FR2630547A1 - COLD MARKING OF MOLECULES OF PEPTIDE OR PROTEIN NATURE - Google Patents

Abstract

The invention relates to a molecule for marking a peptide or a protein, or more generally a compound of peptidic or proteinic nature, the invention being characterized in that said marking molecule comprises 1) at least one unsaturated organic site, particularly with a carbonaceous unsaturation, such as a triple bond C=C or a new aromatic nucleus complexed by an organometallic compound having the formula MxLy wherein Mx represents one or a plurality of optionally different metals and Ly represents one or a plurality of optionally different ligands, complexing the metal or metals, and 2) at least one carboxyl and/or amine function, the carboxyl function being optionally protected or activated and the amine function being optionally protected. The present invention also relates to conjugates comprising the marking molecule of the invention, linked to a peptide or a protein through a peptidic bond, as well as the utilization of conjugates as tracers in the field of dosages for in vitro diagnosis, and as imaging agents for in vivo diagnosis.

Description

 The present invention relates to a new type of cold labeling of peptides, proteins or more generally of molecules of peptide or protein nature, in particular for the purpose of their determination.

 The present invention concerns in fact labeling molecules and conjugates obtained by covalently coupling these labeling molecules with peptides or proteins or more generally molecules of peptide or protein nature.

 The present invention also relates to the use as tracers in assay methods, these peptides or proteins and other compounds of peptide or protein nature or as imaging agents in the case of in vivo diagnostics.

 In any assay, it is essential to determine the distribution between free and bound fractions. For this purpose, a quantity of labeled ligands (or tracers) is incorporated in the system.

The most common markings use isotopes
3 131 125 radioactive (eg 3H, I, I) despite the known drawbacks of this approach (instability of species, difficulties of marking, high costs, legal limitations, concentration of suppliers) insofar as the use of non-radioactive tracers currently proposed offers still too theoretical advantages (defects of precision and sensitivity). However, these drawbacks stimulated the search for cold analysis methods among which the use of fluorescence is the most widely used. The Applicant has however considered a completely new organometallic alternative. It is based on a cluster of recent findings. there is a window in the infrared spectrum of
all the proteins centered on 2000 cm 1.

metal carbonyls (MXCOy) are among the species
the only ones to have vibrator strips
pure and intense in this region.

. a new generation of spectroscopic devices
Fourier transform infrared (IR-FT)
this technique in the detection area of some
MXCOy marker picograms.

 However, this analytical approach, of general scope, can only be very attractive if the methods for introducing MXCOy markers are simplified and generalized.

Certain solutions have already been proposed by the Applicant in French patent applications
FR 82 16 024, FR 87 00 914 and FR 87 04 698 in which tracers consisting of complexes between different bioreactants and an organometallic compound have been described.

 The object of the present invention is to propose other solutions for molecules of peptide or protein nature.

 In the case of peptides and proteins, the use of tracers with external markers is of common use in immunology, whereas the examples with haptens (steroids, drugs) multiply regularly. The isotopes of iodine (particularly 1251) are almost universally used for this purpose because of the high specific activities thus reached 2000 - 4000 Ci / mmol for an iodized steroid against 25-100 Ci / mmol for its tritiated equivalent. Common iodination methods include chloramine T, lactoperoxidase, iodogen, and conjugation labeling.

 This latter method was developed to circumvent the damage of the iocation sometimes observed when 1251 is introduced by direct substitution into the protein and peptide systems. It makes use of an iodization carried out, in a first step on an organic copula, in the absence of the protein under examination.

Then, in a subsequent operation, this labeled reagent forms a covalent adduct with the biological species concerned. This approach is called conjugation tagging to differentiate it from the substitution method.

Despite its qualities, this method of introducing radioactive markers is not without difficulties
the substituted marker is larger than the atom
iodine (itself comparable to benzene in this regard), and
can lead to physiological alterations of the
tracer.

occasional problems of moving by mate
unlabeled (because of the presence of
high affinity anti-body for the binding bridge)
are not to be neglected.

iodotyrosine or iodohistamine can bind
themselves to serum proteins and lead to
arterfacts.

To overcome these drawbacks, not to mention the use of radioactivity, the present invention therefore proposes a new type of labeling of peptides and proteins with an organometallic compound of general formula ML
xy in which
Mx represents one or more metals possibly
different, and
Ly represents one or more ligands complexing the
or metals.

 According to the invention, these organometallic compounds and, consequently, the free or bound parts comprising them, are advantageously detected and assayed by Fourier transform infrared spectroscopy when this compound has at least one free carbonyl ligand, but may also be detected by way of the metal with methods such as atomic absorption spectroscopy or electron microscopy visualization for heavy metals, such as osmium or ruthenium, for example in the context of peptide receptor assays or immunological assays of proteins .

