WO2013084198A1

WO2013084198A1 - Chemical modification and bioconjugation of proteins or peptides using boron compounds

Info

Publication number: WO2013084198A1
Application number: PCT/IB2012/057063
Authority: WO
Inventors: Pedro Miguel PIMENTA GÓIS; Pedro Miguel SARAIVA DUARTE CAL; João Filipe BOGALHO VICENTE
Original assignee: Universidade De Lisboa
Priority date: 2011-12-07
Filing date: 2012-12-07
Publication date: 2013-06-13

Abstract

The invention described herein provides a family of boron compounds for the chemical modification and bioconjugation of proteins and peptides. The method involves the formation of imines of boron reagents with the side chains of lysine residues and the N-terminal of proteins and peptides that are stable due to the coordination of the nitrogen with boron. The invention allows the ligation of boronic acids and esters to the protein, the protein glycosilation and pegylation or the ligation of fluorescent molecules. This method is reversible upon the addition of sugars, catechols and thiols. The present invention can be applied in both Pharmaceutical and Medical industries.

Description

DESCRIPTION

"CHEMICAL MODIFICATION AND BIOCONJUGATION OF PROTEINS OR

PEPTIDES USING BORON COMPOUNDS"

Field of the Invention

The invention described herein provides a method for the chemical modification and bioconj ugation of proteins and peptides via the formation of stable imines.

Summary of the Invention

The invention described herein provides a family of boron compounds for the chemical modification and bioconj ugation of proteins and peptides. The method involves the formation of imines of boron reagents with the side chains of lysine residues and the N-terminal of proteins and peptides that are stable due to the coordination of the nitrogen with boron. The invention allows the ligation of boronic acids and esters to the protein, the protein glycosilation and pegylation or the ligation of fluorescent molecules. This method is reversible upon the addition of sugars, catechols and thiols.

State of the art

1.1 - Protein's modification

Proteins are biomolecules that carry out the majority of cells' functions, acting as their structural blocks, as catalysts in all processes concerning cellular metabolism, and as regulators of cell cycle, differentiation, growth, division, motility, death, etc.. Many of those functions are dependent on post-translational modifications.¹ With the production of recombinant proteins and their constant improvement, these biomolecules have become the most valuable ones on earth (estimated $90+ billion U.S. market share in 2009) . Therefore, procedures that allow their manipulation or introduction of new functionalities are of great matter, having special interest when they allow the possibility of: improving pharmaceutical protein pharmokinetics , studying protein function and distribution, creating artificial post-translational modifications or constructing new biomolecule-based materials, such as drug- delivery systems.²

Protein modifications enable the modulation or the follow up of functions performed by these. In vitro or in vivo monitoring can be made either by fluorescent or affinity tagging. This characteristic is something that, otherwise, would be hard to obtain. Therapeutical or signaling protein conjugates have become very promising for better knowing different pathologies such as: HIV,^3,4 cancer, ^3,5,6 malaria,^3,7 neurodegenerative diseases like Alzheimer's disease,⁷ acromegaly⁵ or Angina Pectoris .⁴ The most ambitious perspective of this project is creating biomarkers relevant for developing new therapeutic molecules or systems.

1.2 - Selective Protein Modification

The mainstream of bioconj ugation reactions rely on amino acids' properties, whether for their reactivity, as much as for their occurrence in proteins or their accessible surface area. Taking this information into account, one can name nine of the canonical amino acids to be chemically active regarding their side chains: acidic residues (aspartic acid and glutamic acid) , aromatic amino acids (tyrosine and tryptophan) , sulfur containing amino acids (methionine and cysteine) and nitrogen containing amino acids (histidine, lysine or arginine, as well as N- terminal function).¹ However the main characteristic that ease amino acids' modification is basicity. For that reason, cysteine⁸ and lysine are the most commonly modified amino acids.¹ Lysine is an amino acid with a primary amine on its side chain which gives it one of the strongest nucleophilic functions of a protein. This amine can be distinguished from other amines on a protein' s surface through its pKa . Associated with this characteristic, one can mention its general disposition on a protein - exposed on their surface - available for modification when unprotonated .

