WO2003093346A1

WO2003093346A1 - Multifunctional polyethylene glycol derivatives: preparation and use

Info

Publication number: WO2003093346A1
Application number: PCT/EP2003/004620
Authority: WO
Inventors: Gian Maria Bonora; Sara Drioli; Maurizio Ballico
Original assignee: Universita' Degli Studi Di Trieste
Priority date: 2002-05-06
Filing date: 2003-05-02
Publication date: 2003-11-13
Also published as: ITMI20020951A0; AU2003229769A1; ITMI20020951A1

Abstract

The present invention refers to complex-structured multifunctional polyethylene glycol derivatives (multiPEG) obtained by assembling single PEG units with a defined molecular weight through a polyfunctional linker. The assembling of single ,PEG units gives polyfunctional multimeric systems characterized by a high molecular weight, low polydispersity and a binding capacity higher than that of a commercial PEG of equal size. Said multiPEG systems may be used as carriers and stabilisers of pharmacologically or biologically active substances as well as of reagents and catalysts for liquid-phase synthesis reactions and as soluble support in synthetic processes.

Description

MULTIFUNCTIONAL POLYETHYLENE GLYCOL DERIVATIVES: PREPARATION AND USE

Field of the invention

The present invention refers to multifunctional polyethylene glycol derivatives (multiPEG), their preparation obtained by assembling PEG units through a polyfunctional linker, and their use. Prior art

Polyethylene glycol (PEG) and the polymeric materials based thereon are arousing great interest in view of their application in the biotechnological and pharmacological' fields [Poly(ethylene glyόol) Chemistry. Biotechnical and Biomedical Applications, J. Milton Harris Ed., Plenum Press, New York, 1992). The major example of said application is the PEGylation of peptides and proteins (F.M. Veronese, Biomaterials, 22, 405-417, 2001). This technology improves the therapeutic efficacy since the molecule attached to PEG, though maintaining its original biological functions, has an increased in vivo stability to degradation processes., In fact the presence of said PEG chains, especially with a high molecular weight, masks the protein surface and prevents the attack from the antibodies or cells involved to their destruction. Furthermore, the increased molecular size reduces kidney ultrafiltration: therefore, it maintains the PEG- conjugated active molecules circulating in the body for a longer time and advantageously modifies their pharmacokinetic properties. Finally, PEG gives its own physicochemical properties to the molecules attached thereto, thus improving their bioavailability and solubility and, in general, facilitating their administration (M.L. Nucci, R. Shorr e A. Abuchowski, Adv. Drug Delivery Rev., 6, 133-151, 1991).

Recent investigations also concerned the PEGylation of oligonucleotide chains capable of behaving as antisense and antigen (G.M. Bonora et al., Farmaco, 53, 634-637, 1998) or ribozymes for the specific hydrolysis of RNA chains (L Gold, J. Biol. Chem., 270, 13581-13584, 1995). In this case, the scarce cell uptake generally exhibited by the charged polar chains of oligonucleotides may be improved by the amphiphilic character of the PEG chains. Furthermore, an increased stability to enzymatic degradation (G.M. Bonora et al. Bioconjugate Chem., 8, 793-797, 1997) and a longer retention time in the blood circulatory system - with an effect that is the higher, the higher is the polymer molecular weight - have been observed. ;

In addition to the above examples, numerous small-sized molecules in the form of PEG-conjugates have been used for the purpose of improving the characteristics connected with the pharmacological administration of same. Out of them it is worth mentioning anticancer agents, such as doxorubicin and taxol, antimalarial drugs such as artemisine, and various enzymatic inhibitors.

Anyway, it is to be stressed that PEG, being a synthetic polymer, is polydisperse and, even in the best cases, the polydispersity value (M_w/M_n) passes from 1.01 in low-molecular-weight samples (3 kDa) to more than 1.2 in the high-molecular- weight ones (>10 kDa). Said polydispersity is a negative feature, which reflects in the conjugate and in its final characteristics. In addition to the above uses of PEG in the modification of biologically active molecules, a further basic feature is its use in the so-called "liquid phase synthesis", or synthesis in solution, supported on soluble polymers. Said method is somehow complementary to Merrifield's synthesis on a solid support, except that - like traditional syntheses - it is carried out in solution. It follows that no problems arise due to the reaction environment heterogeneity. The system may be applied to the support of reagents, to their removal from the solution and, obviously, to the transient support of the molecule in the assembling step.

In general, the polymers used to this end must satisfy some conditions, e.g. commercial availability, good chemical and mechanical properties, presence of appropriate functional groups for the organic molecules attachment, as well as the required solubilising properties. As already mentioned, said polymers should have an as narrow as possible range of molecular weights to avoid the occurrence of different properties with size variation. Obviously, the polymeric support must not interfere with the envisaged reaction conditions; therefore, it usually consists of hydrocarbon- or ether-type chains. In general, the functionalising capacity of the selected polymer - measured as the number of reactive sites/g polymer - must be as high as possible, compatibly with the solubilising capacity. Usually, these two characteristics are mutually exclusive and, therefore, a right compromise between a good solubility and an economically advantageous synthesis is to be reached. Commercial PEGs exhibit most of said characteristics, excepting a reduced size homogeneity and a limited functionalϊsing capacity, obviously limited to the two polymer chain ends only. The major fields of application connected with the use of PEG are the synthesis of peptides (E. Bayer and M. Mutter, Nature, 237, 512-513, 1972), oligonucleotides (G.M. Bonora et al., Nucleic Acids Res., 21, 1213-1218, 1993) and oljgosaccharides (G. Nodosi and J.J. Krepinsky, Synlett, 159, 1996). In addition to the synthesis of the aforementioned biopolymers, an ever increasing number of organic syntheses using PEG as an inert soluble support, was and is proposed, especially for combinatorial chemical processes (C.-M. Sun, Combinat. Chem. & High Throughput Screening, 2, 299-315, 1999). Furthermore, PEG may be also used as a supporting agent with specific catalysts and reagents, whose conjugation allows ease of recovery and recycling of the reagent (P. Wentworth Jr. andK. D. Janda, Chem. Commun., 1917-1924, 1999). The soluble polymeric support most widely used so far for the aforesaid purposes is monomethoxy PEG, generally with a m.w. of 5 kDa. Since, as already said, its linear topology gives only one functionalisable reactive group, its capacity is rather limited, i.e. 0.2 mmole/g polymer. With a view to increasing said capacity, which limits the use of PEG, a linear bifunctional PEG, modified by benzylether dendrons, was proposed (M. Benaglia et al, J.Org.Chem., 63, 8628-8629, 1998). A further approach proposed the so-called PEG-star polymers, based on a polyphosphazenic or polyglyceric nucleus where several PEG chains were introduced (N.N. Reed and K.D. Janda, Org. Lett., 2, 1311-1313, 2000). The advantage of said polyethers lies in their high chemical stability and in the good reactivity of the functional groups in the homogeneous phase, with a capacity 4 to

6 times higher than that of a commercial PEG of equal size. Alternatively, soluble

^' linear polymers bearing a functional group per monomeric unit of the polymer chain, e.g. polyvinyl alcohol (PVA), pojyacrylic acid, and polyacrylamide, have been used. However, PVA is little soluble in an organic solvent and its functionalisation requires rather severe conditions (K.E. Geckeler, Adv.Polym.Sci., 121, 31-79, 1995). Polyacrylamide in the soluble non-cross-linked form has been used in the organic synthesis; however, its use is limited by its low chemical stability especially in reactions with strong bases or organometallic compounds: The so-called ROMP polymers, i.e. obtained by ring-opening , metathesis polymerization, have been recently introduced as supporting polymers. They have a high capacity (approx. 3.3 mmole/g), but exhibit a gel-type behaviour - which places them at the border between the homogeneous and the heterogeneous phase (A.G.M. Barret et al., Organic Lett., 1, 579-582, 1999) - besides exhibiting a heat- and oxygen-sensitive double bond.