 It was interesting to associate the analytical potentialities linked to an organometallic chemistry so as to create an unusual type of cold biosondes, provided, of course, that the structural modification is judiciously introduced so as to preserve the recognition capacities of the peptides vis- to their receivers, for example. The problem to be solved is essentially chemical in nature and makes use of the means for introducing the biosondes - cold - organometallic.

To do this, the subject of the present invention is thus a labeling molecule allowing the labeling of a peptide or a protein or more generally of a compound of peptide or protein nature,
characterized in that said marker molecule com
door
1) at least one unsaturated organic site, especially with.

a carbon unsaturation, such as a triple bond
C = C or an aromatic ring complexed by a compound
Organometallic salt of formula ML and 2) at least one carboxyl and / or amine function, the
carboxyl being possibly protected or activated
and the amine function being optionally protected.

 These amine and carboxyl protecting groups as well as the carboxyl function activating group are well known in peptide chemistry.

formule dans laquelle n est un nombre entier compris entre 1 et 6, de pré-
férence n = 2 M L représente le composé organométallique tel que défini
xy
ci-dessus
Z représente H ou un groupe de protection ou d'activa
tion de la fonction COOH.According to a first embodiment of the present invention, the labeling molecule consists of a molecule of formula (I3

wherein n is an integer from 1 to 6, preferably
n = 2 ML represents the organometallic compound as defined
xy
above
Z represents H or a protecting or activating group
COOH function.

We can mention in particular the molecules for which

R 'is chosen according to the peptide to be labeled,
especially for peptides soluble in water,
preferably will have R '= -SO3Na.

For peptides soluble in THF or
DMF, preferably R '= -H or a lower alkyl
C1 to C7 such as C3
R is a C1 to C7 lower alkyl, preferably
R = -CH3.

The interest of this molecule besides the fact
it is very stable and easily synthesizable, is
its high ability to react with a peptide by virtue of the N-hydroxysuccinimidyl type activation group that it comprises.

que l'on prépare à partir de d'une molécule de formule (I) avec Z = H en présence de de paradiméthylsulfonîophénolméthylsulfonate

We can also mention the case where

which is prepared from a molecule of formula (I) with Z = H in the presence of paradimethylsulfonophenolmethylsulfonate

 This molecule also has a high ability to peptide coupling in aqueous medium and is therefore very interesting for coupling peptides soluble in water.

formule dans laquelle x représente H ou un groupe de protection de la fonction
amine et
Y représente H ou un groupe de protection ou d'activa
tion de la fonction carboxyle et
R1 représente une chaine de dérivation d'un acide aminé
naturel ou non naturel comportant une triple liaison
CC complexée par un composé organométallique du type ML.According to another embodiment of the present invention, the labeling molecule consists of a natural or unnatural amino acid of formula II

formula where x is H or a function protection group
amine and
Y represents H or a protection or activa group
of the carboxyl function and
R1 represents a branch chain of an amino acid
natural or unnatural with triple bond
CC complexed with an organometallic compound of the ML type.

xy
This type of labeling molecule has the additional advantage of being able to be coupled to a peptide or a protein via either the amine function or the carboxyl function of the amino acid with either the carboxyl function or the amine function respectively of the peptide or the protein.

formule dans laquelle
X et Y ont les significations données précédemment.By way of example of a labeling molecule according to this type of embodiment of the invention, there may be mentioned the complexed acetylenic norleucine of formula III

formula in which
X and Y have the meanings given above.

In particular, acetyl groups may be mentioned
BOC for protection of the amine function and C1-C7 lower alkyl groups for protection of the carboxyl function. The carboxyl function can be activated in particular by the succinimidyl group or the nitrophenate group.

 In the organometallic compounds according to the present invention, it is possible to use metals of groups VI, VII, VIII and IX of the periodic table, in particular chromium, molybdenum, tungsten, manganese, cobalt, nickel, technetium, rhenium, ruthenium, and osmium.

 The ligands of these organometallic compounds can be very varied, in particular CO, CS, CSe, CNR1, phenyl, P (R2, R3, R4), cyclopentadienyl (Cp), R1 being in particular an alkyl radical or -COR5 and R2, R3 , R4 and R5 being in particular substituted or unsubstituted phenyl or phenoxy radicals, substituted or unsubstituted C 1 -C 7 alkyl or alkoxy or a halogen atom, R 5 may be -N (CH 2 CH 2 Cl) 2.

ML compounds may have several
xy metals and up to 12 ligands.

Among the organometallic compounds, there may be mentioned for example Co2 (CO) 6, M3 (CO) 10 with X = Ru or
Bone or Mo2Cp2 (CO) 4, W2Cp2 (CO) 4.