Throughout the years, several modifications have been reported in the literature regarding the chemical modification of lysines. Among them, it is possible to select four molecules as being the representative examples of such procedures: NHS (N-hydroxysuccimide) esters, anhydrides, isocyanates and aldehydes (with subsequent reduction) .

NHS esters are among the most used molecules for protein bioconj ugation . These reactive esters form an amide bond in the presence of unprotonated amine, which is obtained when an aqueous environment possesses a pH of 8.5 ± 0.5. It is possible to mention quite a few number of different applications using this molecule, such as: pegylation, ^9-11 making it possible to pegylate important biomolecules like hormones (e.g. insulin which provides an orally bioavailable form¹²) ; cross-coupling of different biomolecules; cell imaging and fluorescence; ' vaccine formulation; antibodies modification ' and other important studies.^19,20

Anhydrides have several examples that allow such procedure, namely the ones reported in a work by Freeman et al . In this work, the protein modification led to changes in the crystals' porosity, that consequently made significant differences in terms of solubility or crystals shape.²¹ Be that as it may, the most used anhydrides, for this purpose, are the maleic and citraconic anhydrides, enabling authors, for instance, to study changes in enzymatic activities.^22-26 Isocyanates or isothiocyanates form an urea or thiourea bond, respectively. This functionality behaves like NHS esters and is a fairly more stable species than these. However, it needs a more basic medium to react with amines (pH 9.0 to 9.5) . A large number of studies were performed m order to justify isocyanates' toxicity, but some recent studies take advantage of this selective reaction to synthesize biodegradable polymers³¹ and to introduce fluorochromes .

Aldehydes react with amines under mild aqueous conditions to form imine intermediates, but these compounds are not stable to hydrolysis and so, these are reduced with mild or strong reducing agents such as sodium cyanoborohydride³³ or under iridium catalyzed transfer hydrogenation . In terms of application, one can state a study made by Alfred et al that allows an important biological function to happen artificially - glycosylation . Through this method, it's possible to couple a protein to a carbohydrate forming a glycoprotein, a functionalization of the utmost relevance in biology.³⁵

Due to the importance of boronic acids and derivatives, over the past five years, several attempts were made in order to incorporate these molecules in proteins. This functionality with different oxidation states is most useful for biological applications because it's abiotic, inter-convertible between the sp2 and sp3 forms, has a strong interaction with diol-containing molecules (such as carbohydrates) and an interesting Lewis acidity, as well as having a unique behaviour upon neutron bombardment.³⁶

In 2002, through a maleimide reaction it was possible to introduce a fluorescent tag containing BODIPY (boron- dipyrromethene) into a protein, due to the high affinity of this molecule with cysteine.³⁷ Several years later, different studies were made to fruitfully incorporate similar dyes into a protein by translational modification .38-40 Nevertheless, these derivatives didn't confer many of the advantages that boronic acids could, so, in 2008, Schultz et al were able to successfully encode a boronate-containmg acid, 41 ' 42 as well as, two years later,

4 ^

doing the same m Mycobacterium tuberculosis .

Another approach uses a semi-synthetic procedure in order to incorporate a boronic acid in the N-terminal function of peptides. Like so, Santos et al was able to find selective inhibitors of human ClpXP - an ATP-dependent serine protease present in the mitochondrial matrix.⁴⁴ It is also mentioned in prior art, the use of phenylboronic acid complexes for bioconjugate preparation.⁴⁵

This molecule allows a very selective reaction to occur, at mild conditions - Suzuki Miyaura Cross- Coupling - and many experiments were made to perform it either in peptides^46,47 or in proteins.⁴⁸ However, this reaction wasn't done with incorporated boronic acid, but by means of incorporated arylhalides (which then reacted with external boronic acids) .

DESCRIPTION OF THE INVENTION

The invention described herein provides a method for modification of the lysine residue side chain and N- terminal of proteins and peptides based on the formation of stable imines in aqueous media and buffers with boron compounds of the general formula ( I ) shown in Fig. 1. In accordance with the present invention, there is therefore a compound of the formula (I) :

wherein,

R¹ and R² represents independently in compound of the general formula (I) : OH, F, C₁-C6 alkyl which optionally may incorporate one further heteroatom selected from nitrogen, oxygen and sulphur, formyl or C₂-C6 alkanoyl, OC¾Ar or OCIH^CHsAr in which the Ar group may be a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring or fused ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur; or the group R^XB R² together represents a 5 to 7 ring optionally incorporating one or more heteroatoms selected from oxygen, nitrogen or sulphur.