The disadvantages of high-capacity linear polymers, such as the low solubility, gel formation, and some particular thermal properties, may be overcome by the creation of branched polymers. The so-called polymeric dendrimers, also commercial type (Astramol, DSM and Starbust, Dendritec), are soluble in many organic solvents and exhibit a large number of functionalisable groups. However, they are rather expensive and less chemically stable than polyethers. Alternatively, it is possible to use the so-called hyperbranched polymers having an intermediate structure between that of regular dendrimers and of linear polymers. They too are commercially available, i.e. under the trademark PEI® from Basf, polyesters under the trademark Boltόrn® from Perstorp and polyesteramides under the trademark Hybrane® from DSM. They generally have a low molecular weight and a very wide and little defined molecular weight distribution (>2); furthermore, their chemical ""stability is limited by the presence in the skeleton of ester and amidic bonds.

A very promising class of high-capacity soluble polymeric supports consists of aliphatic dendritic polyethers and of hyperbranched polyethers-polyols. They were recently proposed using 1,2 and 1,3-diol end groups, and were obtained, by convergent synthesis, from the condensation of propylene glycol and methallyl dichloride growing units, or by divergent synthesis starting from a multifunctional core represented by glycerol which binds glycidol growing units (M. Jayaraman and J.M.J. Frechet, J. Am.Chem.Soc, 120, 12996-12997, 1998; R.Haag et al., J.Am.Chem.Soc, 122, 2954-2955, 2000). -All advantageous physicochemical properties - analogous to PEG's - are retained and the capacity has increased up to 14 mmole/g. However, the multistage preparation is very hard-working , especially if the desired product must have a high molecular weight, i.e. higher than 1.5 kDa, which is the lower limit for each dialysis or ultrafiltration operation often required in intermediate purification processes during the synthetic application of said supports. Recently, A. Sunder et al. (Macromolecules, 39, 253- 254, 2000) proposed an advantageous process for the synthesis of hyperbranched polyethers-polyols, wherein the molecular weights may be as high as 30 kDa with a distribution below 1.5, through a single-stage anionic polymerisation on a multigram scale. In any case, as concerns said multibranched polymeric supports, it is always necessary to find an optimal compromise among solubility, accessibility, and capacity of binding pharmacologically or biologically active molecules as well as reagents and catalysts for the liquid phase synthesis to the functional groups: Although, as already said, thanks to its physicochemical properties, PEG may be an interesting polymer for binding organic molecules, the high-molecular-weight polyethylene glycols exhibit, for said use, some disadvantages deriving from their high molecular weight dispersion and from their reduced functionalising capacity. It is an aim of the present invention to provide new multimeric systems characterised by adequate physicochemical properties, high molecular weight and low polydispersity, as well as by the presence of several functional groups. To be used as carriers and stabilisers of pharmacologically or biologically active substances as well as of reagents and catalysts for liquid phase synthesis reactions, said multimeric systems - considered vs. the polymers with a comparable molecular weight - must have a defined m.w. range and a higher binding capacity of organic molecules. It is a further aim of the present invention to develop processes for the preparation of said new multimeric systems simple, reproducible and of easy application to an industrial scale. Summary of the invention

The problem of obtaining new multimeric systems exhibiting good physicochemical properties and good binding capacity has been advantageously and surprisingly solved by the present invention which refers to complex-structured multifunctional polyethylene glycol derivatives (multiPEG) obtained by assembling various PEG units with a defined molecular weight and a low polydispersity.

It is therefore an object of the present invention multifunctional polyethylene glycol derivatives (multiPEG) having a complex branched structure of formula (I)

X- Y -(R")n-C-(R)_n- Y PEG-Y-R-C-(R")_n-Y' -X'

(I)

z-w m Z-W

A process for the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) having a branched complex structure, comprising at least the following steps:

- selective monoderivatisation with an appropriate protecting group of a functional group or of a PEG unit with a defined m.w. or of the linker consisting of a polyfunctional molecule with hydrocarbon skeleton;

- activation of a residual functional group on the monoprotected PEG unit or on the monoprotected linker;

- condensation reactions of mono- or multimeric PEG units and linkers to obtain multifunctional polyethylene glycol derivatives (multiPEG) with the desired size and complex structure and the products ^"obtained with said process are further objects of the present invention.

Jt is a still further object of the present invention to use complex-structured multifunctional polyethylene glycol derivatives (multiPEG) as carriers or stabilisers of pharmacologically or biologically active substances as well as of reagents and catalysts for liquid phase synthesis reactions and as soluble support in synthetic processes.

Brief description of the drawings Figure 1: ¹H-NMR spectra of A) crude tetraPEG pentaOH; B) crude pentaPEG octaOH; C) crude pentaPEG dodecaOH. - 20mg / 0.5 ml of DMSO d⁶ -

Spectrometer: JEOL 400 MHz.

Figure 2: Analytical GPC on HP 1100 - column: PL aquagel-OH 30 8 micron;

300x7.5 mm - Eluent: water milliQ - Flux: 0.6 ml/min - Detector: KNAUER refractive index - Solution: 20 microliter from a 3mg/ml of A) crude tetraPEG pentaOH; B) crude pentaPEG octaOH; C) crude pentaPEG dodecaOH. Detailed description of the invention

The objects and benefits of the present invention will be apparent from the

Detailed Description that follows.

As known, high-molecular-weight polymers have found extensive application in the biotechnological and pharmaceutical fields as carriers of organic molecules for pharmaco-therapeutic purposes and for organic synthesis. However, as concerns such application, said polymers exhibit some disadvantages, which, in the case of high-molecular-weight PEGs consist in their polydispersity and low binding capacity and, in the case of the conjugation with other polymers, in poor physicochemical properties, such as for example gel formation or poor solubility or extremely complex and expensive synthesis processes.

Said disadvantages may be overcome by means of the multifunctional PEG derivatives prepared from PEG units with a defined molecular weight.