The subject of the present invention is also a process for the preparation of a labeling molecule, characterized in that a molecule comprising 1) an unsaturated organic site, in particular with an unsaturated
carbon ration, such as a triple bond or
aromatic nucleus, and 2) at least one carboxyl and / or amine function
protected or activated, which is complex
especially in TffF or a methanol / acetic acid mixture with
an organometallic compound of the MLy type
The unsaturated organic site, such as the triple bond, is therefore used to form small clusters which have the decisive advantage of providing the obtainted labeling molecule with a high degree of stability, which makes it easy to handle.

 Advantageously, the complexation by the organometallic compound M L in the presence of THF will be after protection of the amine and / or carboxyl functions, the molecule comprising at least one triple bond and at least one amine and / or carboxyl function.

 The subject of the present invention is also conjugates comprising the labeling molecule, linked to a peptide or a protein via a peptide linkage of the amide linkage type between an amine or carboxyl function, optionally protected or activated, of the molecule of labeling and, respectively, a carboxyl or amine function of the peptide or protein.

 The subject of the present invention is therefore also a process for the preparation of a conjugate according to the invention, characterized in that the labeling molecule and the peptide or protein are coupled via an amide bond between the function optionally protected or activated amine or carboxyl of the labeling molecule and, respectively, a carboxyl or amine function of the peptide or protein.

 When the labeling molecule comprises an amine function and a carboxyl function, the coupling with a peptide or a protein will advantageously be effected with the amine function of the protected labeling molecule and its activated carboxyl function, this coupling occurring for example in THF in presence of DCC (dicyclohexylcarbodiimide).

The conjugates according to the invention may also be prepared by coupling a molecule comprising 1) at least one unsaturated organic site, in particular with
a carbon unsaturation, such as a triple bond
CEC or an aromatic ring and 2) at least one carboxyl and / or amine function coupling with a protein or a peptide, the complexation of the organometallic compound of formula MXLy on the triple bond CC, occurring after the coupling.

 The present invention also relates to the use of the conjugates according to the invention, as tracers in the context of assays for in vitro diagnostics or as imaging agents in the context of in vivo diagnostics.

 The present invention will now be explained with the aid of the following detailed examples.

1) Syn~thelse
Un tel complexe a été synthétisé avec R' = H et
M1 = Co(CO)3, Mo(CO)2Cp, W(CO)2Cp, c'est-d-dire à partir de complexe M2L2(CO)4, selon le schéma

EXAMPLE I - COLD MARKER OF THE TYPE

1) Syn ~ thelse
Such a complex has been synthesized with R '= H and
M1 = Co (CO) 3, Mo (CO) 2Cp, W (CO) 2Cp, that is to say from complex M2L2 (CO) 4, according to the scheme

Such a complex has been synthesized with
R '= H and SO3Na.

a)tM13 = Co(COX3 , R' = H
A une solution de 0,5.10-3 mole de Co2(CO)8
(0,t88 g) dans 10 cm3 de THF distillé est ajoutée
goutte à goutte une solutionde 0,5.10-3 mole
d'hexyne-4 oate de N-succinimidyle (0,104 g) dans
le THF (6 cm3). Operating mode
Synthesis of

a) tM13 = Co (COX3, R '= H)
To a solution of 0.5 × 10 -3 moles of Co 2 (CO) 8
(0, t88 g) in 10 cm3 of distilled THF is added
dropwise a solution of 0.5 × 10 -3 mol
N-succinimidyl hexyne-4 oate (0.104 g) in
THF (6 cm3).

Stirring is allowed 4 hours. The solvent is
evaporated and the residue is taken up in 2 cm3 of CH2Cl2
then purified on a silica plate (eluent: CH 2 Cl 2).

 Red crystals are isolated: m = 0.132 g.

b) [M1] = Mo (CO) 2Cp R '= H
To a solution of 0.5; 10-3 moles of Cp2Mo2 (CO) 4
(0.217 g) in 10 cc of distilled THF is added
drip a solution of 0.5.10-3 mole
N-succinimidyl hexyne-4ate (0.104 g) in the
THF (6 cm3). After evaporation of the solvent, the residue
is taken up in CH2Cl2 (3 cm3) and purified on a plate
silica (eluent: CH2Cl2). We obtain crystals
red m = 0.150 g.

c) rM13 = W (Co) 2Cp R '= H
Same procedure with
0.5.10 3mole of Cp2W2 (CO) 4 (0.305)
0.5 × 10 -3 moles of N-succinimidyl hexyne-4-oate
(0.104 g)
After purification, a red solid is isolated
m = 0.14 g.

2) Peptide coupling
The coupling with an amino acid HPheOMe (Phe r phenylalanine) was carried out according to the scheme

 This product has been characterized by NMR.

m), 7,26

m).1 H NMR (CD 2 Cl 2 Brucker 250, 6 ppm): 2.0 (CH 2 m), 2.66 (CH 3 -C = C, s), 3.03 (CH 2, m), 3.15 (CH 2, m) , 3.71 (O-CH31 s), 4.83 (CH, m), 5.24 (2Cp, 2s), 5.88 (HN, d), 7.11

m), 7.26

m).

