side chain.
  • the lysine residue to be labeled is not used in a photolabile manner but in an acid-labile manner.
  • N ⁇ -photolably protected lysine derivatives are used, as described in Example 2.
  • an N ⁇ -photolabile-protected amino acid is used in the last coupling step of the peptide synthesis (position 1 in the peptide sequence).
  • Embodiment 4 Representation of 3 H-propionyl-Lys 4 -NPY ( 3 H-NPY)
  • the neuropeptide Y (NPY) consists of 36 amino acids and contains a free amino group in the Lys side chain in addition to the N-terminus. 1 - 5] It could be shown that a free N-Teminus is required for full biological activity. In contrast, the amino function of the Lys side chain can be modified without loss of affinity for the display of radioligands. ] These are commercially available in the form of 123 I-NPY and H-NPY (Amersham).
  • N-terminal Nvoc-protected NPY was therefore first synthesized and then on the Lys 4 side chain by reaction with 3 H-propionyl -NHS ester radiolabelled. After photochemical cleavage of the Nvoc group and purification by HPLC, the H-NPY thus represented was compared in binding assays with the commercially available H-NPY.
  • the NPY on Lys - "cold" - propionylated was previously after synthesis of Lys 4 (Dde) -NPY, cleavage of the Dde protective group and by subsequent Obtained reaction with propionic anhydride on the resin.
  • the radioactivity profile in the ⁇ -counter was determined by 3 H measurement of aliquots of the collected HPLC fractions. A peak can be seen in the HPLC chromatogram and in the radioactivity profile in fractions 31-36, which contained 0.8 MBq of radioactivity (22%) and thus corresponds to the desired radioactively labeled peptide (FIG. 8). It can therefore be assumed that it is H-propionyl-Lys 4 -NPY.
  • Embodiment 5 Representation of 3 H-propionyl-Lys 13 -PTH (1-34) -amide ( 3 H-PTH)
  • the peptide was synthesized using automated solid phase peptide synthesis.
  • Protected Lys derivatives were used for positions 26 and 27 Dde side chains. After the Dde protecting groups had been split off, the Lys 26 '27 amino groups and the N terminus were newly protected simultaneously with the Nvoc group. After splitting off and purification, a homogeneous product with a purity of> 95% was present. (Fig.
  • Propionylated PTH (1-34) amide was previously obtained after synthesis of Lys 13 (Dde) -PTH (1-34) amide, cleavage of the Dde protective group and subsequent reaction with propionic anhydride on the resin.
  • the radioactivity profile of the collected HPLC fractions was determined by 3 H measurement of aliquots in the ß counter. In the HPLC chromatogram and in the radioactivity profile, a peak can be seen in fractions 19-23 which contained 58.9 ' kBq of radioactivity and corresponds to the desired radioactively labeled peptide. (Fig. 14) It can therefore be assumed that this is 3 H-propionyl-Lys 13 - PTH (1-34) amide.
  • Embodiment 6 Representation of N-terminal Nvoc-protected Lys -hPP
  • the human sequence of the pancreatic polypeptide has an amino group only through the N-terminus. Since PP and NPY belong to the same family of peptides, the N-terminus of PP is also considered to be of particular importance in binding to the Y4 receptor. When designing a radio ligand, it should therefore be unmodified. For this reason, a Lys residue was introduced into the non-conserved position 4 during peptide synthesis. Later radioactive marking is to take place there.
  • Fig. 1 Schematic representation of the synthesis of Nvoc-protected peptides with a free Lys side chain
  • Fig. 2 Schematic representation of the synthesis of Nvoc-protected peptides with a free N-terminus
  • Fig. 3 Schematic representation of the selective radioactive labeling of a
  • Fig. 4 HPLC chromatogram of N-terminal Nvoc-protected NPY
  • Fig. 6 ESI-MS of N-terminal Nvoc-protected NPY.
  • Fig. 7 HPLC chromatogram of the 3 H labeling approach of Nvoc-NPY after UV irradiation.
  • the peak with the retention time of 30.3 min corresponds to unlabelled NPY after the Nvoc cleavage and the peak at 32.7 min corresponds to 3 H-propionyl-Lys 4 -NPY.
  • Fig. 8 Radioactivity profile of the after labeling, irradiation and HPLC
  • Nvoc-NPY fractions obtained (fractions 20-40; dead volume from the detector to the fraction collector approx. 1 min).
  • Fig. 9 Binding curves of the 3 H-propionyl obtained according to the Nvoc strategy
  • Lys 4 -NPY with unlabelled NPY as competitor The displacement curves at A) Yl receptor, B) Y2 receptor and C) Y5 receptor are shown.
  • Fig. 11 DAD spectrum of the HPLC chromatogram of N-terminal
  • Lys 26 '27 Nvoc-protected PTH (l-34) amide in Fig. 10 In the lower part of the spectrum is shown DAD 200-400 nm, at the top of the absorption of the peptide bonds at 220 nm (upper line) and the absorption of the Nvoc group at 350 nm (bottom line). The presence of three Nvoc groups in the molecule results in a stronger absorption for this group compared to FIG. 5 at the same concentrations.
  • Fig. 12 ESI-MS of N-terminal and Lys 26 '27 Nvoc-protected PTH (l-34) - amide.
  • Fig. 13 HPLC chromatogram of the 3 H labeling batch of Nvoc-PTH (1-
  • Fig. 14 Radioactivity profile of the after labeling, irradiation and HPLC
  • Nvoc-PTH (1-34) -amide fractions obtained fractions 10-27; dead volume from the detector to the fraction collector approx. 1 min).
  • the IC 50 value of 3.3 nM obtained at a radioligand concentration of 1 nM indicates an almost identical binding behavior of the radioligand and competitor.
  • Fig. 16 HPLC chromatogram (220 nm) of N-terminal Nvoc-protected
  • FIG. 17 DAD spectrum of the HPLC chromatogram of N-terminal Nvoc-protected Lys-hPP from FIG. 16. The DAD spectrum of 200-400 nm is shown in the lower part, the absorption of the peptide bonds is shown in the upper part 220 nm (upper line) and the absorption of the Nvoc group at 350 nm (lower line) are shown.
  • Figure 18 ESI-MS of N ⁇ -Nvoc-Lys 4 -hPP.
  • Pillai, V.N.R. Photoremovable protecting groups in organic synthesis. Synthesis,
  • N ⁇ nitrogen of the ⁇ -amino group e.g. in lysine