The multifunctional polyethylene glycol derivatives (multiPEG) having a complex branched structure of formula (I)

X- Y -(R^»)_n-C-(R)_n- Y PEG-Y-R-C-(R")_n-Y' -X'

(I)

z-w Z-W m comprise more than one PEG unit assembled through a polyfunctional linker of formula (II)

where: Y and Y, identical or different, are each reactive functional groups, selected out of OH, SH, NH₂, either free or protected by appropriate protecting groups; Z is a third functional group different from Y or Y' selected out of OH, SH, NH₂ and, when present inside the compounds of formula (I), out of -O-, -S-, -NH-; R and R", identical or different, are each a hydrocarbon residue, either cyclic or acyclic, linear or branched, saturated or unsaturated aliphatic, or aromatic, comprising from 1 to 10 carbon atoms; R' can be H or CH₃; n can be equal to 0 or 1 , wherein at least one of the two R and R" is present; m can be equal to 0 or 1 ; when m = 0 X can be the functional group of the PEG unit, either free or protected by an appropriate protecting group or linked to an organic molecule; X' can be a -PEG-X unit; W can be a H or a radical of formula (III)

or -Z-W can be a radical of (IV)

-Y-(R)_n (IV)

where W or -Z-W can have the same meaning above indicated when m = 1

X, X', W, W or -Z-W and -Z-W can be a) H b) a -PEG-X unit c) a radical of formula (V) -PEG-Z- (R)_n o -PEG-Z-(R")_n (V)

d) a radical of formula (VI)

-PEG-Z-(R)_n-C-(R")_n-Y'-PEG-X o -PEG-Z-(R")_n-C-(R)_n-r-PEG-X (VI)

Y-PEG-X Y'-PEG-X

e) a radical of formula (VII)

f) a radical of formula (VIII)

-(R")_n- -Cc--((R))_n-Y-PEG-X -(R)_n-C-(R")_n-Y'-PEG-X (VIII)

Z-W 1 ,2 Z-W 1 ,2 g) a radical of formula (IX)

h) a radical of formula (X)

where W or -Z-W can have the same meaning as a), b), c), d), e), f), g), and h). The PEG unit can be a PEG with a m.w. higher than 500 Da and preferably ranging from 500 to 3000 Da. The protecting group of a functional group of the PEG unit can be any acid- or base-labile group known for the protection of a primary OH functional group, such as for example 4,4-dimethoxytrityl (DMT-CI) or 9-fluorenylmethoxycarbonyl (FmocCI). The protecting group of the Y and Y^' functional groups of the Z[Y-R-C-(R^")_n-Y]R

Jinker of formula (II) can be any known group for the protection of the OH, SH, NH₂ functional groups, such as for example the aforementioned DMT-CI and FmocCI for the OH group, or benzyloxycarbonyl Z for the NH₂ group, and pyridyl disulphide for the SH group. R and R" can be selected out of (CH₂)n', (CH=CH)_n-, (CH≡CH)_n>, (Ph)_n< with n^' ranging from 0 to 2.

Multifunctional polyethylene glycol derivatives (multiPEG) having a complex structure, formed by the repetition of PEG units with a defined m.w. higher than 500 Da assembled by successive condensation reactions through the linker of formula (II), can be prepared with a synthesis process, described in detail hereinafter, in any case characterized at least by:

- selective monoderivatisation with an appropriate protecting group of a functional group or of a PEG unit with a defined m.w. or of the linker consisting of a polyfunctional molecule with hydrocarbon skeleton; - activation of a residual functional group either on the monoprotected PEG unit or on the monoprotected linker;

- assembling by repeated condensation reactions between the activated functional group and a free functional group either of one or more PEG units or one or' more linkers to obtain the desired multifunctional polyethylene glycol derivatives (multiPEG).

The above procedure has been so far hindered by the need to produce, in a pure and controlled ay, adequate amounts of bifunctional polydisperse PEG, selectively protected at one of the two terminal ends. In fact, only in this way it is possible to plan an effective assembling strategy, which, after the final removal of the protections still present, allows the obtainment of a multiPEG with controlled structure, size and functionality, retaining all physicochemical characteristics of the polyether skeleton. Therefore, the monoderivatisation of the starting PEG unit and the purification of the polydisperse PEG chains with a high molecular weight (>2 kDa) are the steps setting a limit on the whole synthesis process. Depending on the complex structure desired, the synthesis may be applied according to three different schemes. 1st synthesis scheme To obtain multifunctional polyethylene glycol derivatives (multiPEG) with a complex branched structure, characterised by the repetition of PEG units assembled by successive condensation reactions through a linker of formula (II), where Y and Y' are identical or different and Z is an OH group, the synthesis scheme adopted may be as represented below (GP = Protecting Group):

The following steps are envisaged: a) selective monoderivatisation with an appropriate protecting group of the starting PEG unit with a defined m.w. and separation of the same; b) activation of the.residual OH functional group of the monoprotected PEG unit; c) assembling of two monoprotected and activated PEG units through a condensation with a linker of formula (II) having the aforesaid characteristics, with formation of a first multifunctional unit containing two starting PEG units; d) starting from the unit obtained in the preceding step, repetition, if any, of the step of activation of the functional group of the linker of the PEG unit, and of step c) in which the linker may be identical with or different from the starting linker, to obtain the desired multifunctional derivatives (multiPEG) containing several PEG units with a defined molecular weight. A detailed description of the aforesaid process follows: a. Selective monoderivatisation of the starting PEG

Commercial PEG, polydiperse type, exhibits two hydroxyl groups at the polymer chain ends, which may be modified by traditional organic synthesis processes. In particular, acid- or base-labile groups, e.g. the already mentioned DMT-CI and FmocCI, may be used. The problem of selective monoderivatisation is solved through the reaction with an adequate excess of the reactive derivative of the protecting group and the successive separation from all un- or bi-reacted polymer chains. Since said modification does not substantially alter the general characteristics of the polymer, the only possible means of purification is chromatographic separation. In this case too, due to the predominant characteristics of the PEG chain, the three basic components of the mixture, OH- PEG-OH, GPO-PEG-OH and GPO-PEG-GPO, cannot be separated as are. It is, therefore, necessary temporarily to introduce a convenient ionisable group allowing the ion-exchange chromatographic separation. The successive removal of same, after chromatographic separation, yields the desired product. b. Activation of the GPO-PEG-OH unit For the introduction of the successive linker molecule with a final chemically stable bond, the residual OH group of PEG is to be activated so as to allow its quantitative modification under conditions not causing the removal of the protecting group. Numerous systems activating said group, on the basis of the PEGylation processes of biologically active macromolecules, are known. The systems that generate an ester carbonate, active for the final obtainment e.g. of urethane bonds with the amino group of a linker molecule, are preferred. c. Formation of a PEG unit comprising two starting PEG units

The reaction of two GPO-PEG-OH activated units obtained in the preceding step with a linker with a further OH group allows the obtainment of PEG dimerisation, without the apparent formation of PEG-linker monomer in detectable amounts. At this point, the residual OH group on the linker represents • the only still free functionality that may be activated for successive polymerisations. d. Formation of a PEG unit comprising four starting PEG units

The successive activation of the OH group of the linker on the dimer obtained to this end and the reaction with a second linker molecule, identical with or different from the previous one, yields a tetrameric PEG still exhibiting a residual reactive

OH group on the linker.

Depending on the multiPEG derivative desired, the process may further proceed according to the strategy just described. Alternatively, according to the synthesis scheme reported below, it is possible to obtain multiPEGs having a different complex structure, wherein the branching structure is modified and the starting PEG unit constitutes the core of the final multiPEG derivative.

