Field of the invention

The present invention relates to certain fluorophor compounds and a method of detecting a phosphorylated peptide or phosphorySated polypeptide using the same Furthermore a method of detecting and differentiating phosphorylated and unphosphorylated peptides or polypeptides using the fluorophor compounds ^s described A kit and a diagnostic composition conπpπs'πg the fluorophor compounds as well as a method of diagnosing a disease are also disclosed

Background of the invention

Detection and quantitation of protetns are standard techniques in molecular biology They may be performed in solution or on supports supports including gels, membranes and filters As regards protein detection on supports in particular on gels, this process ss often referred to as "staining"

While silver- and Coomassie-staining are widely used for total protein staining a number of stains selective for certain functional groups have emerged Glycosylation (1 2), His-tags (3) and pnosphorylation (^ 5) are typical p ^r otetn modifications ta ^r geted by selective gel stasns reported so far

With respect to its biological importance phosphorylation (6, 7) is widely regarded as the most significant post-transiatioπal modification Phosphorylation plays an important role in signaling pathways and it is estimated that 30 % of the ennre proteome becomes phosphorylated at some point (8) Wniie ihere are phosphσ-specrπc antibodfes available (9, 10) they usually are specie for epitopes comprising the proximity of the phosphorylation site As a co ^π seque ⁿ ce not even.' phosohoπ/lafed site in a p ^r oteιn is amerable ÷c detection with a given antibody Alternatively, ³ⁱ P-!abe!ing of the proteins provides a very sensitive ^f oo! for detection of phosphorylation (11 12} However, the handling and disposal of radioactive material are costly, potentially hazardous and increasingly regulated. Therefore, when staining for phosphorylation, fluorescence detection is the method of choice due to its inherent sensitivity. The reported (13) and commercially available (4, 5) fluorescent phospho-specific stains achieve their sensitivity from their binding site specificity. While other fluorescent probes for phosphorylated amino acids have been reported (14). their selectivity has only been demonstrated for peptides without other metal-cheiating amino acids like histidine, tryptophan or cysteine.

Grauer (15) describes organic moϊecules designated as "synthetic receptors" which differentiate between different phosphorylated peptides. The synthetic receptors comprise a bis-Zn"~cyclen complex. Also described are synthetic receptors which are conjugated to further moiety designed to bind to an amino acid in position (i+3) relative to the phosphorylated position. A fSuorophore is not described in Grauer (15).

Mizukami (16) describes fluorescent anion sensors. The described sensor molecuies comprise a mono-metal-cylen complex conjugated to a fluorophore. Cd" is considered the most suitable metal ion, whereas the Zn" complex is described as being unable to act as an ion sensor. The detected anions include pyrophosphate and nucleotides such as AMP and cAWΪP.

in view of the prior art, the technical problem can be considered as the provision of alternative or improved means and methods for detecting phosphorylated peptides and polypeptides.

Summary of the invention

The main aspects of the invention are summarized in the following items;

1. A compound comprising an anion and a cation having the general formula (1).

(!)

wherein:

A is selected from the group consisting of S, O, NH and PH;

E is selected from the group consisting of CH ₂ and CH ₂ CH ₂ ;

M iε selected from the group consisting of Zn, Mn, Fe, Sc, Ti, Zr ₁ Eu, Gd, and Tb;

D is a linking group;

L is a linking group;

G is a substituted or unsubstituted fluorophor group of the general formula (H), (ill) or (IV):

(II)

wherein

Z ¹ is selected from -CH ₂ - and -C(O)-; Z ² is selected from -CH-- and -O- ; and ''"''^ is a single or double bond;

(!») (IV) wherein

Z ³ is -CH ₂ -, -C(O)- or -C(S)-;

R' and R" are independently selected for each occurrence and are selected from the group consisting of H and C ₁ ^ aikyi; and

X is an anion;

n is an integer from 1 to 6; m is 0 or 1, with the proviso that m = 1 if the fluorophor group is substituted or unsubstituted coumarin; and

" " indicates a chemical bond, while " " indicates that the metal M is coordinated by the moiety A.

A method of detecting a phosphorylated peptide or phosphoryiated polypeptide, the method comprising:

(a) bringing into contact said phosphorylated peptide or phosphorylated polypeptide with a compound as defined in item 1 : and

(b) detecting a complex comprising said phosphoryiaied peptide or phosphorylated polypeptide and said compound, thereby detecting said phosphorylated peptide or phosphoryiated polypeptide.

3. A method of detecting a phosphorylated peptide or phosphorylated polypeptide, the method comprising: (a) bringing into contact said phosphorylated peptide or phosphorylated polypeptide with a compound comprising an anion and a caion having the genera i formula

(I):

(I)

wherein:

A is selected from the group consisting of S, O, NH and PH;

E is selected from the group consisting of CH ₂ and CH ₂ CH ₂ ;

M is selected from the group consisting of Zn, Mn, Fe, Sc, Ti. Zr ₅ Eu, Gd, and Tb;

D is a linking group;

L is a linking group;

G is a substituted or unsubstituted fluorophor group of the general formula (H), (Hi) or (IV):

(!!)

wherein

Z ¹ is selected from -CH ₂ - and -C(O)-; Z ² is selected from -CH ₂ - and -O-; and ^^^ is a single or double bond;

(HI) (IV) wherein

Z ³ is "CH ₂ -, -C(O)- or -C(S)-;

R' and R" are independently selected for each occurrence and are selected from the group consisting of H and C _M alky!; and

X is an anion;

π is an integer from 1 to 6; m is 0 or 1 ; and

" " indicates a chemical bond, while " " indicates that the metal M is coordinated by the moiety A; and

(b) detecting a complex comprising said phosphory Sated peptide or phosphoryiated polypeptide and said compound, thereby detecting said phosphorySated peptide or phosphorySated polypeptide; wherein the method is effected on or in a support.

A method of detecting and differentiating phosphoryiated and unphαsphoryiated peptides or polypeptides, the method comprising.

(a) bringing into contact said peptides or polypeptides with a compound as defined in item 1 , and

(b) determining

(i) whether fluorescence emission being characteristic of a complex of said compound with phosphorySated peptides or phosphoryiated polypeptides occurs and (ii) whether fluorescence emission being characteristic of a complex of said compound with unphoεphorylated peptides or unphosphoryiated polypeptides occurs, thereby detecting and differentiating phosphorylated and unphosphoryiated peptides or polypeptides.

5. A method of detecting and differentiating phosphorylated and uπphosphorylaled peptides or polypeptides, the method comprising:

(a) bringing into contact said peptides or polypeptides with a compound as defined in claim 3; and

(b) determining

(i) whether fluorescence emission being characteristic of a complex of said compound with phosphorylated peptides or phosphoryiated polypeptides occurs; and

(ii) whether fluorescence emission being characteristic of a complex of said compound with unphosphoryiated peptides or unphosphoryiated polypeptides occurs, thereby detecting and differentiating phosphorylated and unphosphoryiated peptides or polypeptides; wherein the method is effected on or in a support.

6. Use of a compound as defined in item 1 for detecting a phosphoryiated peptide or phosphorylated polypeptide.

7. Use of a compound as defined in item 3 for detecting a phcsphorylated peptide or phosphorylated polypeptide, wherein the detection is effected on or in a support.

8. Use of a compound as defined in item 1 for detecting and differentiating phosphoryiated and unphosphoryiated forms of a peptide or polypeptide.

9. Use of a compound as defined in item 3 for detecting and differentiating phosphorylated and unphosphoryiated forms of a peptide or polypeptide, wherein the detection is effected on or in a support.

10. A kit comprising one or more compounds as denned in item 1.

1 1. A diagnostic composition comprising one or more compounds ss defined in item 1 12. A method of diagnosing a disease characterized by the presence of an aberrant amount of a phosphorylated peptide or phosphorylated polypeptide, the method comprising:

(a) bringing into contact a sample obtained from a subject suspected to suffer from said disease with a compound as defined in item 1 or 3: and

(b) determining whether the fluorescence emission of said sampte differs from the fluorescence emission of a control sampte.

Preferably the method of diagnosing a disease is effected on or in a support.

Brief description of the figures

Figure 1 : A scheme illustrating the discrimination between phosphoryiated and non- phosphorylated proteins on a solid support.

Figure 2: Gel stained with probe 1. Each lane contains 1 μg BSA (66 kDa). From left to right: lane 1 : 1 μg α-casein (23 kDa) dephosphoryiated, lanes 2 - 8: 1 μg,

500 ng, 250 ng, 125 ng, 62 ng, 31 ng, 15 ng α-casein. The top image was taken on a UV table (λ _ex = 316 nm), the lower image shows CBB R-250 total protein restain.

Figure 3: Ge! stained with probe 2, Each lane contains 1 μg BSA (66 kDa). From left to right: lane 1 : 1 μg α-casein (23 kDa) dephosphoryiated, lanes 2 - 8: 1 μg,

500 ng, 250 ng, 125 ng, 62 ng, 31 ng, 15 ng α-casein. The top image was taken on a UV table (λ _ex = 316 nm), lower image shows CBB R-250 total protein restain.

Figure 4: Normalized emission spectra of gel bands stained with probe 2 acquired through a 455 nm Iσngpass filter (λ _ex = 316 nm). The BSA band showed the same spectrum as dephosphorySated α-caεeϊn (data not shown).

Detailed description of the invention

i he flucrophor compounds of the present invention comprise an anion and a cation having the general formula (I):

(I)

wherein

A is selected from the group consisting of S. O ₁ NH and PH;

E is selected from the group consisting of CH ₂ and CH ₂ CH ₂ ;

M is selected from the group consisting of Zn ₁ Mn, Fe, Sc, Ti, Zr, Eu, Gd, and Tb;

D is a linking group;

L is a linking group;

G is a substituted or unsubstituted fiuorophor group of the general formula (II), (III) or (IV):

(Ir)

wherein

Z' is selected from -CH ₇ - and -C(O)-; Z-" is selected from -CH ₂ - and -O-; and ■ ^" — is a single or double bond;

(!!!) (IV)

wherein

Z ³ is -CH ₂ -, -C(O)- or -C(S)-;

R' and R" are independently selected for each occurrence and are selected from the group consisting of H and Ci_ ₄ alkyl; and X is an anion; and

n is an integer from 1 to 6.