IR (KBr) CO = 1973, 1895, 1823 cm -1 mass.

(Quadrupole Spectrometer Riber-Maf R 10-10 in DCI-NH 3) 708 (M + 1).

 This complex thus makes it possible to couple and hence to label peptides soluble in THF or DMF.

 When R '= SO3Na1 the soluble peptides can be further labeled in calf.

Operating mode
A solution of 0.5 × 10 3 mol of phenylalanine methyl ester hydrochloride (HpheOCH3, HCl) (0.107 g) in DMF (5 cm3) is neutralized with the equivalent amount of triethylamine (0.5 × 10 3 mol). This solution is added to a solution of 0.5 × 10 -3 mol in THF (10 cm 3) of the previously prepared complex with a) CM 13 = Co (CO) 6 R '= H.

After stirring for 4 hours, the solvent
is evaporated, the residue is taken up in CH2Cl2 and purified
on silica plate (eluent CH2Cl2 + ethyl acetate
5%). Red crystals are isolated m = 0.197 g.

b) EM13 = CP Mo (CO) 2 R = H.

After stirring for 6 hours, the solvent is
evaporated, the residue is taken up in CH2Cl2 and purified on
silica plate (CH 2 Cl 2 eluent + 5% ethyl acetate).

 Red crystals are isolated m = 0.15 g.

c) CM13 = Cp W (CO) 2 R '= H
Same procedure as before.

After purification, red crystals are isolated
m - = 0.14 g.

n = - 2 R = CH3 ML =
xy Co2 <CO) 6
The 4-hexanoic acid (10 mole, 1.12 g) and the para-dimethyl sulfonophenol methyl sulfonate (10 mole, 2.66 g) are dissolved in CH 3 CN and the DCC (2.66 g 2 mole) is added to the solution. The mixture is stirred for 12 hours at a temperature below OOC; The dicyclohexylurea is filtered, the solvent evaporated.

An oil is obtained.

 The oil is taken up in 10 cm3 of distilled THF.

A solution of 10 moles of Co2 (CO) 8 (3.42 g) is added.

The mixture is stirred for 2 hours, then the solvent is evaporated. An oil is obtained: M = 2 g.

1) Complexation par le cobalt et le molybdène
a) Synthèse
Ce marqueur a été synthétisé avec
M1 = Co(CO)3 , Mo(CO)2Cp c'est-à-dire à partir de
composés organométalliques M2L2(CO)4

EXAMPLE III - MARKER OF THE COMPLEX AMINO ACID TYPE

1) Complexation by cobalt and molybdenum
a) Summary
This marker has been synthesized with
M1 = Co (CO) 3, Mo (CO) 2Cp, that is to say starting from
organometallic compounds M2L2 (CO) 4

with <SEP>#X<SEP> = <SEP> CH3-CO <SEP> for <SEP> M <SEP> = <SEP> Mo <SEP> and <SEP> L <SEP> = <SEP> Cp
<tb> and
<tb><SEP> Y <SEP> = <SEP> C2H5 <SEP><SEP> for <SEP> M <SEP> = <SEP> Co <SEP> and <SEP> L <SEP> = <SEP> CO
<tb> with returns of 66% and 75%

<tb> and <SEP>#X<SEP> = <SEP> BOC (1) <SEP><SEP> for <SEP> M <SEP> = <SEP> Co <SEP> and <SE> L <SEP> = <SEP> CO
<tb><SEP> Y <SEP> = <SEP> CH3
<tb> with yields of 44%.

 The first, variously protected reaction (Scheme 1) (e.g. N-acetyl and ethyl ester) leads without any structural alteration to the expected cluster.

Schéma 1 a) X = CH3-CO Y = C2H5 M= Mo L = Cp Rdt = 66 %
b) X - CH3CO Y = C2H5 M = Co L - CO Rdt = 75 Z
c) X -BOC Y = CH3 M = Co L = CO Rdt = 44 %
Mode opératoire 1") X = CH3-CO, Y = C2H51 M = Co, L = CO -3
A une solution de Co2(CO)8 (0,155 g ; 0,45.10 mole) dans le THF (10 cm3) est ajoutée goutte à goutte une solution de norleucine acétylénique protégée (X = CH3-CO, Y = C2H5) (0,089 g, 0,45.10 mole) dans le
THF (6 cm3). On laisse agiter 2 heures à température ambiante. Après évaporation du solvant, le résidu-est repris par CH2Cl2. On purifie sur plaque de silice (éluant : Acétate d'éthyle). On isole des cristaux rouges m = 0,169 g.(1) (terbutyloxycarbonyl)