2nd synthesis scheme

To obtain complex-structured multifunctional polyethylene glycol derivatives

(multiPEG) with a different branching structure, the synthesis scheme adopted may be as represented below (GP = Protecting Group):

OH— PEG— OH + 2χ Y + 4x GPO— PEG^{- OH}

In this case, into the starting PEG unit, which is not protected, are introduced ^"linkers of formula (II), where Y and Y' are two groups equal to OH and Z is different, being a higher-nucleophilicity reactive group. In this case, the steps of the synthesis are described in detail hereinbelow: a) activation of the OH functional groups of the PEG unit; b) introduction into the activated PEG unit of two linkers of formula (II) exhibiting the aforementioned characteristics; c) selective monoderivatisation with an appropriate protecting group of another PEG unit with a defined molecular weight; d) modification of OH groups ^"of PEG units obtained in step b) or c) with a higher nucleophilic moiety; e) activation of the residual OH functional groups of the PEG units obtained in step b) or c); f) assembling of one or more PEG units obtained in step c) on the multifunctional core obtained in step e); g) deprotection of the functional group of the PEG unit obtained in step f) and introduction of one or more linkers.

The repetition, if any, of the step of activation of the functional groups of the linkers introduced into the PEG unit as well as of step d) will yield multifunctional derivatives, multiPEGs, with a higher structural complexity.

3rd synthesis scheme

A further synthesis scheme for assembling the polymeric system, object of the present invention, through two different linkers, can be envisaged. The first linker is the same as that used in the 2nd synthesis scheme, while the second is characterised by Y and Y' equal or different from each other and different from Z being Z equal to OH, where one of the two Y or Y' functional groups is temporarily and selectively protected by a protecting group. In this case, complex-structured multiPEG derivatives are obtained, wherein the initial PEG unit represents the core as in the previous case, but branching is different.

The scheme of this synthesis may be represented as follows (GP = Protecting

Group):

OH— PEG— OH + 2x Y- •Y' — PEG— I

Y' a r

The steps of the synthesis are described in detail hereinbelow: a) activation of the OH functional groups of the PEG unit; b) introduction into the activated PEG unit of two linkers of formula (II), in which Y and Y' are equal to OH and Z is a higher-nucleophilicity group; c) selective monoderivatisation of a second linker of formula (II), characterized by

Y and Y' equal or different from each other and different from Z with Z equal to

OH, in which one of the two Y or Y' functional groups is protected by an appropriate protecting group; d> activation of the OH functional groups of the linkers of the PEG unit obtained in b); ^' ", ^' e) introduction of the linkers obtained in step c) into the PEG unit obtained in the preceding step; f) selective monoprotection of other PEG units and activation of the residual functional group; g) deprotection of the functional group of the linker and introduction into the unit obtained in e) of monoprotected and activated PEG units obtained in f). Depending on the multiPEG derivative desired, the process may further proceed according to the strategy just described. Whatever synthesis scheme has been adopted, the simple removal of the various ""protecting groups on the functional groups of the PEG unit or the linker introduction into said groups after activation will give a new multiPEG, whose physicochemical properties will be almost identical with those of PEGs with the same m.w.; however, the binding capacity will increase with the number of assembled PEG units and of linkers. In any case, multifunctional multiPEG derivatives having a different complex structure, characterized by adequate physicochemical properties, by a high molecular weight, by the presence of more functional groups, both on PEG and on the linker capable of binding organic molecules, and by low polydispersity are obtained, while the dispersity characteristics of the starting PEG are maintained. In particular, from PEG units with a m.w. higher than 500 Da multiPEGs with multiple molecular weights in respect of the molecular weight of the starting PEG unit can be obtained. The molecular weight of the multiPEGs of the present invention may be selected depending on the envisaged use of multiPEG. Furthermore, the use of a different linker in the synthesis may cause a variation in the final product characteristics, i.e. an increase in the intermediate chain length or a variation in chemical functionalities. In other words, by adopting e.g. the first synthesis scheme and starting from a PEG with a m.w. of 5 kDa, equal to 0.4 functionalisable mmole per g, after assembling two units, the molecular weight will increase to 10 kDa with a functionality of 0.3 mmole/g instead of 0.2 mmole/g; after assembling four units, with a resulting m.w. of 20 kDa, the functionality will be 0.25 instead of 0.1, and so on, depending on the number of assemblings and on PEG'S initial size. It is also possible to introduce, into the multiPEGs obtained, selected organic molecules, such as for example bioactive molecules, either identical with or different from one another, using in this case the still free functional groups of linker molecules, before the final deprotection of the OH groups present in the initial PEG. The final purification, if required, may be easily obtained by exclusion chromatography or by suitable dialysis or ultrafiltration processes.

The syntheses of some multiPEG derivatives obtainable through the schemes described above are conveyed by way of indication, not of limitation, of the present invention.

Example 1 : Synthesis of tetra-PEG-pentaol (scheme 1) A) Synthesis of DMT-PEGttnnm-Succinate 1. Synthesis of DMT-PEG(3ooo)- H

PEG(3ooo) was co-evaporated twice from anhydrous pyridine in a 250 ml three- necked flask and dried for 30 min by rotary pump. PEG was dissolved in the minimum amount of anhydrous pyridine. Argon was flushed through the side necks by needles. The mixture was added under stirring with 4,4'-dimethoxytrityl chloride (DMT-CI) (powdered; 1.2 equivalents in respect of PEG'S OH groups) and then with 4-dimethylaminopyridine (DMAP) (powdered; 1 equivalent) and triethylamine (TEA) (liquid; 4 equivalents). The mixture was agitated under argon atmosphere, at room temperature, for 4 hrs. The ice-cooled DMT-PEG-OH was precipitated with anhydrous ether (butyl tertiary methyl ether, MTBE) (1 g in approx. 100 ml solvent). The precipitate was ice- cooled under agitation for 30 min and cautiously filtered through a por. 4 Gooch 1β

under vacuum. The precipitate was washed a few times with /-propanol (this operation often prevents the recrystallisation from ethanol, EtOH) and with MTBE; then it was dried over KOH under vacuum.

The presence of DMT-CI residue. was checked by TLC (eluent CHCI₃/EtOH 9:1 , revelation HCIO₄(70%)/EtOH 3:2). The product was recrysallised from hot/cold

EtOH. ,

The degree of functionalisation was estimated by measuring the absorbance of an aliquot of the compound dissolved in H₂CIO₄ (70%)/EtOH 3:2 at 498 nm through the following formula: μmoles/g = A^ x V(HCIO₄/EtOH 3:2, in ml) x 14.3/mg product

The functionalisation was equal to 62%.

2. Succinylation of DMT-PEG₍₃ooo)-OH

DMT-PEG-OH was co-evaporated twice from anhydrous pyridine in a 250 ml three-necked flask and dried for 30 min by rotary pump. The compound was dissolved in the minimum amount of anhydrous pyridine and placed into an ice bath under stirring. The solution was added with succinic anhydride (5 equivalents in respect of the amount of OH still present) and with DMAP (2.5 equivalents), and caused to react under argon atmosphere, at room temperature, overnight, in the dark. ^" he ice-cooled DMT-PEG-succinate was precipitated with MTBE. The precipitate was ice-cooled under agitation for approx. 30 min and then filtered through a por.