In some embodiments (among others, if e.g., the methods are effected in solution), m is O or 1 , with the proviso that m = 1 if the fluorophor group is substituted or unsubstituted coumarin. The coumarin group has the formula:

In those embodiments in which the methods are effected on or in a support, m is O or 1.

in the above formula (I) "-— " indicates a chemical bond, while " " indicates that the metal M is coordinated by the moiety A.

The anion is not specifically limited. A skilled person can chose one or more anions, so that the compound of the present invention has a neutral charge based on the charge of the metal M, Examples of suitable anions are neutral anions such as chloride, perchiorεte _; and nitrate. Preferred anions are chloride and perchlorate The moiety A serves to complex or coordinate the metal atom M in the crown ether or its N-, S- or P-anaiogue. It is selected from the group consisting of S, O, NH and PH ₁ preferably from the group consisting of O and NH: Most preferably A is NH because of its Lewis basicity and its ability to complex the "hard" metal cation.

E is selected from the group consisting Of CH ₂ and CH ₂ CH ₂ and is preferably CH ₂ CH ₂ .

M is selected from the group consisting of Zn, Mn, Fe ₅ Sc, Ti, Zr, Eu, Gd, and Tb. in a preferred embodiment M is selected from the group consisting of Zn and Mn, more preferably M is Zn. The selected metals, especially those of the preferred embodiments, not only have a high bindung affinity but also very Sow non-specific interactions.

D is a linking group. The nature of the linking group is not particularly limited and it can be chosen from any linking group which does not iπterfer with the detection of phosphorylation. Examples of the linking group include

— C —

H

, a 5- to 7-membered carbocycϊic group and a 5- to 7-membered heterocyclic group containing one or more heteroatorns selected from N, P, O and S. The carbocyciic group and heterocyclic group can be saturated or include one or more double bonds. Examples of carbocyciic groups include cyclopentyl, cyclohexy], cycioheptyl and phenyl. Examples of the heterocyclic group include

Preferably D is selected from

Most preferably D is because its inflexible aromatic system increases the bindinα affinity

n is an integer from 1 to 6, preferably π is 2.

m is as defined above. In a preferred embodiment m is 1 because the binding affinity to phosphate is higher. Therefore, the influence of the metal complex on the fluorophor is increased which in turn results in increased fϊuoresceπce when the phosphate group is bound. L is a linking group. The nature of the linking group is not particularly limited and it can be chosen from any linking group which does not iπterfer with the detection of phosphorylation. Examples of the linking group include -X _p -(CH ₂ )q-Y _r - in which

X is selected from -O-, -S- and -NH-, preferably X is -NH-.

p = 0 or 1 , preferably p = 1.

q = 1 to 6, preferably q = 2 to 4. The short chain length promotes the influence of the phosphate coordination on the fjuoreεeπce.

Y is selected from -O-, -S-, -NH-, -C(O)- -SO ₂ NH-, -NHSO ₂ -, -C(O)NH-, -NHC(O)-, -C(O)O- and -OC(O)-. Preferably Y is selected from -SO ₂ NH-, -NHSO ₂ -, -C(O)NH- and -NHC(O)- more preferably Y is selected from -C(O)NH-, and -NHC(O)-. The linking group is particularly stable and easy to synthesize in the preferred embodiments.

r is O or 1 , preferably r is 1.

Mizukami (16) described a fluorescent anion sensor which functioned in neutral aqueous solutions. In such solutions a heteroatom which could coordinate to the metal had to be present in the linker between the cycien and the fluorescent group otherwise the complexes were found to be unsuitable as an anion sensor. The present inventors have found that such a hetercatom is not necessary in the methods of the present invention. Consequently the selection of the linker is less restricted and metal chelates which have a high binding affinity with respect to the phosphate bond can be selected.

The alkylene chain in the group can optionally be interrupted by 1 or 2 -0-, -S- or -NH- groups with the proviso that the -O-, -S- and -NH- groups, if present, are not directly adjacent to each other or the other heteroatom-containing groups X and Y, if present.

Preferred groups -X _F -(CH ₂ ) _C -Y- are -NH-(CH ₂ ) _q -NHC(0)- and -O-(CH ₂ ) _q -O- where q is as defined above. These groups result ensure that the resultant compounds have a preferable solubility and stability.

A more preferred hnker group is -NH-(CH ₂ ) ₀ -N HC(O)- with q = 2 to 4. G is a substituted or unsubstituted fluorophor group. Fiuorophor groups within the meaning of the present invention are fluorophor groups of the general formula (N) ₁ (111) or (IV).

In one embodiment the fluorophor compound has the general formula (II):

01)

wherein

Z ¹ is selected from -CH ₂ - and -C(O)-, and is preferably -C(O)-;

Z ² is selected from -CH ₂ - and -O-, and preferably is -O-; and

^^^ is a single or double bond, preferably a double bond.

Of the groups having the formula (11) substituted and unsubstituted coumarin groups are preferred.

The groups having the formula (II) can be substituted in any available position by one or more substituents. The substituεnts are not particularly limited as long as they do not interfer with the detection of phosphorylation. Possible substituents include C _{1 4} alkyl groups, OH, CF ₃ , NH ₂ , NH(C ₁ -, alkyl), N(C ₁ -, alkyf) ₂ , NO ₂ . Hal. SO ₃ H and ohenyi. Preferred εubstifuents are N(C^ aikyl} ₂ , OH, NO ₂ and SO ₃ H, more preferred substituents are N(C _1- ^ aikyi) ₂ .

Preferred examples of groups hsving the formula (H) are

which are substituted by N(CI _-4 alkyl) ₂ , especially

In a further embodiment the fluorophor compound has the general formula (ill) or (IV):

(IV)

wherein

Z ^z is -CH ₂ -, -C(O)- or -C(S)-, preferably -C(O)-.

R' and R" are independently selected for each occurrence and are selected from the group consisting of H and C _1-4 alkyl.

X can be any suitable anion and is preferably a halogen anion such as chloride.

The groups having the formula (111) or (!V) can be substituted in any available position by one or more substifuentε. The substituents are not particularly limited as long as they do not interfer with the detection of phosphorylation. Possible substituents include C _1-4 alkyi groups, OH, CF ₃ , NH ₂ , NH(C ₁ ^ alkyl), N(C _1-4 alky!),. NO ₂ , Hal, SO ₃ H and phenyl. Preferred εubstiiuentε are Hal, C ₁ -* alkyl and SO ₃ H. Most preferably the groups having the formula (N!) are unsubstituted. The groups having the formula (!V) are preferably unsubsiituted or are substituted by Cu alkyi.

Preferred cations which are suitable in the present invention include

The compounds of the formula (I) can be synthesized as follows,

in reaction (i) compound 1 is reacted with compound 2. i he reaction is uεuaiSy conducted in an aprotic solvent. Examples of aprotic solvents induce THF and dicxane. Preferably dioxaπe is chosen. The reaction is usually conducted at a temperature in the range of 60 to 120 ⁰ C ₁ preferably from 800 to 100 ⁰ C. A base is typically employed in this reaction step. Examples of typical bases include DIEA, KOH, NaOH and K ₂ CO ₃ . preferably K ₂ CO ₃ is used. The reaction product is usually isolated before it is employed in reaction (ii) The isolation procedure is not particularly limited. Typically the reaction product will be separated from the solvent by removing the solvent under reduced pressure and subsequent column chromatography.

In reaction (if) compound 3 or compound 1 is reacted with compound 4. The reaction is usually conductedj in an aprotic solvent Examples of aprotic solvents include THF and dioxane. Preferably dioxane is chosen The reaction is usually conducted at a temperature in the range of 100 to 140 ⁰ C, preferably from 100 to 120 ^C C. A base is typically employed in this reaction step Examples of typical bases include DIEA, KOH, NaOH and K ₂ CO ₃ , preferably K ₂ CO ₃ is used The reaction product is usually isolated before it is employed in reaction (iii). The isolation procedure is not particularly hinted Typically the reaction product will be separated from the solvent by removing the solvent under reduced pressure and subsequent column chromatography.

in reaction (iii) the protecting group PG ² of the linker is removed if D contains a primary amine, otherwise no protecting group is required. Protecting groups can be Z, Boc, and Fmoc Preferably, the protecting group is Boc. The Boc group can be removed by addition of acid Acids can be HCI, TFA and H ₂ SO ₄ . Preferably, the acid is TFA The reaction is usually conducted in an aprotic solvent Examples of aprotic solvents include DCM, Et ₂ O and dioxane Preferably DCM is chosen. The reaction is usually conducted at room temperature The reaction product is usually isolated before it is employed in reaction (ιv) The isolation procedure is not particularly limited. Typically the reaction product will be separated from the solvent and the acid oy removing the solvent and the acid under reduced pressure

In reaction (ιv) compound 4 or compound 7 is reacted with compound 8 Preferably, the coupling is conducted between a primary amine and a carboxyiate, in L and G, respectively The reaction can be performed using HOBt or HOAt, DiEA ana HATu, PyBop, 3op, JBTU or hBTU. Typically, HOBt, TBTU and DiEA are employed The reaction is usually conducted in an aprotϊc solvent Examples of aprotic solvents include THF and dioxane Preferably dioxane is chosen The reaction is usually conducted at a temperature in the range of -10 TO 25 ^C C preferably from 0 TO 10 ⁰ C The reaction product is usually isolated before it is employed m reaction (v) The isolation proceαure is not particularly limited Typically the reaction product wiH be separated from the solvent by removing the solvent unαer reduced pressure and subεeαuent column chromatography In reaction (v) the protecting groups PG are removed if A is a secondary amine, otherwise no protecting group is required Protecting groups can be Z, Boc and Fmoc Preferably the protecting group is Boc The Boc group can be removed by addition of acid Acids can be HCl, TFA and H ₂ SO ₄ Preferably, the acid is TFA The reaction is usually conducted in an aprotic solvent Examples of aprotsc solvents include DCM, Et ₂ O and dioxane Preferably