Figure 1 a) X = CH3-CO Y = C2H5 M = Mo L = Cp Yield = 66%
b) X - CH3CO Y = C2H5 M = Co L - CO Rdt = 75 Z
c) X -BOC Y = CH3 M = Co L = CO Yield = 44%
Procedure 1 ") X = CH3-CO, Y = C2H51 M = Co, L = CO-3
To a solution of Co2 (CO) 8 (0.155 g, 0.45.10 mol) in THF (10 cm3) is added dropwise a solution of protected acetylenic norleucine (X = CH3-CO, Y = C2H5) (0.089 g). , 0.45.10 moles) in the
THF (6 cm3). Stirring is carried out for 2 hours at room temperature. After evaporation of the solvent, the residue is taken up in CH 2 Cl 2. Purified on a silica plate (eluent: ethyl acetate). Red crystals are isolated m = 0.169 g.

20) X = CH3-CO, Y = C2H5 t M = Mo, L = Cp
Same procedure as before.

 To a solution of Cp2Mo2 (CO) 4 (0.195 g, 0.4S.10 3 mol) in THF (10 cm3) is added dropwise a solution of protected acetylenic norleucine (0.089 g, 0.45 × 10 3 mol) in THF. (6 cm3). The mixture is stirred for 2 hours at room temperature. The solvent is evaporated, the residue is taken up in CH 2 Cl 2 and purified on a silica plate (eluent: ethyl acetate). Red crystals are isolated: m = 0.191 g.

3) X = Boc, Y = CH3, M = Co, L = CO
Same procedure as in 10).

 After purification, red crystals m = 0.1-04 g are obtained.

 The products obtained are very easily purified by thin layer chromatography and with suitable yields. All spectral characteristics (N.M.R., I.R. mass) agree with the expected structure.

 Analytical results of the compounds obtained.

2b Anal. Elem. C 39,75, H 3,10 N 2,89, Co 24,43
trouvé 39,69 H 3,25 b N 2,81 Co 24,66
RMN- 1H (CDC13 Varian T 60, TMS stand . 6 en ppm)

2c R.M.N.IH (CDC13, Brucker 250, TMS stand. 6ppm) 6 ::
1,43(CH3)3-C,s), 2,63(CH3-C5C,8), 3,48(CH,m)

2a Anal. Elem. : C 45.66, H 3.96 N 2.22 Mo 30.42
found: C 45.38, H 4.07 N 2.23 MB 29.56
NMR-HR (CDC13, Varian T60) -TMS stand. (# In ppm) 6 = 1.45

2b Anal. Elem. C 39.75, H 3.10 N 2.89, Co 24.43
found 39.69 H 3.25 b N 2.81 Co 24.66
1H-NMR (CDC13 Varian T 60, TMS stand 6 in ppm)

2c NMRH (CDC13, Brucker 250, TMS stand 6ppm) 6 ::
1.43 (CH3) 3-C, s), 2.63 (CH3-C5C, 8), 3.48 (CH, m)

mass (quadrupole spectrometer Riber Mag R 10-10 in DCI - NH3)
545 (MnH, +), 528 (M + 1) 527 (M) b) Peptide Coupling
Coupling was performed with HPheOCH3.

avec les groupes d'activation suivants

it can be done by two different ways which are represented on diagrams 2 and 3 below and which illustrate the two possibilities. either coupling after complexation, or coupling before complexation of the mar molecule
quage with the organometallic compound

with the following activation groups

We obtain by these two approaches yields that are appreciably similar but have not been optimized. The clusters are characterized by NMR,
IR and mass spectrometry following.

a) ComElexation
A une solution de Co2(CO)8 (0,218g
0,63.10-3 mole) dans le THF (10 cm3) est ajoutée
goutte à goutte une solution de norleucine acétylé
nique activée (0,17 g ; 0,63.10 3mole) dans le THF
(6 cm3). Le mélange est agité 2 heures.Operating mode

a) ComElexation
To a solution of Co2 (CO) 8 (0.218g
0.63 × 10 -3 mol) in THF (10 cm 3) is added
drip a solution of acetylated norleucine
activated (0.17 g, 0.63 × 10 3 mol) in THF
(6 cm3). The mixture is stirred for 2 hours.

b) Coupling ~ with ~ HpheOCH3 ~
Hydrochloride of methyl ester of -3
phenylalanine (HpheOCH3, HCl) (0.63.10 mol)
0.13 g) dissolved in 3 cm3 of DMF is neutral
by the equivalent amount of triethylamine (88 l). This solution is added to the previous one.

Stirring is allowed 3 hours. After filtration and evaporation of the solvent, the residue is taken up in 3 cm 3 of CH 2 CH 2 and purified on a silica plate (eluent ethyl ether 95. ethyl acetate: 5). Red crystals are isolated: m = 0.11 g.