4 Gooch under vacuum. The precipitate was washed a few times with /-propanol and ether, and dried over KOH under vacuum. The product was recrystallised from hot/cold EtOH. B) Purification of DMT-PEGf.^nnn Succinate

Purification of DMT-PEG-succinate was carried out by anion exchange liquid chromatography, using a QAE Sephadex A-50 resin, and a 20.0 x 2.5 cm column for approx. 0.5 g product. ^~

The resin was equilibrated with a 20 mM solution of 1 ,3-diaminopropane in bidistilled H₂O at pH 9.00. A flow of approx. 1.1 ml/min was maintained. DMT-

PEG(3ooo)-succinate (300-450 mg) dissolved in a buffer solution (approx. 3-4 ml) at pH 9.00 was added. Fractions of 5 ml were collected and the absorbance of group DMT at 260 nm was read. Then the fractions relevant to each peak were combined and evaporated to dryness. The products were extracted by taking up the residues with acetonitrile (AcCN), and agitated for 30 minutes. The solutions were filtered by elimination of the undissolved salt and dehydrated with anhydrous Na₂SO₄. The AcCN solutions were concentrated by rotavapor to approx. 5 ml and the ice-cooled products were precipitated with anhydrous ether, filtered through a 4 por. Gooch, washed with ether, and dried over KOH under vacuum. ¹H-NMR analysis revealed that the main chromatographic peak corresponds to the desired DMT-PEG-succinate. O Succinate removal

The combined DMT-PEG-succinate samples were dissolved in cone. NH₄OH (50 ml; 30% v/v) in a 250 ml flask and agitated at room temperature for 4 hrs. Ammonia was evaporated to dryness by rotavapor and the residue was taken up with AcCN. The mixture was agitated, at room temperature, for 30 min, the undissolved salt residue was filtered and the AcCN solution was dehydrated with Na₂SO₄.

The solution was concentrated by rotavapor to approx. 5 ml, and the ice-cooled product was precipitated with anhydrous ether, filtered through a por. 4 Gooch under vacuum, washed with ether, and dried over KOH under vacuum.

The product was analysed by ¹H-NMR and its final functionalisation was determined (<50%). The compound was essentially DMT-PEG-OH containing PEG residual trace amounts. D) Synthesis of DMT-PEGnnn -COO-pNOo-Phenyl DMT-PEG-OH was co-evaporated from anhydrous toluene in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in anhydrous dichloromethane (DCM) (3.6 ml), placed into an ice bath, and stirred. NO₂- phenyl-chloroformate (in a 2:1 ratio in respect of the PEG derivative) and TEA (in a 1:1 ratio in respect of chloroformate) were added. The ice bath was removed. The solution pH was initially controlled and brought to 8 with TEA. The product was caused to react under agitation , at room temperature, for 24 hrs. The ice-cooled product was precipitated with MTBE, recovered by filtration through a por. 4 Gooch under vacuum, washed with /-propanol and ether, and dried over KOH under acuum. The product was recrystallised from hot/cold EtOH. The product, analysed by ¹H-NMR, proved to be DMT-PEG-COO-pNO -Phenyl containing PEG-di(pNO₂-phenyl-carbonate)^' in trace amounts.

E) Synthesis of di(DMT-PEGnnn )

1 ,3-Diamino-2-propanol (in 10% excess in respect of PEG derivative) in a 1:1 mixture of AcCN and dimethylformamide (DMF) (1 ml) was dissolved in a 250 ml flask and stirred. The pH value was brought to 8 with 1 drop glacial acetic acid (AcOH).

DMT-PEG-COO-pNO₂-Phenyl was added portionwise within the span of 2 hrs, while the pH value was maintained at 8 with TEA. The mixture was agitated, at room temperature, for 3 days. The solution turned yellow due to the pNO₂-phenol released. The product was precipitated in an ice bath with MTBE, washed with /-propanol and ether, and dried over KOH under vacuum. The compound was recrystallised from hot/cold EtOH.

The analysis of the product with trinitrobenzene sulphonic acid (TNBS) proved that no free amino groups were present: therefore, there was no trace of '"rnonoderivative. The ¹H-NMR analysis of the compound in CDCI₃ and DMSO showed the presence of unreacted DMT-PEG-OH.

F) Synthesis of di(DMT-PEG-COO-pNO Phenyl di(DMT-PEG) was co-evaporated once from anhydrous toluene in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in anhydrous DCM (2.5 ml), placed into an ice bath and stirred. pNO -phenyl-chloroformate (in a 2:1 ratio in respect of the PEG derivative) and TEA (in a 1 :1 ratio in respect of chloroformate) were added. The ice bath was removed. The solution pH was initially controlled and brought to 8 with TEA. The product was caused to react under agitation, at room temperature, for 24 hrs. The ice-cooled product was precipitated with MTBE, recovered by filtration through a por. 4 Gooch under vacuum, washed with /-propanol and ether, and dried over KOH under vacuum. The product was recrystallised from hot/cold EtOH. The product, analysed by ¹H-NMR, proved to be a mixture of di(DMT-PEG-COO- pNO₂-Phenyl) and DMT-PEG-COO-pNO₂-Phenyl. G) Synthesis of tet^'ra(DMT-PEGnnnrη^')

1 ,3-Diamino-2-propanol (in 10% excess in respect of a 1:1 mixture of mono- and di-derivative) in a 1 :1 mixture of AcCN and DMF (1 ml) was dissolved in a 250 ml flask and stirred. The pH value was brought to 8 with 1 drop of glacial acetic acid (AcOH). di(DMT-PEG-COO-pNO₂-Phenyl) was added portionwise within the span of 2 hrs, while maintaining the pH value at 8 with TEA. The mixture was agitated, at room temperature, for 3 days: the solution turned yellow due to the pNO₂-phenol released.

The product was precipitated with MTBE in an ice bath, washed with /-propanol and ether, and then dried over KOH under vacuum. The compound was recrystallised from hot/cold EtOH. The analysis of the product with TNBS proved that no free amino groups were present. H) Purification

Purification was carried out by molecular exclusion chromatography using a Bio- Gel P 100 resin, on a 2.5 x 55 cm column. The final sample (0.1 g) was eluted with an aqueous solution at pH 9.0 and a 0.15 ml/min flow. The fractions were collected depending on their absorption at 260 nm due to the DMT group present. In fig. 1-A the ¹H-NMR of the crude product and in fig. 2-A the GPC chromatography of the same are reported. The chromatogram showed the presence of tetraPEG, along with a given amount of lower m.w. derivatives. The chromatographic analysis was confirmed by ¹H-NMR .

From the fractions collected, treated as customary, it was possible to collect tetraPEG in an amount of 60% in respect of the quantity fed to the column. At the end, the DMT groups were eliminated by 5-min treatment with 6% trichloroacetic acid (TCA) in DCE. The final product was precipitated with MTBE, filtered and dried.