DCM is chosen The reaction is usually conducted at room temperature The reaction product is usuaily isolated before it is employed in reaction (iv) The isolation procedure is not particularly limited Typically the reaction product wtll be separated from the solvent and the acid by removing the solvent and the acid under reduced pressure

in reaction (vι) the free ligand is compiexed by the metal cation M M can be introduced in the form of soluble salts such as nitrates or perchiorates Preferably, M is introduced as the perchlorate The reaction is usually conducted in a prone solvent Examples of protsc solvents include water and C<_ ₄ aikanols such as EtOH and MeOH Preferably water is chosen The reaction is usually conducted at a temperature in the range of 60 to 100 ⁰ C, preferably from 80 to 90 ^D C The reaction product is usually isolated by removing the solvent under reαuced pressure

The present invention furthermore relates to a method of detecting a phosphoryiated peptide or phosphoryiated polypeptide the method comprises (a) bπnging into contact said phosphoryiated peptide or phosphoryiated polypeptide with a compound according to the snvention, and (b) detecting a complex comprising said phosphoryiated peptide or phosphoryiated polypeptide and said compound thereby detecting said phosphor/lated peptide or phosphoryiated polypeptide

The term "oeptide" as used herein describes a group of molecules which consist of 30 or ieεs than 30 ammo acids The term "polypeptide" as used herem describes a group o ^f molecules which consist of more than 30 amino acids in accordance with tne invention the group of polypeptides comprises "proteins" as long as the proteins coπs'st of a single pol ^y peptide Also in line with tne Definition tne term ' polypeptide" describes fragments of proteins as long as these fragments consist of more than 30 amino acids Polypeptides may furtner form multimers such as dime ^r s, trimerε and higher oligomers, i e consisting of more than one polypeptide molecule Polypeptide molecules forming such dimers trimers etc may be identical or nor-identica! The corresponding higher order structures of such mjltimsrε are, consequently, iermed homo- or heterodimerε, homo or heterotπmers etc By detecting a peptide or polypeptide which ss comprised in such a multimer, the method of the invention also serves to detect such multimers where applicable The terms "polypeptide" and "protein" also refer to naturally modtfied polypeptides/protems wherein the modification is effected e g by giycosylation, acetylation, phosphorylation and the like Such modifications are well known in the art.

The term "phσsphorylated" in conjunction with peptides, polypeptides and proteins is known in the art As stated above, the term refers to modified forms of peptides and polypeptides, the modified forms being characterized in that one or more phosphate moieties are attached, usually covalentjy attached, to the peptide or polypeptide Common sites of attachment are the amino acids bearing hydroxyl groups, in particular Ser, Thr and Tyr Phosphorylation involves the formation of a phosphate ester between the phosphate and the hydroxy! group of Ser, Thr and Tyr In case more than one residue amenable to phosphorylation is comprised in a peptide or polypeptide, different phosphorylated forms may occur which differ in the number of phosphate moieties For example a single amsno acid, a subset of residues amenable to phosphorylation, or all residues amenable to phosphorylation may be phosphorylated The method of the invention is suitable to detect a!! these forms, wherein sensitivity of the method may increase with the number of phospnate moieties being bound to the peptide or polypeptide Preferably, also the degree of phosphorylation, i e the (average) number of phosphates per peptide or polypeptide can be determined Generally, fluorescence intensity will be proportional to the number of phosphoryiated sites Where necessary, the method of determining the degree of phosphorylation can be calibrated without further ado Generally binding of the compounds according to the invention will preferentially occur to surface-exposed residues As a rule, ammo aaόs bearing hydroxyl groups owing to the: ^r hydropπdicity are preferentially surface exposed on proxeins DOlyoeDtiαes and peptides

The term "detecting" according to the invention relates to determining presence or absence of the analyte It may furthermore comprise Quantitation Quantitation may rely on the linear relation between fluorescence errvssion intensity ana concentration, which generally applies with good approximation over a wide range of concentrations Moreover the skilled person can cal'brate the quantitation process without fjrther aαo, for example by using dilution series of standard compounds

^" "he ^r ecιted "br.ng ^p g into contact" is perormed under conditions which allow rormation of a complex comprsing the phosphorylated peptide and the compound of the invention, or of a complex comprising the phosphorylated polypeptide and the compound of the invention, respectively In other words, "bringing into contact" refers to bringing about conditions under which peptide or polypeptide molecules and compounds of the invention are in such spatia! proximity that complex formation may occur Such condmons are known in the art or can be determined by the skilled person without further ado In particular, suitable conditions include aqueous solutions of the peptide or polypeptide, for example buffered aqueous solutions, to which the compounds according to the invention are added

Buffers are well known in the art and the skilled person is aware of appropriate buffers in dependency of the substances being assayed Common buffers comprise (ρK _a values in brackets) H ₃ PO ₄ / NaH ₂ PO ₄ (pK _{a 1} = 2 12), glycine (pK _{a i} = 2 34), acetic acsd (4 75), citric acid (4.76) MES (6 15), cacodylic acid (6 27), H ₂ CO ₃ / NaHCO ₃ (ρK _{s 1} = 6 37) bis-Tπs (6 50), ADA (6 60), bis-Tris propane CpK ₈ - = 6 80), PIPES (6 80), ACES (6 90), imidazole (7 00), BES (7 15), MOPS (7 20), NaH ₂ PO ₄ / Na ₂ HPO ₄ (pK _{s 2} = 7.21 ), TES (7 50), HEPES (7 55), HEPPSO (7 80), tπethanoiamine (7 80), tπcsne (8 10), Tris (8 10), glycine amide (8 20), bicine (8 35), gSycylglycine {ρK _{a 2} = 8 40), TAPS (8 40), bis-Tπs propane (pK _{a 2} = 9 00), boric acid (H ₃ BO ₃ / Na ₂ B ₄ O ₇ ) (9 24), CHES (9 50), glycine (pK _{a 2} = 9 50), NaHCO ₃ / Na ₂ CO ₃ (ρK _{a 2} = 10 25), CAPS (10 40) and Na ₂ HPO ₄ / Na ₃ PO ₄ (pK _{≥ 3} = 12 67) Furthermore, ionic strength may be adjusted e g , by the addition of sodium chloride and/or potassium chloride Preferred concentrations of sodium chloride are from 0 to 2 M, preferably from 100 to 200 mM Examples of buffers comprising sodsum chloπde include PBS (phosphate buffered saline) containing 1 37 M NaCl, 27 rriM KCI ₁ 43 mM Na ₂ HPO ₄ -7H ₂ O and 14 mM KH ₂ PO ₄ in the 10-fold aqueous stock solution which is adjusted to pH 7 3, SSC containing 3 M NaCI and 0 3 M sodium citrate in 20-fo!d aqueous stock solution which is adjusted to pH 7 0 and STE (saline TΠΞ EDTA) containing 10 mM Tris base, 10 mM NaCi and i mM EDTA (acsdj Alternatively sodium chloπde is absent from the buffer preparation Examples for common buffer preparations without sodium or potassium chϊonαe are TAE (Tπs acetate EDTA) containing 2 FVi Tris acetate and 0 1 M EDTA ir the 50-fold aqueous siock soluiion at pH 8 5, TBE (Tn s borate EDTA) coniaining 0 89 M Tπs base 0 89 M bone acid and 0 02 M EDTA in the 10-fold aqueous stock solution at pH 8 0 ana TE (Tπs EDTA) containing 10 mM Tπs base and 1 mM EDTA (acid) at pH 7 5

Also and as descπoed in the eyamoles the compouncs o ^f the invention rray be dissolved in de-ionizsd water To the extent the peptide or polypeptide is soluble in de-ionized water, suitable conditions acco ^r ding to the invent or also include solutions of the peptide or polypeptide and the compound o ^f !ne invention in de-iσruzed water Typically, the polypeptide or peptide is comprised in a sample The sample may be taken from an organism, a cell in culture, from the environment or from a patient or subject suspected to suffer from a disease The sample may be a crude cellular extract or may be puπfied to different degrees (for details see below) The purified sample may be an aqueous solution or aqueous buffered solution of the polypeptide or peptide.

The polypeptide or peptide may also be part of a ceil, tissue or organism The cell may be a cell in culture The tissue may be in vitro, ex vivo or part of an organism The organism may be a plant or an animal, animals including humans When applied to cells, tissue or organisms, the methods according to the invention may aiso provide information as regards the spatial distribution of pnosphoryiaied polypeptides or peptides When performed repeatedly at different poinis in lime to the same cell, tissue or organism, the methods according to the invention may aiso provide information as regards the time-dependency of phosphorylation. Moreover, spatio-temporal patterns of phosphorylation may be determined

The term "complex" is known in the art and refers to non-covaleπt association of two or more molecules In the context of the method according to the invention, the term "complex" refers to a complex comprising or consisting of a peptide moiecuie or polypeptide molecule ana one or more compounds according to the invention, i e , one or more probe molecules As stated above, the peptide or polypeptide may be comprised in a multsmer of polypeptides The number of probe molecules comprised in the complex will depend on the amount of the compound being used and the number of phosphorylated sites which are accessible In case more than one probe molecule associates with a peptide or polypeptide these probe molecules may be of the same or different molecular species

The detecting of the compiex may be performed by any means and methods suitable to detect formation of a compiex Such methods iπcluαe for example the determination of the change of amount or concentration of the free (uncompiexed) form of peptide, polypeptide and/or compound according to the invention

Preferred metnodε of detecting the complex make use of the fluorescent properties of the compounds of the indention For example, if daieciion s perfo'meα on or sn a support such as a gel (for details see further below) the peptides or polypeptides may not be distributed homogeneously within tie gel for example after performing e ectrophoresis After staining of tne gel with compounds of the invention washing may be performed in order to remove unbound compounds In such a case, detection involves detecting the fluorescent region or band on the gei The fluorescent region or band corresponds to a phosphoryiated peptide or polypeptide