Same procedure as previously with Cp2 Mo2 (CO) 4: (0.63 × 10 -3 mol, 0.273 g) -3
Norleucine: (0.63.10 mol, 0.17 g)
HpheOCH3 HCl: (p, 63.10-3 mol, 0.13 g).

After purification, red crystals are isolated: m = 0.14 g.

Same procedure as before with
Co2 (CO) 8: (0.63 × 10 -3 mol, m = 0.218 g)
Activated Norleucine: (0.63 × 10 3 moles, m = 0.180 g)
After purification, crystals are obtained
red: m = 0.15 g.

4) Complexation of a protected dipeptide
To a solution of (0.59.10 3 mol, 0.205 g)
of Co2 (CO) 8 in THF (10 cm3) is added dropwise
dropwise a dipeptide solution (0.59 × 10 -3 mol;
0.236 g) in THF (6 cm3). The mixture is agitated
4 hours. The solution is evaporated, the residue is
taken up with CH2Cl2 and purified on a silica plate
(eluent: CH2Cl2). Red crystals are isolated:
m = 0.11 g).

3a: Anal. Elem. C 46.77 H 3.59 N 4.54
found 47.60 3.76 4.72
H-NMR (CDCl3, Bruker 250, TMS booth. #Ppm): "
1.94 (CH3-CO, s), 2.59 (CH3-CC-, s), 3.25 (2CH2, m),
4.54 (CH, m), 4.84 (CH, m), 6.35 (NH, d), 6.89 (NH, d), 7.25 (C6H5)

mass (quadrupole spectrometer Riber mag R-10-10 in DCI-NH3)
617 (M + 1) 634 (M + NH4b) 3H NMR (CDCl3, Bruker 250, TMS booth 6ppm): 6
1.97 (CH3-CO, s), 2.67 (CH3-CC, s), 3.0 (2CH2, m), 3.71
(CO2-CH3, s), 3.97 (CH, m), 4.80 (CH, m), 5.18-5.24 (2Cp),

masse (spectrometre quadrupôleRiber mag R-10-10 en DCI - NH3) 691(M-1+NH4+), 675(M+1), 674(M) C) Couplage avec un dipeptide
Enfin, le couplage peptidique d'un aminoacide complexé avec un dipeptide conduisant à un tripeptide complexé a été réalisé selon le schéma

mass (quadrupole spectrometer Riber-Mag R-10-10 in DCI-NH3)
765 (M + 1) 4H NMR (CDCl3, Brucker 250, TMS stand., #Ppm): "
1.40 ((CH 3) 3 C, s), 2.53 (CH 3 -C ##), 3.45 (2CH 2, m),
3.76 (-CO2-CH3, s), 4.40 (CH, m), 4.64 (CH, m), 5.01 (NH, d),

mass (quadrupole spectrometer Riber mag R-10-10 in DCI-NH3) 691 (M-1 + NH4 +), 675 (M + 1), 674 (M) C) Coupling with a dipeptide
Finally, the peptide coupling of an amino acid complexed with a dipeptide leading to a complexed tripeptide was carried out according to the scheme

Operating mode
a) Complexation of mono acetylenic norleucine
protected.

To a solution of C-t67.10 4 moles
Cp2 Mo2 (CO) 4 (0.029 g) when the THF (10 cm3) is
added dropwise a solution of 0.67.10 mol
of acetyl-norleucine acetylenic (0.011 g) in the
THF (6 cm3). The mixture is stirred for 2 hours.

b) To this solution is added 0.67 × 10 -4 mole of
DCC (0.0139g). Let it shake for 1 hour, then
add a solution of the dipeptide Asp (OBz) CONH2
(0.67 × 10 -4 mol, 0.024 g) in THF (2 cm). The
The mixture is stirred for 12 hours at room temperature.

After evaporation, the residue is taken up in 2 cm3 of
CH2Cl2 and purified on a silica plate (eluent
ethyl acetate). Red crystals are obtained
m = 0.016 g).

1H NMR (acetone d6, Brucker 250, 6 ppm): 6
1.98 (CH3-CO, s), 2.85 (CH3-C # C, s), 3.15 (CH2, m),
4.16 (CH, m), 5.34 (2Cp, 2L), 7.43 (NH, d), 11 (COOH,
s).

 IR (KBr) θ C O = 1972, 1909, 1833.

mass (quadrupole spectrometer Riber-Mag R-10-10 in
DCI-NH3): 604 (M + 1).

s), 5,35 (2 Cp, 2 s), 6,57 (NH2, s), 6,86 (NH2, s), 7,25

m), 7,55 (NH, d), 7,7 (NH, d), 8,0 (NH, d). 6 H-NMR (d6 acetone, Brucker 250, 6 ppm): #: 2.05 (CH3-CO, s), 2.87 (CH3-CEC, s), 3.0 (CH2, m) 4 (CH3-CO, s), , m), 4.64 (CH, m), 4.66 (CH, m), 5.098 (CH 2)

s), 5.35 (2 Cp, 2 s), 6.57 (NH 2, s), 6.86 (NH 2, s), 7.25;

m), 7.55 (NH, d), 7.7 (NH, d), 8.0 (NH, d).