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Abstract

L'invention concerne un procédé de marquage sélectif de peptides rendant possible en particulier l'opération de marquage radioactif sélectif de groupes amino individuels des peptides en solution. Des peptides peuvent ainsi faire l'objet d'un marquage radioactif sélectif indépendamment de leur séquence dès l'ordre du microgramme (ñg). Le procédé selon l'invention consiste à placer, lors de la synthèse peptidique, à au moins un emplacement devant être marqué ultérieurement un élément acide aminé comportant un groupe amino qui est protégé par un groupe se différenciant fonctionnellement des groupes protecteurs au niveau des groupes amino ne devant pas être marqués. Après la synthèse peptidique, le groupe protecteur est éliminé sélectivement à l'emplacement devant être marqué et le peptide est séparé du support. Dans le peptide sélectivement déprotégé ainsi obtenu, un marquage est introduit, par l'intermédiaire de substances aminoréactives, en solution à l'emplacement devant être marqué. Les groupes protecteurs au niveau des groupes amino ne devant pas être marqués sont éliminés de manière simple après le marquage, de préférence par rayons UV, et le peptide sélectivement marqué ainsi obtenu est purifié par une méthode connue.
PCT/DE2003/002534 2002-07-23 2003-07-23 Procede de marquage selectif de peptides Ceased WO2004014941A1 (fr)

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JP2013522352A (ja) * 2010-03-24 2013-06-13 メディカル リサーチ カウンシル 方法

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US5889146A (en) * 1997-11-26 1999-03-30 Institute Of Nuclear Energy Research Method for synthesis of bifunctional chelating agents-peptides
WO2002028890A1 (fr) * 2000-09-29 2002-04-11 The Scripps Research Institute Peptides etiquetes et procedes et intermediaires servant a leur fabrication

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US5143854A (en) * 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
US5889146A (en) * 1997-11-26 1999-03-30 Institute Of Nuclear Energy Research Method for synthesis of bifunctional chelating agents-peptides
WO2002028890A1 (fr) * 2000-09-29 2002-04-11 The Scripps Research Institute Peptides etiquetes et procedes et intermediaires servant a leur fabrication

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CHERSI A ET AL: "Selective 'in synthesis' labeling of peptides with biotin and rhodamine", BBA - GENERAL SUBJECTS, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 1474, no. 2, 6 April 2000 (2000-04-06), pages 196 - 200, XP004276556, ISSN: 0304-4165 *
GARIEPY JEAN ET AL: "A simple two-step approach for introducing a protected diaminedithiol chelator during solid-phase assembly of peptides", BIOCONJUGATE CHEMISTRY, vol. 13, no. 3, May 2002 (2002-05-01), pages 679 - 684, XP002259014, ISSN: 1043-1802 *
LELIEVRE D ET AL: "On-Line Solid-Phase Synthesis of a Peptide Bi-Derivatized with Biotin and 4-Azido Salicylic Acid", TETRAHEDRON LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 36, no. 51, 18 December 1995 (1995-12-18), pages 9317 - 9320, XP004026744, ISSN: 0040-4039 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013522352A (ja) * 2010-03-24 2013-06-13 メディカル リサーチ カウンシル 方法

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