Detection may be performed with the nake or eye and/or with the help of photographic devices, optionally in combination with microscopic devices Analysis of the obtaining images may be performed with the aid of computers having suitable software implemented thereon Computer-based image analysis is we!! known in the art and commonly referreα to as image analysis

In a preferred embodiment, the detecting of said complex comprises determining whether, upon said bringing into contact, (ι) fluorescence emission of said compound increases, and/or (ιι) the wavelength of the fluorescence emission maximum of said compound is shifted, wherein an increase of fluorescence emission and/or shift of the wavelength of the fluorescence emission maximum is indicative of the presence of said complex The effect according to (ή is an overall change in fluorescence intensity The effect according to (ιi) is change of the fluorescence emission spectrum

Tne present invention furthermore provides a method of detecting and differentiating phosphoryiated and unphoεphorylated peptides or polypeptides, the method comprising (a) bringing into contact said peptides or polypeptides with a compound and fb) determining (ι) whether fluorescence emission Deing characteristic of a complex of said compound with phospnorylated peptides or phosphoryiated polypeptides occurs; and (ιι) whether fluorescence emission being characteristic of a complex of said compound with unphcsphoryiated peptides or unphospncyssted polypeptides occurs, thereby detecting and differentiating phosphorylafed and unphosphoryiated peptides or polypeptides

The ternn 'differentiating" refers to distinguishing between phosohorylated and unphoεphorylated peptides and polypeptides Phosphory.ated and unphosphorylateα polypeptides (or peptides) may be polypeptides (or peptides) with different amino acid seαuences or may be polypeptides (or peptides) w-th the same amino acid sequence In the latter cεse ohosohoryla^ed and unphosphorvSated forms of an otherwise identical at least with regard to the ammo acsd sequence, polypeptide (o- peptide) are differentiated The term "fluorescence emission betng characteristic of a complex [ ]" refers to a reference state for comparison This reference state may be the fluorescence emission of a particular peptide or oolypeptide in phoεphorylated and unphoεphorylated form, respectively This may be particularly useful for those embodiments where differentiating between phosphorylated and unphosphoryiated forms of this particular peptide or polypeptide is desired However, the reference state is not so limited The reference states may be mixtures of polypeptides (or peptides), or polypeptides (or peptides), wherein phosphorylated and unphosphoryiated reference states have different ammo acid seαuences

This embodiment exploits the following properties of the compounds of the invention The compounds of the invention are capable of forming a complex also with binding sites on a peptide or polypeptide which are not phosphorylated sites The complexes of compounds of the invention with non-phosphate binding sites differ from complexes with phosphorySated sites with regard to fluorescence emission

In preferred embodiments and depending on the choice of the fluorophore, the overall fluorescence emission of the unphosphoryiated peptide or polypeptide is different sn intensity, preferably lower in intensity as compared to the phoεphory'afed polypeptide or peptide and/or the fluorescence emission, while besng of similar magnitude, exhibits an emission maximum at a different wavelength The latter phenomenon, i e , the dependency of the fluorescence emission spectrum on the environment, is also referred to as solvatochromic effect Fiuorophores according to formula (II) exhibit a solvatochromic effect whereas fiuorophores according to formula (!Ii) exhioit lower fluorescence intensity when bound to a binding si ^τ e whicn is not a phoεphoryiaied site Without wishing to be bound by a specific theory the latrer effect is attributed to αuenching which occurs at πon-phoεohaie binding sites

Accordingly, in a preferred embodiment of the method of detecting and differentiating Dhoεpborylated and unpnosphoryiated peptides or polypeptides fluorescence enrssion according to (i) differs from fluoiescence erri'ssion according to (ιι) with regard to the wavelength of the fluorescence emission maximum !π a particularV preferred ernoodiment, the f'uorophor comρr ^< sed in said compound is of the genera ¹ foTnu'a (I ¹ )

In prefered eπDαdments of the methods cf tne invention, the methods are effected on or in a support Supports include any type of solid material including glass polymers or metals or combinations thereof Supports may have the form of fiat sheets such as membranes, or may be three-dimensional objects with a certain thsckness such as gels A support may serve to immobilize peptides or polypeptides for example on one or more sites or areas within the support in the latter case, polypeptides or peptides are not distributed homogeneously within the support Preferably said peptide or polypeptide is capable of binding or adhering to said support Binding or adhering of peptides or polypeptides to the support material generally occurs non-covaSently Peptides and polypeptides may be bound at the surface of the support (e.g , of a membrane) or witnin the support, e g , within a gel By immobilizing the polypeptide or peptide, a support offers distinct advantages over detection in solution Polypeptides or peptides are confined to their respective sites of immobilization and cannot diffuse freely If immobilization occurs at specific sites or areas the locations of polypeptides or peptides may correspond to or reveal information on further properties of the polypeptides or peptides, including their identity For example, in those embodiments where the support is an array, polypeptides or peptides are deposited at defined sites or areas within the support Knowledge of the site where a polypeptide or peptide has been deposited provides knowledge of its identity Detection of fluorescence at those sites by methods of the invention a'lows information on the phosphorylation states of the polypeptide or peptide immobilized at that particular site to be obtained Alternatively polypeptides or peptides may be confined to particular areas of a support as a consequence of a separation process such as electrophoresis, see below in that case, the location of the polypeptide or peptide generally is determined by and corresponds to its molecular weight Also, and in particular the support is a membrane or filter the polypeptide or peptide may be confined to a particular area as a consequence of a process of transfer from another support wherein the ooiypeptide or peptide is coπfired TO a particular area within sa'd another support Sucn process of transfer is known in the art and includes olotting When blotting precedes performing the method of the invention this embodiment of the methoc. is also referreα to as On-bιot"

Supports furthermore include cells tissues or nort-πuman ojganf≤ms wherein said celis, tissues or non-human organssms are attached to or bound to a support Celis tfssues or organisms may be fixated Fixation is a means of preparing cells tissues or organisms and SS known in the art Preferred embodiments of cells, t'ESjes and organisms are described herein above Tissues rray be provided as crcss-sectionε When performing the method of the invention on or in a support, the step of bringing into contact may also be referred to as "staining". Preferred or exemplified conditions for staining, in particular for staining of gel, can be found in the enclosed examples.

Preferred supports are selected from (a) gels; (b) membranes; (c) filters; and (d) arrays. Gels, membranes and filters are commonly used in the field of molecular biology and analytics.

A gel is a solid, jelly-like material that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute crosstinked system, which exhibits no flow when in the steady-state. By weight, gels are mostly liquid, yet they behave like solids due to a three-dimensional crossϋnked network within the liquid. It is the crossiinks within the fluid that give a gel its structure (hardness) and contribute to stickiness (tack).

Preferably, the gel is a polyacrylamide gel. A polyacrylamide gel is a separation matrix used in electrophoresis of biomolecules, such as proteins or nucleic acids. A polyacrylamide gel may comprise a stacking ge! and a resolving gei. Typically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the weils and defines the lanes where proteins, sample buffer and ladders will be placed) is inserted. The percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample. The smaller the known molecular weight, the higher the percentage that should be used.

Alternatively, the gel may for example be an agarose gel.

Gels such as polyacrylamide gels may comprise a deπaturant. Denaturants include sodium dodecy! sulphate (SDS), urea and guanidinium chloride. Furthermore, agents which reduce the disulfide bonds present in many polypeptides may be used as denaturants. Agents reducing disulfide bonds include 2-mercaρtoethaπol and dithiothreitoi.

Denaturants may be used to unfold polypeptides or reduce the amount of tertiary structure. As a consequence, the migration behaviour of the polypeptide in the gel is predominantly, or at least to greater extent than in the folded state, governed by the molecular weight.

Membranes and filters are usualfy provided as flat sheets made of various materials, preferably of materia! to which polypeptides or peptides can adhere or bind. Preferred membranes are polyvinylidene difluoπde (PVDF) membranes and nitrocellulose membranes These membranes are known in the art, commonly used for Western blotting, and are available from various manufacturers

In preferred embodiments of methods using a gel, electrophoresis of said peptide or polypeptide is performed prior to. concomitantly with, or after step (a). Gel electrophoresis is well known in the art and is a technique used for the separation of biomofecuJes, including peptides and polypeptides, using an electric current applied to a gel matrix. Gel electrophoresis permits the separation of peptides and polypeptides according to their size In certain instances, also different degrees of phosphorylation of a peptide or polypeptide may give rise to discernible differences in migration during gel electrophoresis. This embodiment of the invention is also referred to as "in gel" method

The term "array" ss well known in the art In the context of the present invention it refers to a support having different species of polypeptides or peptides immobilized thereto. Preferably, each polypeptide or peptide has its own distinct site of immobilization, thereby facilitating assignment of position on the array to identity of the polypeptides or peptides. Supports suitable for the [mmbiiization of polypeptides or peptides are known in the art (see e g , Stoll, D., Bachmann, J., Tempim, M. F , Jooε, T O (2004) Microarray technology: an increasing variety of screening tools for proteomic research Targets 3. 24-31 Cahill, D J., Nordhoff, E. (2003) Protein arrays and their role in proteomics. Adv Biochem Eng Biotechnol 83i 77-87., Gershon, D. (2003) Proteomics technologies' probing the proteome Nature 424 581-7

in further embodiments of the methods of the invention, the methods are effected in solution. Performing the methods in solution ;s an alternative to per ^' orming them using a support.

The present invention furthermore provides the use of a compound of the present invention for detecting a phospnoryiated peptide or phosphory.afed polypeptide.