Example 2: Synthesis of penta-PEG-octaol (scheme 2) A) Synthesis of DMT-PEG nnr -Succinate 1) Synthesis of DMTrPEG₍₂ooo)-OH

PEG(2ooo) was cό-evaporated twice from anhydrous pyridine in a 250 ml three- necked flask and dried for 30 min by rotary pump. PEG was dissolved in the minimum amount of anhydrous pyridine. Argon was flushed through the side necks by needles. The mixture was added under stirring with 4,4'-dimethoxytrityl chloride, (DMT-CI) (powdered; 1 equivalent in respect of the OH groups present in PEG), 4-dirηethyiaminopyridine (DMAP) (powdered; 1 equivalent), and triethylamine (TEA) (liquid; 4 equivalents). The mixture was agitated under argon atmosphere, at room temperature, for 18 hrs. The ice-cooled DMT-PEG-OH was precipitated with anhydrous ether (1 g in approx. 100 ml solvent). The precipitate was ice-cooled under agitation for approx. 30 min and cautiously filtered through a por. 4 Gooch under vacuum. The precipitate was washed a few times with ethyl ether and dried over KOH under vacuum. The presence of DMT-CI residue was checked by TLC (eluent CHCI₃/EtOH 9:1, revelation HCIO₄(70%)/EtOH 3:2).

The degree of funptionalisation was estimated by measuring the absorbance of an aliquot of the compound dissolved in H₂CIO₄ (70%)/EtOH 3:2 at 498 nm through the following formula: ^""^* μmoles/g = A₄98 x V(HCIO₄/EtOH 3:2, in ml) x 14.3/mg product

The functionalisation was equal to 69.0%. The products were analysed by ESI-MS and ¹H-NMR. 2. Succinylatiόn of DMT-PEG(2ooo)-OH DMT-PEG-OH was co-evaporated twice from anhydrous pyridine in a 250 ml three-necked flask and dried for 30 min by rotary pump. The compound was dissolved in the minimum amount of anhydrous pyridine and placed into an ice bath under stirring. The solution was added with succinic anhydride and DMAP (solid): The following ratios were used:

DMT-PEG-OH: succinic anhydride:DMAP = 1 : 2.5 : 1.7 The solution was caused to react under argon atmosphere, at room temperature, overnight, in the dark. The ice-cooled DMT-PEG-succinate was precipitated with anhydrous ether (1 g in approx. 100 ml solvent). The precipitate was ice-cooled under agitation for approx. 30 min and cautiously filtered through a por. 4 Gooch under vacuum. The precipitate was washed a few times with ethyl ether and dried over KOH under vacuum. The resulting product was analysed by ESI-MS and ¹ H-NMR.

B) Purification of DMT-PEG/?nn -Succinate

Purification of DMT-PEG-succinate was carried out by anion exchange liquid chromatography (IE-LC), using a QAE Sephadex A-50 resin, and a 28 x 1.5 cm column for approx. 0.5 g product. The resin was equilibrated with a 20 mM solution of 1 ,3-diaminopropane in bidistilled H₂O at pH 9.00. A flow of approx. 1.1 ml/min was maintained. DMT-

PEG₍₂ooo)-succinate (500 mg) dissolved in a buffer solution (approx. 3 ml) at pH

9.00 was added.

Fractions of 5 ml were collected and the absorbance of group DMT at 260 nm was read. The fractions corresponding to each peak were combined (those with superimposed peaks were discarded) and evaporated to dryness by rotavapor.

The products were extracted taking up the residues obtained with acetonitrile

(AcCN), and agitated for 30 minutes. The solutions were filtered by elimination of the undissolved salt and dehydrated with anhydrous Na₂SO₄- The AcCN solutions '"'Were concentrated by rotavapor to approx. 5 ml and the ice-cooled products were precipitated with anhydrous ether, filtered through a por. 4 Gooch, washed with ether and dried over KOH under vacuum.

All DMT-PEG-succinate samples obtained from the synthesis of DMT-PEG-OH were purified under said conditions. ¹H-NMR and ESI-MS analyses revealed that the main chromatographic peak corresponded to DMT-PEG-succinate.

C) Succinate Removal

The combined DMT-PEG-succinate samples were dissolved in cone. NH₄OH (50 ml; 30% v/v) in a 250 ml flask and agitated at room temperature for 4 hrs. Ammonia was evaporated to dryness by rotavapor (a 500 ml flask was used to avoid splashes and product loss) and the residue was taken up with AcCN. The mixture was agitated at room temperature of 30 min, the solid left (consisting of salts) was filtered and the AcCN solution was dehydrated with anhydrous Na₂SO₄. The solution was concentrated by rotavapor to approx. 5 ml, and the ice-cooled product was precipitated with anhydrous ether, filtered through a por. 4 Gooch under vacuum, washed with ether, and dried over KOH under vacuum. The product was recrystallised from dichlorbmethane/ether.

The product was .analysed by ¹H-NMR and ESI-MS, and its functionalisation degree was determined (51.22%). The compound was essentially DMT-PEG-OH containing DMT-PEG-DMT in residual trace amounts.

D) Synthesis of DMT-PEG/7nnm-COO-pNO9-Phenyl DMT-PEG₍₂ooo)-OH was co-evaporated from anhydrous toluene in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in the minimum amount of anhydrous dichloromethane (DCM), placed into an ice bath and stirred. pNO₂-phenyl-chloroformate (solid, in a 2:1 ratio in respect of the PEG derivative) and TEA (liquid, in a 1:1 ratio in respect of chloroformate) were added. The ice bath was removed. The solution pH was initially controlled and brought to 8 with TEA. The product was caused to react under agitation, at room temperature, for 24 hrs.

The ice-cooled product was precipitated with anhydrous ethyl ether (1 g in approx. 100 ml solvent), recovered by filtration through a por. 4 Gooch under vacuum, ""Washed with ether, and dried over KOH under vacuum. The product was recrystallised from dichloromethane/ether. The compound, analysed by ¹H-NMR and ESI-MS, proved to be DMT-PEG-COO-pNO₂-Phenyl containing DMT-PEG- DMT in trace amounts.

E) Synthesis of (DMT-PEG(?nnn))-1.3-diaminopropane DMT-PEG₍₂ooo₎-COO-pNO₂-Phenyl was co-evaporated twice from anhydrous dichloromethane in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in the minimum amount of anhydrous dichloromethane (DCM) and stirred. 1 ,3-Diaminopropane (liquid, in a 3:1 ratio in respect of the PEG derivative) was added. The product was caused to react under agitation, at room temperature, for 24 hrs.

The ice-cooled product was precipitated with anhydrous ethyl ether (1 g in approx. 100 ml solvent), recovered by filtration through a por. 4 Gooch under vacuum, washed with ether, and dried over KOH under vacuum. The product was recrystallised from dichloromethane/ether.

The compound, analysed by ¹H-NMR and ESI-MS, proved to be DMT-PEG-1,3- diaminopropane containing DMT-PEG-DMT in trace amounts. F) Synthesis of PEG/_?nnnrdi(pNO?-phenyl carbonate)

PEG(₂ooo) was co-evaporated twice from anhydrous dichloromethane (DCM) in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in the minimum amount Of anhydrous dichloromethane (DCM), placed into an ice bath and stirred. pN02-phenyl chloroformate (solid, in a 2:1 ratio in respect of the OH groups present in PEG) and triethylamine (TEA) (in a 1 :1 ratio in respect of chloroformate) were added. The ice bath was removed. The solution pH was initially controlled and brought to 8 with TEA. The product was caused to react under agitation, at room temperature, for 24 hrs. The ice-cooled product was precipitated with anhydrous ethyl ether (1 g in approx. 100 ml. solvent), recovered by filtration through a por. 4 Gooch under vacuum, washed with ether, and dried over KOH under vacuum. The product was recrystallised from dichloromethane/ether and its structure confirmed by ¹ H-NMR and ESI-MS. G) Synthesis of PEG^nn -tetraol PEG₍₂ooo)-di(pN0₂-phenyl carbonate) was co-evaporated twice from anhydrous dichloromethane in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in the minimum amount of anhydrous dichloromethane (DCM) and added with 2-amino-1 ,3-propanediol (solid, in a 3:1 ratio in respect of pNO₂-phenyl carbonate groups present in the PEG derivative). The product was caused to react under agitation, at room temperature, for 18 hrs.