R³ and R⁴ represents independently in compound of the general formula (I) : H, C¾, C₁-C6 alkyl which optionally may incorporate one further heteroatom selected from nitrogen, oxygen and sulphur, C₂-C6 alkanoyl, C¾Ar or CH₂CH₃Ar in which the Ar group may be a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring or fused ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur; or the group R³-C=C-R⁴ together represents a 5 to 7 ring optionally incorporating one or more heteroatoms selected from oxygen, nitrogen or sulphur, an aromatic ring optionally substituted with fluorescent groups, sugars and polyethylene glycol chains.

R⁵ represents in compound of the general formula (I) : C¾, C₁-C6 alkyl which optionally may incorporate one further heteroatom selected from nitrogen, oxygen and sulphur, C₂ ^_ C6 alkanoyl, CH₂Ar or CH₂CH₃Ar in which the Ar group may be a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring or fused ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur, a 4 to 7 ring optionally incorporating one or more heteroatoms selected from oxygen, nitrogen or sulphur, an aromatic ring optionally substituted with fluorescent groups, sugars and polyethylene glycol chains.

The aforementioned boron compounds of the general formula (I) , in contact with the lysine residue and N-terminal of proteins and peptides generates imines due to stabilization induced by the coordination of the nitrogen with the boron atom either in water, aqueous media or in the presence of buffers such as KPi (K₂HPO₄/KHPO₄ ; pHs 6 up to 9) as shown in fig . 2.

The following examples in which butylamine is used as a model primary alkylic imine and 2-formylphenylboronic acid as an example of a boron compound of the general formula (I), highlight some of the conditions which improve the imine formation. Considering the influence of pH (Table 1), the optimum level of imine formation is obtained between pH 6 and 9 using KPi (50mM) as buffer in concentrations ranging from 25% to 100% (Table 2) .

Table 1. Influence of pH on the generation of imines between alkylic primary imines and boron compounds of the general formula (I); Y = yield. pH Y

6, 01 45%

7,05 37%

8, 02 52%

9, 17 48% Table 2. Influence of buffer dilution on the generation of imines between alkylic primary imines and boron compounds of the general formula (I); S = solvent; Y = yield.

The reaction may be carried out in neat or deuterated water and buffers such as KPi but not exclusively (Table 3) , in a reagents concentration range between 5 and 166 mM (Table 4) .

Table 3. Influence of solvent in the generation of imines between alkylic primary imines and boron compounds of the general formula (I); S = solvent; Y = yield

Table 4. Influence of reagents concentration on the generation of imines between alkylic primary imines and boron compounds of the general formula (I); C = concentration; Y = yield

c Y

5mM 13%

50mM 25% lOOmM 73%

166mM 58%

As shown in table 5, the presence of substituents can alter the reactivity of the boron compound of the general formula (I), which can be due either to a deficient solubility of the compound in water and buffer solutions or to electronic an effect which inhibits the imine formation or favours its hydrolysis (Table 5) .

Table Influence of the boron compound structure on the generation of imines between alkylic primary imines and boron compounds of the general formula (I); BA = Boronic Acid; Y = yield

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. General formula of boron compounds for the modification of lysine residues and N-terminal of proteins and peptides.

Figure 2. General concept of the formation of stable alkylic imines in water, aqueous media or in the presence of buffers with boron reagents of the general formula (I) .

Figure 3. General reaction with 2-Formylbenzeneboronic acid and butylamine (ε-amine model for lateral chain of lysine) .

Figure 4. Stability of alkylic imines formed between primary amines and boron compounds of the general formula (I) .

Figure 5. General reaction with 2 -Acetylbenzeneboronic acid and butylamine (ε-amine model for lateral chain of lysine) .

Figure 6. Influence of pH on the generation of imines between alkylic primary imines and boron compounds of the general formula (I) where R5 represents an alkylic group.

Figure 7. Kinetics of the imine formation between alkylic primary imines and boron compounds of the general formula (I) where R5 represents an alkylic group and stability towards hydrolysis.