Also provided is the use of a compound of the present invention for detecting and Gifferennating phosphorySated and unphosphorylatec forms of a peptide oi polypeptide

The invention furthermore relates to a kit comprising one or more compounds of the present invention. Preferably the kit further comprises a manual, the manual comprising instructions for performing at least one of the methods according to the invention

The present invention furthermore relates to a diagnostic composition comprising one or more compounds of the present invention The diagnostic composttion may furthermore comprise a carrier, diluent or excipieπt For example, the diagnostic composition may be an aqueous solution or buffered aqueous solution of one or more compounds of the invention Buffers are wet! known in the art and further detailed herein above

Also provided is a method of diagnosing a disease characterized by presence of an aberrant amount of a phosphoryiated peptide or phosphorylated polypeptide, the method comprising (a) bringing into contact a sample obtained from a subject suspected to suffer from said disease with a compound of the present invention, and (b) determining whether the fiuorεscence emission of said sample differs from the fluorescence emission of a control sample

A "control sample" may be a sample wnich has been taken from a heaϊthy individual or a sample which reflects the phosphorylation status of the polypeptide in a healthy individual The term "aberrant amount" refers to both an aberrant absolute amount of the peptide or polypeptide in question wherein the ratio of phosphoryiated and unphosphorylated form do not deviate from the ratio found in heaithy individuals, as well as to aberrant ratios (relative amounts) The latter phenomenon includes hyper- and hypophosphoryiation

Preferably the sample is purified prior to performing step (a) Purification preferably includes removal of mεoiub'e mateπai ^f ormed upor iyεts of the ceils of the sample Purification may furthermore comprise removal of nucleic acids and/or enrichment of polypeptides or certain groups of polypeptides A ¹ SO envisaged 'S purήcatron which delivers the polypeptide in question be it phosphoryiated or not, phosphoryiated to different degrees or mixtures thereof in substantially pure form Suitable means and methods for purification are well known to the pe ^r son sksllec in art and nclϋde chromatography such as size exclusion chromatography, ion exchange chromatography and affini+y chromatography

Cellular signaling involves numerous phophorylation and de-phoεphorySafion events Enzymes capable of adding phosphate residues to polypeptides and peptides include kinases ana form a prominent class of drug targets Aberrant growtn factor signaling, for example, may give rise to aberrantly high kinase activity, which in turn may entail aberrant amounts of phosphorylated peptides or phosphoryiated polypeptides as defined above. Since aberrant growth factor signaling relates to hyperproiiferation and neoplasms, cancer is one of the diseases which may be diagnosed on the basis of such aberrant amounts,

The present invention will now be illustrated by the following examples. However, the present invention is not to be construed as being limited thereto.

EXAMPLES

Example 1 : Synthesis of probes 1 and 2

Ail reactions were performed under an inert atmosphere of N ₂ using standard Schlenk techniques if not otherwise stated.

A Varian Gary B)O 50 UV/ViS/NiR Spectrometer was used. A 1 cm quartz cell was purchased from Hellma and Uvaεol solvents were obtained from Merck or Baker. Absorption and emission maxima are given in nm, molar absorptivϊiies (ε) are given in M ^'1 cm ^"1 .

IR spectra were recorded on a Bio-Rad FT-IR FTS 155 and a Bio-Rad FTS 2000 EvIX FT-IR using a Specac Golden Gate Mk SI ATR accessory where stated.

NMR spectrometers used were: Bruker Avance 600 ( ¹ H. 600.1 MHz. ¹³ C; 150.1 IvIHz ₁ T - 300 K) ₁ Bruker Avance 400 ( ¹ H: 400.1 MHz, ^"3 C: 100 6 MHz T - 300 K) and Bruker Avance 300 ( ¹ H- 3OC.1 MHz, ¹³ C: 75 5 MHz, T = 300 K). The chemical shifts are reported in δ [ppm] relative to external standards (solvent restduai peak). The spectra were analyzed by first order, the coupling constants are given in Hertz [Hz]. Characterization of the signals' s = singlet, d = doublet, t - triplet, q = quartet, m = multiplet, bs = broad singlet, psq = pεeudo quintet, dd = double doublet, dt = double triplet, add = double double doublet. Integration is determined as the relative number of atoms. Assignment of signals in ¹³ C-spectra was determined with DEPT-technique (pulse angle: 135 ^r ) and given as (+) for CH ₃ or CH, (-) for CH ₂ and (C _qoat ) for quaternary C. Error of reported values: chemical shift: 0.01 pprn for ¹ H- NMR, 0.1 ppm for ¹³ C-NMR and 0.1 Hz for coupling constants. The solvent used is reported for each spectrum.

Mass spectra were recorded on Varian CH-5 (E!), Finnigan MAT 95 (CJ; FAB and FD) and Finnigan MAT TSQ 7000 (ESl). Xenon served as the ionization gas for FAB.

Melting Points were determined on a Bϋchi SMP-20 melting point apparatus and are uncorrected. TLC analyses were performed on siiica gel 60 F-254 with a 0.2 mm layer thickness. Detection was via UV light at 254 nm / 366 πm or by staining with ninhydrin in EtOH.

For preparative column-chromatography, Merck Geduran Si 60 silica ge! was used.

Commercially available solvents of standard quality were used. Unless otherwise stated, purification and drying was done according to accepted general procedures (15, 16). Compounds 3 (17) and 5 (18) were synthesized according to literature known procedures.

Synthesis of probes 1 and 2

Regioisomeric 1,4, 7-ϊri-tert-bufyl 10. 1O'-{6-[2-(3' 6'-dihydroxy-3-oxo-3H-spιro[ιsob&πiofuran- 1,9'~xanthene]'5/6-ylcarboxamido)ethylamino]-1 3 5-triazine-2, 4~diyl}bis(1,4.7, 10- tetraazacyclododecane-1.4, 7-tricarboxyiate) (6):

In a nitrogen flushed round bottom flask, regioiεomerϊc 5-,6-carboxyfiuorescein (0.15 g, 0.40 mmol) was dissolved in 10 m L of a mixture of dichloromethane and DMF (2:1 ), then DiPEA (0.17 g, 180 μL, 1 .32 rnmoi) and HOBt monohydrate (0.07 g, 0.48 mmol} were added. While stirring, the mixture was cooled to 0 ⁰ C in an ice water bath to add TBTU (0.15 g, 0.48 mmol). After 30 min, 1 ,4.7-tri-tert-butyi 10,10'-(6-(2-aminoethylamino)~1 ,3,5-triazine-2,4-diyl) bis (I ,4,7,10-tetraa2acyclo-dodecaπe-1 ,4,7-tπcarboxyiate) (3) (0.47 g, 0 44 mmo!) was added in portions to the flask and the reaction mixture was heated to 40 "C for 2 h. Subsequently, the solvents were evaporated and the crude product was purified by column chromatography (CHCI ₃ : MeOH = 15 : 1 ) on fiash-siiica-ge! yielding compound 6 as an orange amorphous solid (0 34 g. 0.23 mmol, 59%)

>: 207 ⁰ C 1H-MMR (600 MHz, MeOD COSY _: ROESY, HSQC, HMBC) ^' δ [ppm] = 8 36 (s, 0.7H ₁ 22a), 8.13 (d. ⁵ J _K H = 7 91 Hz ₁ 0.7H. 20a), 8 07 (d, ³ J _KH = 8 08 Hz, 0 3H ₁ 21 b), 8.03 (d, ³ J _{H t} - 3.04 Hz, 0.3H, 22b). 7.61 (s, 0 3H, 19b), 7 24 (d, ³ J _{H H} = 8 02 Hz, 0.7H, 19a), 6.68 (del, ³ J _HM ~ 1.75 Hz, ^E J _H ,H = 1.75 Hz, 2H ₁ 2+ 12), 6 57 <dd, "J _{H H} = 8 71 Hz, ^: 'J _{h H} = 8.71 Hz. 2H, 5+9), 6.52 (dd, ^! Jπ.H = 8.72 Hz, %, _H = 2.31 H∑, 2H. 3+19), 3 99-3.20 (m, 36H, 25a/b. 26a/b, 32+33+35+36), 1 45-1 37 (m. 54H, 41 +45)

¹³ C-NSiR (150 MHz, MeOD. COSY, ROESY. HSQC, HMBC): δ [ppm] = 170.4 (C _quat , 0.3C, 16b). 170.3 (C _αua! , 0.7C, 163), 168.5 (C _{ouat l} 0.7C. 23a). 168.3 (C _quat , 0.3C, 23b), 157.8 (C _qu£! , 2C, 30), 167 5 (C _ouat . 0.7C, 28a), 157.3 (C _quβt , 0.3C, 28b), 161.3 (C _{qua! l} 2C, 4+10), 158.0 (C _quat , 6C ₁ 38+42), 156.7 (C _quat , 0.7C, 1 Sa) ₁ 154.4 (C _qιat , 0.3C ₁ 18b), 153.9 (C _{quat l} 2C, 6+8), 142.4 (C _quaLl 0.3C, 20b), 138.0 (C _quat , 0.7C, 21 a), 135 5 (+, 0,7C ₁ 20a), 130.2 (+, 0.3C, 21b), 130.1 (+, 2C ₁ 5+9), 128.5 (C _quaLl 1 C, 17a/b), 126.1 (+, 0.3C ₁ 22b), 125.6 (+, 0.7C, 19a), 124.8 (+, 0.7C, 22a), 124.0 (+, 0.3C ₁ 19b), 1 13.7 (+, 2C ₁ 3+11), 1 10.8 (C _αuat , 2C, 1 + 13), 103.7 (+, 2C, 2+12), 81.5 (C _quat , 2C, 38), 81.4 <C _{quat i} 4C ₁ 42), 79.4 (C _ouat , 1C. 14), 51 4 (-, 16C, 32+33+35+36), 41.7 (-, 0.7C, 25a), 41.5 (-, 0.3C ₁ 25b), 40.9 (-, 0.7C, 26a), 40.8 (-, 0.3C, 26b), 29.9 (+, 6C ₁ 41), 28.8 (+, 12C, 45)

IR (ATR) [cm ^"1 ]: v - 3318, 2971 , 2935, 1766, 1689, 1665, 1539, 1456, 1410, 1365, 1247. 1157, 1024, 852, 31 1 , 751 , 665 ES-MS (H ₂ OZMeOH + 10 mmoi/L NH ₄ Ac): m/z {%) = 720.0 (100) [M ÷ 2H ⁺ ], 1439.0 (5) [MHl UV (MeCN): λ (ε) = 454 (2175), 481 (1876)

Regioisomeήc N~{2-[4, 6-di( 1, 4, 7, 10-tetraazacyclododecan- 1 -y!)- 1, 3, 5-tria∑irh2~ylaminoJ- ethyt}-3', 5'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1.9'~xanthene]-5-c arboxamide ocϊachioride (7):

Compound 6 (80 mg 0.05 mmol) was dissolved in as little dichloromethane as possible and cooieα to 0 C before hydrochloric acid saturated diethyl ether (1.06 mL) was added slowly to this solution. Stirring WΞS continued for 15 h white the reaction mixture was allowed to reach room temperature. Formation of a yellowish precipitate wss observed. The desired product was obtained by evaporation of the solvent in vacuum as a red amorphous solid (50 mg, 0.05 mmoi, quant. ), which was deprotonated by ion exchange column on weakly basic anion exchanger reεin.