The ice-cooled product was precipitated with anhydrous ethyl ether (1 g in approx. 100 ml solvent), recovered by filtration through a por. 4 Gooch under vacuum, washed with ether, and dried over KOH under vacuum. The compound was recrystallised from dichloromethane/ether, dissolved in the minimum amount of milliQ water, and extracted with dichloromethane. The organic phase was concentrated and dehydrated with anhydrous Na₂SO₄. The product was precipitated with anhydrous ether, recovered as previously described, and then analysed by ¹ H-NMR and ESI-MS. H) Synthesis of PEGf?nn -tetra-OSu

PEG₍₂ooo₎-tetraol was co-evaporated twice from anhydrous dichloromethane in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in a mixture of anhydrous solvents - DCM, acetonitrile and pyridine (in a 3:2:1 ratio) - which was added with N,N'-disuccinimidyl carbonate (solid, in a 3:1 ratio in respect of the OH groups present in PEG). The product was caused to react under agitation, at room temperature, for 18 hrs. The ice-cooled product was precipitated with anhydrous ethyl ether (1 g in approx. 100 ml solvent), recovered by filtration through a por. 4 Gooch under vacuum, washed with ether, and dried over KOH under vacuum. The product was recrystallised from dichloromethane/ether and then analysed by ¹ H-NMR. I) Synthesis of tetra-DMT-(PEG?nnn)κ PEG₍₂ooo₎-tetra-OSu was co-evaporated twice from anhydrous dichloromethane (DCM) in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in anhydrous dichloromethane and very slowly added, under stirring, with DMT-PEG(₂ooo₎-1,3-diaminopropane (in a 1.1 :1 ratio in respect of the OSu groups present in PEG-tetraol). "The product was caused to react under agitation, at room temperature, for 72 hrs. The ice-cooled product was precipitated with anhydrous ethyl ether (100 ml), recovered by filtration through a por. 3 Gooch under vacuum, washed with ether, recrystallised from dichloromethane/ether, and dried over KOH under vacuum. The analysis of the product with trinitrobenzene sulphonic acid (TNBS) indicated the presence of 15% free amino groups.

¹ H-NMR, ESI-MS and GPC analyses of the compound revealed also the presence of unreacted DMT-PEG-1 ,3-diaminopropane and of traces of DMT-PEG-DMT. The degree of functionalisation of DMT was equal to 94% (calculated as described under para. A-1 above). J) Synthesis of tetra-hvdroxy-fPEG^nnn^

An ice-cooled solution of tetra-DMT-(PEG2ooo)s in anhydrous dichloroethane (DCE) was very slowly added dropwise, under magnetic stirring, with a 6% solution of trichloroacetic acid (TCA) in dichloroethane (DCE). Once the addition was complete, stirring was continued, at room temperature, for 30 minutes.

The ice-cooled product was precipitated with anhydrous ethyl ether (100 ml), recovered by filtration through a por. 3 Gooch under vacuum, washed with ether, and dried over KOH under vacuum.

The procedure of DMT recovery was repeated a second time under the conditions described above.

Finally, the product was recrystallised from dichloroethane/ether and dried over

KOH under vacuum. The ¹H-NMR, ESI-MS and GPC analyses revealed also the presence of PEG-1,3- diaminopropane and of traces of bifunctional PEG.

K) Synthesis of tetra-OSu-(PEGgnnn)*

Tetra-Hydroxy-(PEG2ooo)5 obtained in step J) was co-evaporated twice from anhydrous dichloromethane in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in a mixture of anhydrous solvents - DCM, acetonitrile and pyridine (in a' 3:2:1 ratio) - which was added with N,N'-disuccinimidyl carbonate (solid, in a 3:1 ratio in respect of the OH groups present in PEG). The product was caused to react under agitation, at room temperature, for 18 hrs.

The ice-cooled product was precipitated with anhydrous ethyl ether (100 ml), recovered by filtration through a por. 3 Gooch under vacuum, washed with ether, and dried over KOH under vacuum. The product was recrystallised from dichloromethane/ether and then analysed by ¹H-NMR.

L) Synthesis of octa-hvdroxy-fPEGsnnnYq

Tetra-OSu-(PEG₂ooo)5 was co-evaporated twice from anhydrous dichloromethane (DCM) in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in anhydrous dichloromethane, and the solution was added with 2- amino-1 ,3-propanediol (solid, in a 3:1 ratio in respect of the OSu groups present in

PEG). The product was caused to react under agitation, at room temperature, for

18 hrs. The ice-cooled product was precipitated with anhydrous ethyl ether (100 ml), recovered by filtration through a por. 3 Gooch under vacuum, washed with ether, and dried over KOH under vacuum. The product was recrystallised from dichloromethane/ether.

¹ H-NMR, and GPC analyses were carried out on the product (Fig. 1-B; fig. 2-B). Example 3: Synthesis of penta-PEG-dodecaol (scheme 3) The synthesis of multiPEG, containing 5 PEG units and 12 OH functional groups, denominated penta-PEG-dodecaol, was substantially identical with that described in Example 2, steps A, B, C, D, F, G, H, with the exception of step E.

A) Synthesis of DMT-PEG₍?nnn₎-Succinate

1) Synthesis of DMT-PEG₍₂ooo₎-OH as described in Example 2.

2) Succinylation of DMT-PEG₍₂ooo)- H as described in Example 2.

B) Purification of DMTrPEGf?nnn₎-Succinate as described in Example 2. C) Succinate Removal as described in Example 2.

D) Synthesis of DMT-PEG n -COO-pNO?.Phenyl as described in Example 2.

E) Synthesis of PEG ^nnr difoNQp-phenyl-carbonate) ^'"^s described in Example 2, step F.

F) Synthesis of PEGf?nnn)-tetraol as described in Example 2, step G.

G) Synthesis of PEG^nnm-tetra-OSu as described in Example 2, step H. H) Synthesis of N-BOC-1.3-diamino-2-propanol

1 ,3-Diamino-2-propanol (500 mg; 5.55 mmoles) was dissolved in anhydrous methanol (MeOH) (20 ml) in a 100 ml flask under stirring, and added with TEA (258 μl; 1.85 mmoles). The_ resulting solution, placed into an ice bath, was very slowly added dropwise, with magnetic stirring, with a solution of di-t-butyl- bicarbonate (BOC₂0; 403 mg; 1.85 mmoles). After a 18-hr reaction, the solvent was eliminated by rotary evaporator and the residue was taken up with a 10% citric acid solution (25 ml). The aqueous phase was extracted with^'AcOEt (3 x 20ml) to eliminate the di-BOC derived, if any.

The aqueous phase was basified to pH 9 with a 4M NaOH solution and extracted with AcOEt (4 x 30ml). The organic phase was dried over anhydrous Na₂SO₄, and the solvent was elimihated by rotary evaporator.