Figure 8. General reaction with several boronic acids and Boc-Lys-0CH₃ DETAILED DESCRIPTION OF THE INVENTION

According to a general reaction between 2- formylbenzeneboronic acid and a model for mimicking the lateral chain of lysine (with a similar primary amine) butylamine - several experiments were performed in order to scope the reaction's conditions (Figure 3) .

1 - Influence of pH

2-Formylbenzeneboronic acid (50 mg, 0.33 mmol) was added to a lOmL round bottom flask and then dissolved in 2mL of KPi buffer (50mM) at different pH's. Afterwards, 1 equivalent of 1-butylamine (33yL) was added to the same flask and these compounds reacted for 18 hours at 25°C. At that time, one drop of reaction mixture was taken from the media and dissolved in D₂O in order to perform a ¹H-NMR and evaluate the reaction's yield (Table 1) .

2 - Influence of buffer dilution

2-Formylbenzeneboronic acid (50 mg, 0.33 mmol) was added to a lOmL round bottom flask and then dissolved in 2mL of solvent (KPi buffer (50mM) pH 8.02 mixed with water at different percentages) . Afterwards, 1 equivalent of 1- butylamine (33yL) was added to the same flask and these compounds reacted for 18 hours at 25°C. At that time, one drop of reaction mixture was taken from the media and dissolved in D₂O in order to perform a ¹H-NMR and evaluate the reaction's yield (Table 2) .

3 - Influence of Solvent

2-Formylbenzeneboronic acid (50 mg, 0.33 mmol) was added to a lOmL round bottom flask and then dissolved in 2mL of solvent. Afterwards 1 equivalent of 1-butylamine (33yL) was added to the same flask and these compounds reacted for 18 hours at 25°C. At that time, one drop of reaction mixture was taken from the media and dissolved in D₂0 in order to perform a ¹H-NMR and evaluate the reaction's yield (Table 3) .

¹R NMR (400 MHz, D₂0) δ 8.47 (s, 1H, -CHCHNHCH₂- ) , 7.55 (d, J = 7.5 Hz, 1H, Ar) , 7.51 - 7.45 (m, 2H, Ar) , 7.25 (d, J = 8.9 Hz, 1H, Ar) , 3.62 (t, J = 7.3 Hz, 1H, -NHCH2CH2 - ) , 1.84 - 1.66 (m, 2H, - CH2CH2CH2CH₃ ) , 1.42 - 1.22 (m, mixture, - CH2CH2CH2CH3 ) , 0.98 - 0.75 (m, mixture, - CH2CH2CH2CH3 ) .

For the reaction in dichloromethane, the solvent was evaporated and the ¹H-NMR performed in CDCI₃. Due to a quantitative reaction it was possible to characterize the product. ¹R NMR (400 MHz, CDC1₃) δ 8.52 (s, 1H, -CHCHNHCH₂- ), 7.73 (d, J = 7.1 Hz, 1H, Ar) , 7.62 (d, J = 7.5 Hz, 1H, Ar) , 7.44 (t, J = 7.0 Hz, 1H, Ar) , 7.37 - 7.21 (m, 1H, Ar) , 3.73 (t, J = 7.3 Hz, 2H, -NHCH2CH2 - ) , 1.87 - 1.70 (m, 2H, - CH2CH2CH2CH₃ ) , 1.50 - 1.32 (m, 2H, - CH2CH2CH2CH₃ ) , 0.99 - 0.81 (m, 3H, - CH2CH2CH2CH₃ ) .

4 - Influence of reagents concentration

5mM Concentration: 2-Formylbenzeneboronic acid (1.52 mg, 1,01 x 10^~5 mol) was added to a lOmL round bottom flask and then dissolved in lmL of solvent (25% of KPi buffer (50mM) pH 8.02 mixed with deuterated water) . Afterwards 1 equivalent of 1-butylamine (lyL) was added to the same flask and these compounds reacted for 18 hours at 25°C. At that time, 0.5mL of reaction mixture was taken from the media and performed a ¹H-NMR to evaluate the reaction' s yield (Table 4) .