¹ H-WMR (600 MHz, D ₂ O, COSY, HSQC, HMBC); δ [ppm] = 8.^3 (s, 2H, 24), 7.97 (s, 0.6H ₁ 22a), 7.93-7.8B (d, ^S J _KH = 7.37 Hz, 0 4H, 21b), 7.65-7.79 (d, % _,h = 7.40 Hz, 0 4H, 22b), 7.74 (bs, 0.6H, 20a), 7 44 (s, 0 4H, 19b), 7 02 (bε, 0.6H, 19a), 6.99-6.70 (m, 2H, 2+12), 6.61-S.41 (m, 2H, 3+11), 6.41-6.13 (m, 2H. 5+9), 3.87-3,39 (m, 12H, 25+26+36), 3.27-2.88 (m, 24H, 32+33+35) 1 ³ C-NMR (150 MHz. D ₂ O, COSY, HSQC ₁ HMBC): δ [ppm] = 179.9 (C _quaLl 2C, 30), 174.2 (C _quat , 04C, 1 Sb), 173.2 (C^ ₁ 0.6C, 16a), 171.5 (C _quβL , 1C, 28), 170.1 (C _{0UBt l} 0.4C. 23b) _r 169.5 (Cq ₁₁₃₁ , 0 6C, 23a), 167.2-1S6 3 (C _{qurt l} 2C, 4+10), 158.6-158.4 (C _Q , _al , 2C, 6+8), 157.9 (CqU ₈₁ , 0.6C, 18a), 157.2 (C ₀₁₁₈₁ , 0.4C, 18b), 143.7 (C ₀₁₁₈ ; , 0.4C, 20b), 140 7 (C _{ourt l} 0.SC ₁ 21a), 135.6 (C _{αuat l} 0 4C ₁ 17b), 135.2 (C _quat , 0.5C, 17a), 131.9-131.5 (+, 2C ₁ 2+12), 130 7 (+, 0.6C ₁ 19a), 129.5 (+, 0.4C, 22b), 129.2 (+, 0.4C, 21b), 128.7 (+, 0.4C ₁ 19b), 128 4 (+, 1.2C 20a+22a), 123.3 (+, 2C, 3+11), 104 4 (+, 2C, 5+9), 46.5-45.2 (-, 12C, 33-^35+36), 43.9 (-, 4C, 32), 40.B-40.6 (-, 1 C, 25a/b), 39.9 {-, 1 C, 26)

ES-tøS (MeCNfFFA): m/z (%) = 280.1 (100) [M + 3H ^" ], 419.8 (63) [M + 2Hl, 838.5 (4) [MH ^* ]. 874 4 (3) [MH ⁺ + HC!]

Probe 1.

Deprotonated compound 7 (48 mg, 0.06 mmo!) was dissolved in 2 mL of warm water, then zinc(l!)-chloride (16 mg, 0,12 mmol) was added. After pH-adjustment to 7-8 with saturated NaHCO _{3, aqα} , the mixture was heated to 100 ⁰ C for 2 h while stirring. The desired product was isolated by iyophiliεation of the reaction mixture as a dark orange, amorphous solid (48 mg, 0.04 mmol, 75%).

¹ H-NMR (600 MHz, D ₂ O, COSY, HSQC, HMBC): δ [ppm] = 8.14-6.06 (m, 9H ₁ aryf), 4.25-

2.62 (m, 35H, amide-NH+CH ₂ )

¹¹ C-NMR (150 MHz, D ₂ O, COSY, HSQC, HWiBC): δ [ppm] = 174.1-103.2 (C _quat &-, 24C, 1-

23+28+30), 46.3-38.7 (-, 18C, 25+26+32-36)

ES-MS (H ₂ OZMeOH + 10 mmol/L NKAc): m/z (%) = 51 1.4 (70) [M ⁴⁺ - H ⁺ + CH ₃ COOl

481 .3 (37) [M ⁴⁺ - 2H ^÷ ]

UV (40 mM TRIS-buffer. 10 mM MgCI ₂ , 2 mM DTE, pH 7.6): λ (ε) = 321 (6430), 497 (56320}

Fluorescence (40 mM TRIS-buffer, 10 mM MgCI ₂ , 2 mM DTE, pH 7.6): exc. 494 nm, emission max. 526 nm

7'Diethylamino-2-oxo-2H-chromene-3-caώoxylic acid {2-[4, 6-bis-(1, 4, 7, 10-tetraaza- cycloόodec-1-yiy[1,3,5]triazin-2-yl]-ethyi}-1,4, 7-tήcarboxync acid tri-tert-buty! ester (8):

/ ^■ -Dιethyiamιno-2-oxo-2H-chrornen&-3-carDθxyi!C acid (150 mg, 0 56 mmo!), DIPhA (386 μl_, 2 24 mmo!), TBTU (203 mg, 0.63 mmol). and HOSt (97 mg, 0.63 mmo!) were dissolved under nitrogen atmosphere in dry DMF (4 mL) under ice cooling and stirred for 1 h.

Subsequently amine 3 (635 mg, 0.59 mmol) dissolved in DMF (2 mL) was added drop wise. The reaction was allowed to warm to room temperature and was stirred 30 min at room temperature and 3 h at 40 ⁰ C. The reaction progress was monitored by TLC (chioroform/MeOH 97.5/2.5). After compfetion of the reaction the solvent was removed and the crude product was purified by flash column chromatography on flash siiica gei (chloroform/MeOH 97.5/2,5; R _f (EE) = 0.60) yielding compound 8 (684 rng, 0,52 mmol, 93 %) as a yeilow solid.

MP: 147 ⁰ C

¹ H-NMR (400 MHz: CDCI ₃ ): δ (ppm) = 1.21 (t, ³ J = 7.1 Hz, 6 H, HSQC ₁ COSY: C ¹ H ₃ ), 1.40 (s, 18 H, HSQC ₁ COSY: C ²² H ₃ ), 1.42 (s, 35 H, HSQC, COSY: C ²² H ₃ ), 3.11-3.76 (m, 36 H, C ¹⁴ H ₂ , C ¹⁵ H ₂ , cyclen-CH ₂ ), 3.42 (q, ³ J = 7.1 Hz, 4 H, HSQC, HMBC, COSY: C ² H ₂ ), 5.02 (bs, 1 H, NH ¹⁶ ), 6.46 (d, ⁴ J = 2.3 Hz, 1 H, HSQC, HMBC, COSY: C ⁵ H), 6.82 (dd, ³ J = 8.9 Hz, ⁴ J = 2.4 Hz, 1 H, HSQC, HMBC ₁ COSY: C ⁴ H), 7.39 (d, ³ J = 8.9 Hz, 1 H, HSQC ₅ HMBC, COSY: C ¹¹ H), 8.65 (s, 1 H ₁ HSQC, HMBC, COSY: C ⁷ H), 8.90 (m, 1 H, HSQC ₁ COSY: NH ¹³ ).

¹³ C-NMR (100 MHz; CDCI ₃ ): δ (ppm) = 12.4 (+, 2 C, HSQC, HMBC: C ¹ H ₃ ), 28.41 , 28.45 (+, 18 C, HSQC, HMBC: C ²² H ₃ ), 39 6 (-, 1 C, HSQC, HMBC. C ¹ H) ₁ 40.8 (-, 1 C, HSQC, HMBC; C ¹⁵ H ₂ ), 45.0 (-, 2 C, HSQC ₁ HMBC, COSY: C ² H ₂ ), 50.2 (-, 16 C ₁ HSQC, HMBC: cyclen-CH ₂ ), 79.6 (C _q. 6 C, HSQC, HMBC: C ²¹ ), 96.5 (+, 1 C, HSQC, HMBC, COSY: C ⁵ H), 10S.3 (C, 1 C, HSQC, HMBC: C ⁶ ), 109,9 (+, 1 C ₁ HSQC, HMBC, COSY: C ⁴ H), 110.1 (C _q, 1 C, HSQC, HMBC: C ^B ), 131.1 (+, 1 C, HSQC, HMBC, COSY: C ¹¹ H), 148,0 (+, 1 C ₁ HSQC, HMBC ₁ COSY: C ¹¹ H), 152.5 (C _q, 1 C ₁ HSQC, HMBC: C ³ ), 156.2 (C, _, 6 C, HSQC ₁ HMBC: C ²⁰ ), 157.6 (C _q. 1 C ₁ HSQC, HMBC: C ¹⁰ ), 162.5 (C ₁ , _, 1 C, HSQC ₁ HMBC: C ⁹ ), 163.7 (C ₁ , _, 1 C ₁ HSQC, HMBC: C ¹² ), 165.8 (C _q , 3 C, HSQC, HM8C: C' ⁷ , C ¹⁸ , C ¹⁹ ). ΪR (ATR) [cm ¹ ]: ^ - 2974, 2933, 1686, 1619, 1584, 1532, 1512. 1466, 1409, 1364, 1246.

1 158, 971. 751.