 IR (KBr) ν C = O = 1975, 1903, 1829.

mass (quadrupole spectrometer Riber-Mag R-10-10 in
DCI-NH3): 955 (M + 1).

 These results are directly extrapolable to the labeling of peptides of biological interest; tuftsin Tyrs-Lys-Pro-Arg (macrophage inhibitor) then enkephalins: Tyr-Gly-Gly-Phe-Leu, finally substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe -Sar-Leu Met-NH2.

2) Complexation with ruthenium and osmium
The labeling of protected acetylenic norleucine was carried out by grafting a cluster of osmium and ruthenium on the acetylenic function. The synthesis can be schematized as follows:

 The reagents Ru3 (CO) 11 (CH3CN) and Os3 (CO) 11 (CH3CN) were prepared according to the methods described in the literature (Ru3 (CO) 11 (CH3CN): G.A. Foulds, B.F.G.

Johnson and J. Lewis, J. Organometal. Chem., 1985, 296, 147.

Os3 (CO) 11 (CH3CN): BFG Johnson, J. Lewis and DA Pippard,
J. Chem. Soc., Dalton, 1981, 407).

2.1) Complexation with Ru3 (CO) 11 (CH3CN)
Ru3 (CO) 11 (CH3CN) is prepared from 300 mg of Ru3 (CO) 12 (4.7.10 mol), in 220 ml of dichloromethane, 15 ml of acetonitrile and 0.9 ml of a trimethylamine solution. oxide in methanol at 50 g / l (4.8 × 10 -4 mol). The solvent is then removed by evaporation under reduced pressure and replaced with 10 ml of
THF.

100 mg (5.46 × 10 -4 mole) of protected acetylenic norleucin 1 is added to this solution. After stirring for two hours, the product is purified by plate chromatography (eluent: CH 2 Cl 2 / cyclohexane / CH 3 CN: 35/60/10). After extraction of the product with THF and evaporation, 250 mg of complex 7a (M = Ru) (yield: 60%) are collected. Recrystallization from dichloromethane / pentane gives orange crystals,
F 1000C.

IR (KBr) vCO in cm -1 = 2097 m, 2048 F, 2025 F, 1998 F, 1879 f.

1 H NMR (CDCl 3, 250 MHz, TMS boiling, 5 ppm): 2.04 (CH 3 CO 3 s), 2.31 (CH 3 C = C, s), 2.50-3.00 (CH 2, broad mass), 3.78 (CH3COO, s), 4.88 (CH, m), 6.02 (NH, bulk).

Mass (electronic impact): 766 (M +), 738 (M + CO), 710 (M + 2 CO),
682 (M +/- 3CO), 654 (M +/- 4CO), 626 (M +/- 5CO), 598 (M +/- 6CO).

2.2) Complexation with Os3 (CO) 11 (CH3CN):
To 200 mg of Os3 (CO) 12 (2.2.10 4 mol) dissolved in 100 ml of dichloromethane and 10 ml of acetonitrile are added 0.8 ml of a solution of trimethylamine oxide in methanol at 40 g / l ( 4.3 × 10 -4 moles). The progress of the reaction is monitored by TLC (eluent:
THF / pentane: 1/3). When all the starting material has disappeared, the solvent is evaporated under reduced pressure.

 The yellow solid of Os3 (CO) 11 (CH3CN) obtained is redissolved in 7 ml of THF. 100 mg (5.46 × 10 -4 mole) of the protected acetylenic norleucine 1 is added to. this solution. After two hours of stirring, there is still starting material but the reaction no longer evolves. The compound formed is separated by chromatography on silica gel plates (eluent: THF / pentane: 1/3). After extraction of the product with dichloromethane and evaporation, 50 mg of complex 7b (M = Os) are collected (yield: 25%).

Recrystallization from dichloromethane / pentane gives orange crystals, F 1140C. Spectrum
1H NMR reveals the presence of two complexes of norleucine
1 H NMR (CDCl 3, 250 MHz, TMS booth, 6 ppm):
-Majority complex (70%):
2.02 (CH3CO, s), 2.46 (CH3C6C, s), 2.59 and 2.82- (CH2, m, m), 3.77
(CH3COO, s), 4.75 (CH, m), 6.05 (NH, bulk).

-Complex minority (30%)
2.01 (CH3CO, s), 2.54 (CH3C = C, s), 2.59 and 2.82 (CH2, m, m), 3.77
(CH3COO, s), 4.75 (CH, m), 5.90 (NH, solid).