50mM Concentration: 2-Formylbenzeneboronic acid (7.6 mg, 5.06 x 10^~5 mol) was added to a lOmL round bottom flask and then dissolved in lmL of solvent (25% of KPi buffer (50mM) pH 8.02 mixed with deuterated water) . Afterwards 1 equivalent of 1-butylamine (5yL) was added to the same flask and these compounds reacted for 18 hours at 25°C. At that time, 0.5mL of reaction mixture was taken from the media and performed a ¹H-NMR to evaluate the reaction' s yield .

lOOmM Concentration: 2-Formylbenzeneboronic acid (15.2 mg, 0.101 mmol) was added to a lOmL round bottom flask and then dissolved in 2mL of solvent (25% of KPi buffer (50mM) pH 8.02 mixed with deuterated water) . Afterwards 1 equivalent of 1-butylamine (10yL) was added to the same flask and these compounds reacted for 18 hours at 25°C. At that time, 0.5mL of reaction mixture was taken from the media and performed a ¹H-NMR to evaluate the reaction's yield.

166mM Concentration: 2-Formylbenzeneboronic acid (50 mg, 0.33 mmol) was added to a lOmL round bottom flask and then dissolved in 2mL of solvent (25% of KPi buffer (50mM) pH 8.02 mixed with water) . Afterwards 1 equivalent of 1- butylamine (33yL) was added to the same flask and these compounds reacted for 18 hours at 25°C. At that time, one drop of reaction mixture was taken from the media and dissolved in D₂O in order to perform a ¹H-NMR to evaluate the reaction's yield.

5 - Influence of boronic acid substitution

The boronic acid (0.33 mmol) was added to a lOmL round bottom flask and then dissolved in 2mL of KPi buffer (50mM) pH 8.02. Afterwards, 1 equivalent of 1-butylamine (33yL) was added to the same flask and these compounds reacted for 18 hours at 25°C. At that time, one drop of reaction mixture was taken from the media and dissolved in D₂O in order to perform a ¹H-NMR and evaluate the reaction' s yield (Table 5) .

6 - Imine formation and stability monitored by NMR

Regarding the imine formed between a model primary alkylic imine and 2-formylphenylboronic acid as an example of a boron compound of the general formula (I), they were shown to be quite stable as they endure 24h without noticeable hydrolysis (Table 6 and Figure 4) .

Table 6. Stability of alkylic imines formed between primary amines and boron compounds of the general formula (I); t = time (h = hours) ; A = 2-formylbenzeneboronic acid; B = 3- fluoro-2-formylbenzeneboronic acid; C = 2 , 3-methylenodioxy- 2-formylbenzeneboronic acid.

The boronic acid (0.33 mmol) was added to a lOmL round bottom flask and then dissolved in 2mL of dichloromethane . Afterwards, 1 equivalent of 1-butylamine (33yL) was added to the same flask and these compounds reacted for 16 hours at 25°C. At that time, the solvent was evaporated and a ¹H- NMR was performed in CDCI₃ determining a quantitative yield. After isolating the product a study of dissolution and stability was performed. The imine (6.67 x 10^~5 mol) was dissolved in 0,4mL of D₂0 and a ¹H-NMR was performed at determined times.

As an example of the positive impact of the R5 group in the reactivity of the boron compound of the general formula (I) in the generation of stable alkylic imines in the presence of water, the 2-acetylphenylboronic acid yielded the imine in up to 88% (table 7) whereas the 2 -formylphenylboronic acid in the same conditions yielded the imine in just 49% (table 3) . Using the 2-acetylphenylboronic acid as a model example of boron compounds of the general formula (I) substituted in R5 with and alkylic group, an optimum reaction was obtained at pH levels of 7 up to 9 (Table 8), at pH 8.2 using KPi (50mM) as buffer, the imine was obtained in 81% after just 20-min reaction at room temperature (Table 9) and the formed imine was shown to be stable for more than 23 hours in the presence of water (Table 9) .

Table 7. Influence of group R5 on the generation of imines between alkylic primary imines and boron compounds of the general formula (I); V = Variable; Y = yield

Table 8. Influence of pH on the generation of imines between alkylic primary imines and boron compounds of the general formula (I) where R5 represents an alkylic group; d = day(s) pH 1 d 7 d

6.01 67% 67%

7.05 79% 80%

8.02 84% 88%

9.17 87% 87% Table 9. Kinetics of the imine formation between alkylic primary imines and boron compounds of the general formula (I) where R5 represents an alkylic group and stability towards hydrolysis; t = time (min = minutes); Y = yield

An important aspect of the chemical modification and bioconj ugation of proteins and peptides is the reversibility of the modification which opens the possibility of designing delivery systems based on this property. The imine formed with butylamine which is used as a model primary alkylic imine and 2 -acetylphenylboronic acid as an example of a boron compound of the general formula (I) was shown to be reversible by the addition of sugars (e.g. fructose), thiols (e.g. glutathione) and catechols (e.g. dopamine) (Table 10).