UV (CHCi ₃ ): λ _max (c) = 419 (41020)

MS (ESt(+), DCM/MeOH + 10 mmoi/L NH ₄ Ac): m/z (%) = 1324.0 (100) [MH"), 612.5 (12)

[M + 2 H ^~ f ⁺ , 562.5 (25) [M + 2 H ^" - bocf, 512.4 (51 ) [M ÷ 2 H ^* - 2 DoC] ²⁺ , 462 3 (55) [M + 2 H ^* - 3 bocf ⁴ , 412.3 (34) [M + 2 H ⁺ - 4 DOc] ²⁺ , 362.2 (17) [M + 2 H ⁺ - 5 DOc] ²⁺ .

7-Diethy!amiπo-2-oxo-?H-chromene-3-carboxy!ic acid {2-[4,6-bis-(1 4. 7, IQ-tetraara- cyc}ododec-1-yl)-[1,3, 5]triazin-2-y!amino]-ethyl}-amide (9):

Compound 8 (275 mg, 0.21 mmol) was dissolved in dichioromethane (4 mL) and cooled to

0 °C. Subsequently 4.5 mL of HCi saturated diethylether were added. The solution was stirred 15 miπ at 0 ⁰ C and an additional 20 h at room temperature. The solvent was removed in vacuo, the residue was redissolved in water and extracted with dichioromethane (3x). Subsequently the combined organic layers were dried over MgSO ₄ and the solvent was evaporated yielding the protonated hydrochloride of compound 9 as a yellow solid in quantitative yield (213 rng, 0.21 mrnol).

¹ H-NMR (400 MHz; CDCI ₃ )- δ (ppm) = 1 13 (t, ³ J = 7 3 Hz ₁ 6 H. HSQC, COSY: C ¹ H ₃ ), 2.97- 3.54 <m, 24 H, HSQC, HMBC ₅ COSY: cyclen-CH ₂ ), 3.57-3.72 (m, 6 H, HSQC, HMBC: C ³ H ₂ ; HSQC, HMBC, COSY. C ² H ₂ ), 3.75 (t, ³ J = 5 6 Hz, 2 H, HSQC. COSY, C ¹⁴ H ₂ ), 3 89 (bs, 8 H ₁ HSOC, HMBC, COSY' cyc!en-CH ₂ ), 7.37-7.56 (m, 2 H, HSQC ₁ HMBC: C ⁴ H, HSQC ₁ HMBC: C ^& H) 7.95 (d, ³ J = 8 4 Hz, 1 H, HSQC, HMBC' C ¹¹ H), 8.72 (s. 1 H, HSQC HMBC: C ⁷ H).

¹³ C-NMR (100 UHz; CDCI ₃ ): δ (ppm) = 10.1 (+, 2 C ₁ HSQC, COSY: C ¹ H ₃ ), 39.0 {- 1 C. HSQC, HMBC: C ¹³ H ₂ ), 39.9 (- 1 C, HSQC, HMBC. C ^1ή H ₂ ), 44.0 (- 2 C, HSQC, HMBC, COSY. cyclen-CHa). 44 3 {- 6 C, HSQC, HMBC, COSY. cyclen-CH ₂ ), 46.1 (- & C, HSQC, HMBC ₁ COSY: cyclen-CH ₂ ), 47.7 (-. 2 C, HSQC, HMBC, COSY: cyclen-CH,), 48 3 (-, 2 C, HSQC, HMBC, COSY. cyclen-CHj), 52 6 {-, 2 C ₁ HSQC, HMBC. COSY: C ² H ₂ ). 108.8 (+,

1 C, HSQC, HMBC. C ⁶ H), 117 6 (C _ς, 1 C ₁ HSQC _: HMBC: C ^L ), 1 17.9 (C _q 1 C. HSQC, HMBC: C ⁸ ), 118, 1 (+, 1 C, HSQC, HMBC C ⁴ H), 132.5 '+ 1 O HSQO HWiBC C ¹¹ H) 143 2 (C, _, 1 C HSQC, HMBC: C ³ ), 147 6 (+ 1 C. HSQC, HMBC C ⁷ H), 154 9 (C _q . 1 C ₁ HSQC, HMBC: C ¹⁰ ), 155.2 {C _q , 1 C ₁ HSQC HMBC: C ¹⁶ ), 155 8 (C _q, 1 C, HSQC. HMBC: C ¹⁵ ), -561.4 (C _q 1 C, HSQC, HMBC: C ⁹ ), 163 6 (C _q, 1 C. HSQC ₁ HMBC: C ¹⁷ ), 164.1 (C _q 1 C ₁ HSQC ₁ HMBC: C ⁸ ). MS (ESI(H-), MeCN/TFA) m/z (%) = 362 3 (100) [M + 2 H ⁺ J ²⁺ , 723 5 (10) [MH ^' ]

To obtain the free base of compound 9 a weakly basic ion exchanger rεsm was swollen for 15 mm in water and washed neutral with water A column was charged with resin (473 mg 40 0 mmol hydroxy equivalents at a given capacity of 5 mmol/g) The hydrochloride salt {60 mg, 59 μmol) was dissolved in water put onto the column and eluated with water The eiution of the product was controlled by pH indicator paper (pH > 10) and was completed when pH was again neutral The eiuate was concentrated and lyophihsed to yield 43 mg (59 μmol, 100 %) of free base 9 as a yellow solid

MP 169 ^C C

¹ H-NiViR (600 MHz D ₂ O) δ (ppm) = 1 04 (t, ³ J = 7 1 Hz 6 H HSQC COSY C ¹ H ₃ ) 3 02 (bs, 8 H, cycien-CH ₂ ), 3 12 (bs, 16 H cyclen-CH ₂ ) 3 27 (q ³ J = 7 0 Hz 4 H, HSQC, HMBC C ² H ₂ ), 3 33 (t, ^C J = 4 5 Hz, 2 H HSQC, HMBC C ¹⁵ H ₂ ), 3 48 (t, ³ J = 5 2 Hz, 2 H, HSQC, HMBC- C ¹⁴ H ₂ ), 3 62 (bs S H, cyclen~CH ₂ ) 6 13 (d, ⁴ J = 1 B Hz, 1 H, HSQC, HMBC, ROESY C ¹ H), 6 58 ⁽ d, ³ J = 9 0 Hz, 1 H, HSQC, HMBC, ROESY C ⁴ H) ₁ 7 24 (d, ~J = 9 2 Hz ₁ 1 H HSQC, HMBC, ROESY C ⁵ H), 8 09 (s, ⁴ J = 1 8 Hz, 1 H ₁ HSQC, HM8C ROESY C ⁷ H), 8 28 (bs, 1 H, HSQC, NH) 1 ³ C-MRZlR (150 MHz, D _? O) ό (pprn) = 11 8 (+ 2 C, HSQC HMBC C ¹ H ₃ ), 39 4 (- 1 C HSQC HMBC C ¹⁴ H ₂ ) 40 1 (-, 1 C HSQC, HMBC C ¹³ H ₂ ), ^A 2 4 (- 2 C, HSQC, HMBC cyclen-CH ₂ ), 42 8 {-, 2 C HSQC, HMBC cycien-CH ₂ ), 44 7 (-, 4 C, HSQC, HMBC cycien- CH ₂ ), 45 1 (- 2 C, HSQC HMBC C ² H ₂ ), 45 7 (~ 4 C HSQC, HMBC cyden-CH ₂ ), 46 1 (-, 4 C, HSQC, HMBC cyclen-CH ₂ ), 95 5 (+, 1 C HSQC HMBC, ROESY C ^" Η), 106 0 (C _q 1 C, HSQC HMBC C ⁸ ), 107 6 (C _q 1 C HSQC HMBC C ⁸ ), 1 1 1 4 (+, 1 C, HSQC, HWiBC ROESY C ⁴ H), 131 7 (T 1 C, HSQC HMBC ROESY CΗ) 148 1 (+ 1 C HSQC HfVlBC ROESY C ⁷ H), 153 6 (C _q 1 C HSQC, HMBC C ³ ) 157 0 (C _c 1 C HSQC, HMBC C ¹⁰ ) 163 1 {C _q 1 C HSQC, HMBC C ⁸ ) 165 3 (C _q 1 C HSQC HMBC C ⁹ ) 166 7 (C _q 1 C, HSQC HMBC C ¹⁵ ), 166 8 (C _q 2 C HSQC HMBC C ⁶ C' ⁷ }

IR (ATR) [Cm ¹ J * ^" = 3352 3315 2968, 2732 1694 1576, 1537, 1506 1417 1350, 1231 , 1 134 1079 809

UV (CHCh) A tø = 260 nm (12474) 426 '43341 )

MS (ESt(+), TFA/MeCN) m/z (%) = 362 2 (100) [M + 2 H ⁺ J ²⁺ 241 7 (10) [M + 3 Hψ 723 5

(6; [MH ^÷ ] 837 4 (3 G) [VH ⁺ - TFA]

HRMS Calcd for C ₃₅ H ₅₈ N ₁₄ O ₃ 723 4895, Found 723 4915

Probe 2:

Compound 9 (43 mg, 59 μmoi) was dissolved in 1 mL of water and heated to 65 ⁰ C to get a clear yellow solution. Subsequently zinc(ll)-perchiorate (44 mg, 1 19 μmol) dissolved in 1 m[ of water was added slowly. The reaction mixture was stirred for an additional 28 h at 65 °C. The solvent was removed in vacuo and the residue was redissoived in water and lyophjiizεd. Probe 2 (74 mg, 59 μmol, 100 %) was obtained as a yellow solid.