 These two complexes are currently not separable by conventional plate chromatography.

IR (KBr) νC0 cm1-2099 f, 2060 F, 2054 F, 2021 m, 2005 m, 1845 tf.

Mass (electronic impact) 1033 (M +), 1005 (M + CO), 977 (M + 2CO), 949 (M + 3CO), 921 (M + 4CO), 893 (M + 5CO), 865 (M + 6CO) ), 837 (M +/- 7CO), 809 (M +/- 8CO).

Claims (14)

 1. Marking molecule for labeling a peptide or a protein, or more generally a compound of a peptide or protein nature, characterized in that said labeling molecule comprises 1) at least one unsaturated organic site, in particular with  a carbon unsaturation, such as a triple bond  C = C or an aromatic ring complexed by a compound  organometallic compound of formula M L in which Mx  represents one or more metals possibly different  and  L represents one or more ligands  there  different, complexing the metal (s), and (2) at least one carboxyl and / or amine function, the  carboxyl being possibly protected or activated  and the amine function being optionally protected.  2. Molecule of marking according to claim 1, characterized in that it corresponds to the general formula (I)

 formula in which n is an integer between 1 and 6, of meadow  n = 2 M L represents the organometallic compound as defined  xy  previously  Z represents H or a protecting or activating group  of the carboxyl function.  3. Marking molecule according to claim 1 or 2, characterized in that it corresponds to the formula

 formula in which n is an integer between 1 and 6, of  preferably n = 2 M L represents the organometallic compound as  xy  previously defined R 'is chosen especially from -H, -SO3Na, the alkyls  lower in C1 to C7, and R is selected from C1 to C7 lower alkyls.  4. -Molécule marking according to claim 1 or 2, characterized in that it responds to the General Eormule

 5. Molecule according to claim 1, characterized in that it corresponds to the general formula II

 formula in which X represents H or a protection group of the  amine function Y represents H or a protection or activa group  the carboxyl function R1 represents a derivatization chain of an acid  natural or non-natural amine and  triple bond C = C complexed by an organometallic  type M L  6. Marking molecule according to claim 5, characterized in that it corresponds to the general formula III

 wherein X, Y and M L have the meanings given above.  xy  7. Marking molecule according to one of Claims 5 or 6, characterized in that the protection group of the amine function is chosen from the acetyl group and the BOC group, the protection group of the carboxyl function is a lower alkyl by C1 to C7, the carboxyl function activating group being selected from succinimidyl group and nitrophenate group.  8. Marking molecule according to one of the preceding claims, characterized in that the metals of the organometallic compound are selected from the metals of groups VI, VII, VIII and IX of the periodic table, including chromium, molybdenum, tungsten , manganese, cobalt, nickel, technetium, rhenium, ruthenium and osmium.  9. Marking molecule according to one of the preceding claims, characterized in that the ligands L of the organometallic compound are chosen from CO, CS, CSe, CNR1, P (R2R3R4) cyclopentadienyl (Cp), thinyl R1 being in particular an alkyl radical or -COR5. and R 2, R 31, R 4 and R 5 being in particular phenyl or substituted or unsubstituted phenoxy radicals, substituted or unsubstituted C 1 to C 7 alkyl or alkoxy radicals or a halogen atom, R 5 may be -N (CH 2 CH 2 Cl) 2.  10. Process for the preparation of a labeling molecule according to one of the preceding claims, characterized in that a molecule comprising at least one unsaturated organic site is reacted, in particular with a  carbon unsaturation, such as a triple bond or  aromatic nucleus, and 2) at least one carboxyl and / or amine functional group,  protected or activated species which is complex on the unsaturated site, in particular in the THF or a methanol / acetic acid mixture, with an organometallic compound of the MxLy type  11.Conjugate comprising the labeling molecule according to one of the preceding claims, linked to a peptide or a protein via an amide linkage type peptide bond between the amine or carboxyl function optionally protected or activated the marking molecule. and respectively a carboxyl or amine function of the peptide or protein.  12. Process for the preparation of a conjugate according to claim 9, characterized in that the optionally protected or activated amine or carboxyl function of the labeling molecule is coupled with a carboxyl or amine function respectively of the peptide or of the protein.  13. Process for the preparation of a conjugate according to claim 9, characterized in that a molecule comprising at least one unsaturated organic site, such as a C liaisonC triple bond and at least one carboxyl function and / or amine via said carboxyl and / or amine function to a peptide or a protein via respectively one of its amine or carboxyl functions, the unsaturated organic site being complexed by an organometallic compound of formula ML after said coupling .  14. Use of the conjugates according to claims 11 and 12, as a tracer for assays for in vitro diagnostics, and as imaging agents for in vitro diagnostics, and as agents for imaging for in vivo diagnostics.