Table 10. Reversibility studies of imines formed between alkylic primary imines and boron compounds of the general formula (I); t = time (min = minutes); A = Glucose; B = Fructose; C = Glutathione; D = Lactose; E = Sucrose; F = Adenine; G = Cytosine; H = Thymine; I = Ephedrine; J = Dopamine t

A B C D E F G H I J

(min)

0 94 87 81 87 89 82 93 90 86 86

1 - - 21 84 87 80 91 89 86 51

30 - - 16 84 87 82 91 85 84 50

45 82 44 - - - - - - - -

60 - - 11 84 87 82- 91 86 84 24

105 81 41 - - - - - - - -

120 - - 8 84 87 81 91 86 84 22

150 82 44 - - - - - - - -

480 81 42 - - - - - - - -

1440 81 44 7 84 87 81 91 86 84 21

According to a general reaction between 2- Acetylbenzeneboronic acid and a model for mimicking the lateral chain of lysine (with a similar primary amine) butylamine - several experiments were performed in order to scope the reaction's conditions (Figure 5) .

1 - General Reactions

2-Acetylbenzeneboronic acid (in variable amounts) was added to a lOmL round bottom flask and then dissolved in 2mL of solvent. Afterwards, 1-butylamine (33yL, 0.33 mmol) was added to the same flask and these compounds reacted for different times at 25°C. At those pre-determined times, one drop of reaction mixture was taken from the media and dissolved in D₂0 in order to perform a ¹H-NMR and evaluate the reaction's yield (Table 7) .

2 - Influence of pH

2-Acetylbenzeneboronic acid (55 mg, 0.33 mmol) was added to a lOmL round bottom flask and then dissolved in 2mL of KPi buffer (50mM) at different pH's. Afterwards, 1 equivalent of 1-butylamine (33yL) was added to the same flask and these compounds reacted for 16 hours at 25°C. At that time, one drop of reaction mixture was taken from the media and dissolved in D₂0 to perform a ¹H-NMR and evaluate the reaction's yield (Table 8 and Figure 6) .

3 - Influence of Time - NMR Study

2-Acetylbenzeneboronic acid (11 mg, 6.66 x 10^~5 mol) was added to a NMR tube and then dissolved in 0.4mL of D₂O. Afterwards, at room temperature, 1 equivalent of 1- butylamine (7yL) was added to the same flask and several ¹H-NMR were performed to study the reaction' s kinetics (Table 9 and Figure 7) .

4 - Reversibility with Endogenous Molecules

2-Acetylbenzeneboronic acid (8 mg, 5.0 x 10^~5mol) was added to an eppendorf tube and then dissolved in 0.3mL of D₂0. Afterwards, 1 equivalent of 1-butylamine (5yL) was added to the same tube and these compounds reacted for 16h at 25°C. Subsequently, a ¹H-NMR was performed to evaluate the conjugation rate. Then, 0.1 mL of D₂0 containing leq of different molecules were added to the tube. Several ¹H-NMR were performed from that moment on, in order to study the reaction's reversibility (Table 10).

The chemical modification and bioconj ugation of proteins and peptides using boron compound of the general formula (I) requires the formation of the imine with the lysine residue side chain or the N-terminal. Therefore the imine was shown to be easily formed with the protected lysine amino acid Boc-Lys-OCH₃ in yields up to 71% (Figure 8) and, more importantly, the modification of a model protein such as lyzosyme, was accomplished using 2 -formylphenylboronic acid and 2-acetylphenylboronic acid as examples of boron compounds of the general formula (I) . The modification was determined based on ESI mass spectrometry and up to 6 modifications were detected. The lyzosyme reacted with 2- formylphenylboronic acid and 2-acetylphenylboronic acid as model compounds of the general formula (I) in water and buffer acetate solutions at various pHs . In addition to this, the reversibility of the modification was demonstrated by addition of glutathione, dopamine and fructose. After which, the bioconj ugates formed between lyzozime and 2-formylphenylboronic acid were destroyed and an increase of the free protein was detected.