MP; 166 ⁰ C

¹ H-NMR (300 MHz; CD ₃ CNI): δ (ppm) =1.19 (t, ³ J = 7.0 Hz, 6 H, ethyi-Ci-y, 2.45 (bs, 4 H ₁ cycien-CH ₂ ), 3.02 (bs, 8 H, cyclen-CH ₂ ), 3.25 (bs, 12 H, cyclen-CH ₂ ), 3.37 (bs, 2 H, ethylene diarnine-CH ₂ ), 3.49 (q, ³ J = 7.0 Hz, 6 H, ethyl-CHs, ethylene diamine-CH ₂ ), 3.71 - 4.50 (m. 8 H. cyclen-CH ₂ ), 5 35-6.65 (m, 6 H, cyclen-NH), 6.55 (s, 1 H, CH), 6.80 (d. ³ J = 9.1 H∑, 1 H. CH), 7.58 (d, ³ J = 9.1 Hz, 1 H ₁ CH), 8.72 (s, 1 H, CH) ₁ 9.23 (bs, 1 H, MH). iR (ATR) [cm ^'1 ]; ^ = 3246, 2938, 2S64, 1558, 1457, 1421. 1342, 1287 1083, 968, 793. UV (HEPES pH 7 4, 25 mM): λ (ε) = 432 (25177),

MS (ESI{+), TFA/MeCN): m/z (%) = 485.5 (100) [M ⁴⁺ + 2 CH ₃ COO ^' ] ^{2 +} , 455.7 (50) [M ⁴⁺ - H ^* + CH ₃ COO f\ 425 1 (28) [M' ⁺ - 2 hT] ^:+ .

Example 2: Dephosphorylation by λ- ^D Pase treatment Bovine α-caseιn (40 μg purchased from Sigma-Aidπch) was treated with 400 U of λ-PPase (purchased from New England Bioiabs) in Tπs-HCI (50 mM), NaC! (100 mM) dithiothreitol (2 mM), MnCS ₂ (2 mM) EGTA (0 1 mM), 0 01 % Bπj 35 pH 7 5 at 30 ⁰ C for 6 h

SDS-PAGE

Proteins were resolved on mini gels under denaturating and reducing Laemmii conditions on a PeqLab 45-1010-1 apparatus The gels consisted of a 4 % acrylamide (w/v), 120 mM Tπs-HCI {pH 6 S) 0 1 % SDS (w/v) stacking gel and a 15 % acry.amide (w/v) 375 mM Tπs- HCI (pH 8 8) 0 1 % SDS (w/v) running ge! A 25 mM Tris 1 S2 mM glycine, 0 1 % SDS (w/v) running buffer (pH 8 3) was used Protein samples were heated to 70 °C for 10 mm with ^r educing and denaiurafing RoisLoad 1 sample buffer (purchased from Carl Roth, Germany) before being loaded onto the ge! The geis were run a! 150 V until ihe proteins entered the running gel, then the voltage was increased to 250 V Water cooling was used duππg the enttre run Fixation was accomplished by treating the gels with 50 % MeOH / 10 % AcOH twice, for 30 mm and overnight, respectively

Staining and imaging

The gels were soaked in deionszed water (4 x 10 mm) before being treated with a solution of probe 1 or 2 in αeiomzed water for 1 h with a probe concentration of 10 ⁷ M We found destaining was not strictly necessary at this concentration, however when the probes were used at higher concentrations the gels could be αestamed by wasning with deionszed water until a nonfiuoresceπt background was obtained Due to ths'r roπ-covaSent binding mode 19) me probes could De completely -emoved by repeated washing of the gel with viaier Conveniently removal of the probes was not necessary for Coomasεse reεtaining

Tne gels were wrapped in cling film to prevent them from αrysng out and placed on a PeσLab Superbnght UV tabte (κ _& = 316 nm) Images were iaYer jsing eithe ^r a Pentax

K10D or a Traveler DC 8500 Emission spectra of individual protein banαs we ^r e obtained using the same UV table and a Hamamatsj PMA- 11 photonic multi-channel analyzer Data we ^r e acqi" ^rp d using the supplied ^D MΛ Ootic software £ ^ 55 "m lo^gpaεε f?ter A as p'aced on top of the gel to prevent the UV light saturating the αetector Longpaεs filte ^r s with a shorter cutoff proved unsuitable as tney showed a strong fluorescence when subjected to the UV light After fluorescence imaging, a restain for total protein was accomplished with 0.1 % Coomasεie R-250, 50 % MeOH ₁ 10 % AcOH for 1 h. Destaining was accomplished in 7 % AcOH, 10 % MeOH over night. The geis were again wrapped in ciiπg film and scanned using an office scanner.

Results and Discussion

Probes 1 and 2 were used for staining of phosphoproteins (Cαunteπons are not shown),

To evaluate the phospho-εtaimng selectivity and sensitivity of our probes, a dilution series of phcsphorylated bovine α-casein was elecfrophoretscaliy resolved from the non- phosphorylated protein BSA. In addition a sample of H-casem was dephosphorytated using λ-PPase and used as a controi to ensure the emission response would not depend on the ammo acid composition of the protein. After fixation, the gels were stained and destained when necessary until little or no background was visible.

Probe 1 showed a distinct emission in the bands of phosphoryiated α-casein, whereas the bands of dephosphorylated α-casein and BSA are barely visible (Figure 2). Bis-zinc(ll)- cycien tnazine compiexes coordinate phosphate groups strongly but we aiso expect an affinity of the probe to non-phosphorylated proteins due to the coordination of htstidine by the bιs-zιnc(il)-cyclen tπazine (17, 20) or further unεpecific interactions However, these interactions do not interfere with the specific detection of phosphorylation The emission of the probe is quenched when bound to non-phosphorylated amino acid residues and the emission remains, when bound to phosphoryiated ammo acsd residues Similar emission qjenchmg effects have been previously reported for the interaction of riboflavin with a zιnc(!!)-ιmιdazole complex (21) and for zsnc(ll)-porphyπn with histidme (22) To prove that the observed effects originate from the coordination of the bιs-zinc(li)-cyclen tπaziπe complex and not from the binding of the fluorophore itself, a control gel was prepared and treated with carboxyfluorescein No staining could be observed in this experiment

When bound to phosphoryiated σ-casem, probe 2 showed a strong redshift in the emission compared to unphoεphorylated α-caseιn and BSA (Figure 3) We attribute this spectral change to the different electronic environments when the probe rnofecule fs either unspecificafiy interacting with πon-phosphoryiated ammo acid residues such as histidme (unphoεphorySated σ-casem and BSA) or is coordinating a negatively charged phosphoryiated amtno acid residue (phosphoryiated o-casein) These findings are in agreement with the reported redεhsft in emission of a mono-zιnc(l!)-cyclen coumann comoiex upon coo ^r αιnatιoπ to inorganic phosphate ions (23) To quantify this change in emission fluorescence spectra of the gel bands were ootained using a photonic multichannel analyzer equipped with a fiber optic (Figure 4) As with probe 1 a control gel was treated with the fiuorophore itself and again no staining was observed

Wrt ^h both p ^r obes the dilution ser-es proved that 62 ng of phosphoryiated α~caseιn are still αetectaole on a noma! UV-tabte by the unaided eye (whch was protected f ^r om UV light) while imaging was performed with common digital cameras Hence even without the αse of speαaitzed equipment like 'aεer-illuminated ge! scanners or cooied camera detectors as desc ^p bed in the protocols of commεrc.ally available pbosphoprotein ge! stains our probes reach similar limits of defection

Conclusion

The non-covalent reversible and fluorescent SDS-PAGE probes used in the present examples are capaole of indicating protein ohosohorylanor The probes show oifferent fluorescence responses discriminating phosphorated frcm non-phosphorylated proteins While probe 1 signals binding to a phosphoryiated protein by a significant increase of emission intensity, probe 2 changes its emission spectrum upon binding to a phosphoryiated protein, The probes achieve their selectivity through a combination of the specificity of the diπuclear metal chelate binding site towards phosphate oxoanions and a modulation of the chromophore emission due to the proximity of the phosphoryiated amino acid. The environment-sensitive fluorophore ailows a clear distinction between phosphoryiated and non-phosphoryiated proteins on SDS-PAGE and allows the detection of 62 ng of phosphoryiated α-casein on a normal UV-tabie.

Example 3:

Use of other fluorophores

In order to underline the importance of the properties of the fluorophore in the design of probes 1 and 2, we elucidated the emission response of analogous probes 10 to 12 bearing dansyi, pyrene and Cy3 fiuorophores, respectively. The synthesis of compounds 10 to 12 was identical to 1 and 2, apart from attachment of the fiuorophore to precursor 3 which can be deduced by a person skilled in the art.

Synthesis

chloride changer

Synthesis of dansyl-ϊabeSed compound 10

In reaction (ϊ), the precursor 3 was reacted with dansyl chloride in DCM at room temperature. After removing the solvent under reduced pressure, treatment with TFA in DCM (1 :1) (ii) removed the Boc groups. Deprotonation by a basic ion exchanger (iii) was foliowed by compiexation with Zn(CIO ₄ ) ₂ in water at 80 ⁰ C (iv) The solvent was removed by lyophilization

t) pyreπe carboxyiic acsd / HOBt / T3TU / DEA ii) TFA ni) ion exchanger iv) Zn^ ⁺

Synthesis of pyrene-labeSed compound 11

in reaction (i), the precursor 3 was reacted with pyrene carboxyiic acid in DCM at room temperature using HOBt, TBTU and DiEA as coupling reagents. After removing the solvent under reduced pressure, treatment with TFA in DCM (1 :1 ) (ii) removed the Boc groups. Deprotonation by a basic ion exchanger (iii) was followed by compiexatton with Zn(CiO ₄ ) ₂ in water at 80 °C (iv). The solvent was removed by lyophilizatton,

0 Cy3-NHS ii) TFA in) ton exchanger

IV) Zn ²⁺

Synthesis of Cy3-Safae!ed compound 12

In reaction (i), the precursor 3 was reacted with CyS-NHS in DCM at room temperature. After removing the solvent under reduced pressure, treatment with TFA in DCM (1 :1 ) (ii) removed the Boc groups. Deprotonation by a basic ion exchanger (iii) was followed by compiexation with Zn(CIO4) ₂ in water at 80 ^D C (Iv). The solvent was removed by lyophilization.

Discussion

The labeled metai chelates 10 to 12 were tested for their fluorescence response when bound to phosphoryiated and nonphosphorylated protein as described in example 2. None of the compounds allowed a distinction between phosphoryiated and non-phosphoryiated proteins. The emission did not change either compared to the free probes in solution. These results indicate that the fluorophore is a significant component in the design of probes 1 and 2.

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