5 - Reactions with Boc-Lys-OCH₃

Boc-Lys-OCH₃ (40mg, 0.15 mmol) was added to an eppendorf and dissolved in 0.92mL of D₂0. Afterwards, leq of each boronic acid was left to react for 16 hours at 25°C. At the end, a ¹H-NMR was performed and the yield determined (Table 11) .

Table 11. Imines formed between Boc-Lys-OCH₃ and boron compounds of the general formula (I); BA = Boronic Acid; Y = yield.

6 - Reactions with Lyzosyme

2-Formylbenzeneboronic and 2-Acetylbenzeneboronic acids (1 and lOmM) reacted with ΙΟμΜ of Lyzosyme in water and in 20mM of acetate buffer (at various pHs) at room temperature (Figure 9) . After 10 minutes of reaction the solutions were evaluated by performing an ESI-MS and the conjugated species were detected, up to 6 modifications.

To study reversibility of the link formed, equal quantities of reduced glutathione, dopamine hydrochloride and L- fructose were added to the modified lyzosyme and the results were evaluated through ESI-MS, detecting an increase of unmodified lysosyme, thus proving its reversibility .

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Claims

1. Method for the modification and bioconj ugation of proteins and peptides characterized in that it comprises the formation of an imine by the contact of the protein or peptide with a boron reagent of the general formula (I), a compound of the formula (I), wherein :

R¹ and R² represents independently OH, F, Ci^~C6 alkyl which optionally may incorporate one further heteroatom selected from nitrogen, oxygen and sulphur, formyl or C₂-C6 alkanoyl, OCH₂Ar or OCIH^CHsAr in which the Ar group may be a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring or fused ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur; or the group R^XB R² together represents a 5 to 7 ring optionally incorporating one or more heteroatoms selected from oxygen, nitrogen or sulphur; R³ and R⁴ represents independently H, CH₃, C1-C6 alkyl which optionally may incorporate one further heteroatom selected from nitrogen, oxygen and sulphur, C₂-C6 alkanoyl, CH₂Ar or CH₂C¾Ar in which the Ar group may be a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring or fused ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur; or the group R³-C=C-R⁴ together represents a 5 to 7 ring optionally incorporating one or more heteroatoms selected from oxygen, nitrogen or sulphur, an aromatic ring optionally substituted with fluorescent groups, sugars and polyethylene glycol chains; R⁵ represents CH₃, C₁-C6 alkyl which optionally may incorporate one further heteroatom selected from nitrogen, oxygen and sulphur, C₂-C6 alkanoyl, CH₂Ar or CH₂CH₃Ar in which the Ar group may be a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring or fused ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur, a 4 to 7 ring optionally incorporating one or more heteroatoms selected from oxygen, nitrogen or sulphur, an aromatic ring optionally substituted with fluorescent groups, sugars and polyethylene glycol chains; and in that the aforementioned boron compounds of the general formula (I) , in contact with the lysine residue and N-terminal of proteins and peptides generates imines due to stabilization induced by the coordination of the nitrogen with the boron atom either in water, aqueous media or in the presence of buffers.

Method according to claim 1 characterized in that the imine is formed with one or more lysine residues at the surface of the protein or the N-terminal.

Method according to claims 1 and 2 characterized in that the imine formation takes place in a solution or in suspension in a polar protic solvent.

Method according to claims 1 and 2 characterized in that the imine formation takes place in the presence of potassium carbonate or phosphate buffers, preferably KPi (50mM) as buffer in concentrations ranging from 25% to 100%, with pH values ranging from 6 up to 9.

5. Method according to claims 1 and 2 characterized in that it comprises a subsequent step of reduction of the imine to an amine.

6. Method according to claims 1 and 2 characterized in that the boron reagent of the general formula (I) is to produce a protein or peptide with sugars, polyethylene glycol chains and fluorescent groups attached .

7. Method for the hydrolysis of the imine according to claims 1 and 2 characterized in that it is used upon the addition of sugars, thiols and catechols.