The pure product obtained was analysed by ¹H-NMR and ESI-MS. h Synthesis of PEG(_?nnn)-tetra-BOC-tetraol

PEG₍₂ooo)-tetra-OSu was co-evaporated from anhydrous dichloromethane in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in anhydrous dichloromethane (DCM) and the solution was added, under magnetic stirring, with N-BOC-1 ,3-diamino-2-propanol (in a 3:1 ratio in respect of the OSu groups present in PEG). The product was caused to react under stirring, at room temperature, for 18 hrs. The ice-cooled product was precipitated with anhydrous ethyl ether, recovered by filtration through a por. 4 Gooch under vacuum, washed with ether, recrystallised from dichloromethane/ether, and dried over KOH under vacuum.

The product was analysed by ¹ H-NMR and ESI-MS.

J) Synthesis of PEG ^nn -tetra-amino-tetraol PEG₍₂ooo₎-tetra-BOC-tetraol was dissolved in a 50% solution of trifluoroacetic acid

(TFA) in dichloromethane and stirred for 30 minutes.

Once the volume of the ' solution was partially reduced, the product was precipitated with ether according to the usual procedure. In spite of the several attempts made to achieve the recrystallisation and co-evaporation from ether, no crystalline product was obtained.

In any case, the product was characterised by ¹ H-NMR and ESI-MS.

K) Synthesis of tetra-DMT-tetrahvdroxy-(PEG7nnn)s

PEG₍₂ooo₎-tetra-amino-tetraol was co-evaporated from anhydrous dichloromethane in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in anhydrous dichloromethane (DCM). The pH value of the solution, which was acid due to the presence of TFA in trace amounts, was adjusted to pH 9 with TEA.

Then the solution was very slowly added, under magnetic stirring, with DMT- PEG₍₂ooo)-p-nitrophenyl-carbonate (in a 1.1:1 ratio in respect of the NH₂ groups present in PEG). The product was caused to react under stirring, at room temperature, for 72 hrs.

The ice-cooled product was precipitated with anhydrous ethyl ether (100 ml), recovered by filtration through a por. 4 Gooch under vacuum, washed with ether, recrystallised from , dichloromethane/ether, and dried over KOH under vacuum.

The product was analysed by ¹ H-NMR and ESI-MS. Furthermore, the analysis of the product with trinitrobenzene sulphonic acid (TNBS) indicated the presence of

4.8% free amino groups. The degree of functionalisation of DMT was equal to 78% (calculated as described under para. A-1 ).

L) Synthesis of octa-hvdroxy-(PEG?nnn)R

An ice-cooled solution of branched tetra-DMT-(PEG₂ooo)s-tetraol in anhydrous dichloroethane (DCE) was very slowly added dropwise, under magnetic stirring, with a 6% solution of trichloroacetic acid in dichloroethane (DCE). Once the addition was complete, stirring was continued, at room temperature, for 30 minutes.

The ice-cooled product was precipitated with anhydrous ethyl ether, recovered by filtration through a por. 4 Gooch under vacuum, and washed with ether. "The DMT recovery procedure was repeated a second time under the conditions described above.

Finally, the product was recrystallised from dichloromethane/ether and dried over

KOH under vacuum.

The product was analysed by ¹ H-NMR. M) Synthesis of tetra-hvdroxy-tetra-OSu (PEGpnnn)*

The synthesis was carried out under the same conditions as described under para.

G).

N) Synthesis of dodeca-hvdroxy-(PEG?nnn)s

Branched tetra-hydroxy-(PEG2ooo)s -tetra-OSu was co-evaporated from anhydrous dichloromethane in a 250 ml flask and dried for 30 min by rotary pump. The residue was dissolved in anhydrous dichloromethane (DCM) and the resulting solution was added with 2-amino-1 ,3-propanediol (in a 3:1 ratio in respect of the OSu groups present in PEG). The product was caused to react under agitation, at room temperature, for 18 hrs.

The ice-cooled product was precipitated with anhydrous ethyl ether, recovered by filtration through a por. 4 Gooch under vacuum, washed with ether, dried over KOH under vacuum, and recrystallised from dichloromethane/ether. The product was analysed by ¹H-NMR and GPC (Fig.1-C; Fig. 2-C).

The use of multifunctional PEG derivatives (multiPEG) is based on their capacity of binding organic molecules either reagents and catalysts or with a biological and pharmacological activity to residual functional groups of PEG units and of linkers. Therefore, they can be used in supported synthesis processes of biopolymers and complex molecules, both free and PEG-conjugated; syntheses of supported reactive agents; syntheses of supported catalytic systems operating in the homogeneous phase and recyclable and as soluble support in synthetic processes; as carriers and solubilisers of drugs, biologically active molecules such as diagnostic markers, antigens and antibodies, sequences of DNA, and contrast means; in vivo stabilisation of biologically active - natural and synthetic - molecules. For diagnostic, prophylactic and therapeutic purposes, multifunctional PEG derivatives may be used either in the free state or conjugated with the aforementioned molecules in compositions with known or new pharmaceutically acceptable diluents and excipients, suitable for the selected purposes and ways of administration.

Claims

1. Multifunctional polyethylene glycol derivatives (multiPEG) having a complex branched structure of formula (I)

X- Y -(R")_n-C-(R)_n- Y - PEG-Y-R-C-(R")_n-Y' -X'

(I)

Z-W Z-W m comprising more than one PEG unit. assembled through a polyfunctional linker of formula (II)

where: „Y and Y , identical or different, are each reactive functional groups, selected out of OH, SH, NH₂, either free or protected by adequate protecting groups; Z is a third functional group different from Y or Y' selected out of OH, SH, NH₂ and, when present inside the compounds of formula (I), out of -O- -S-, -NH-;

R and R", identical or different, are each a hydrocarbon residue, either cyclic or acyclic, linear or branched, saturated or unsaturated aliphatic, or aromatic, comprising from 1 to 10 carbon atoms;

R' can be H or CH₃; n can be equal to 0 or 1 , wherein at least one of the two R and R" is present; m can be equal to 0 or 1 ; when m = 0 X can be the functional group of the PEG unit, either free or protected by an appropriate protecting group or linked to an organic molecule;

X' can be a -PEG-X unit; W can be a H or a radical of (III)

-(R)_n-C-(R")_n-Y'-PEG-X (R")_n-C-(R)_n-Y-PEG-X

(III)

Z-W 1 ,2 Z-W 1 ,2

or -Z- W can be a radical of formula (IV)

(IV)

where W or -Z-W can have the same meaning; when m = 1

X, X', W, W or -Z-W and -Z-W can be a) H b) a -PEG-X unit c) a radical of formula (V)

-PEG-Z-(R)_n o -PEG-Z-(R")_n (V)

d) a radical of formula (VI)

PEG-Z-(R)_n- (VI)

e) a radical of formula (VII)

-(R")_n- (R)_n-V -(R)_n-C-(R")_n-Y

(VII)

Z-W 1 ,2 Z-W 1 ,2

f) a radical of formula (VIII)

(VIII)

g) a radical of formula (IX)

h) a radical of formula (X)

where W or -Z-W can have the same meaning as a), b), c), d), e), f), g), and h).

2. Multifunctional polyethylene glycol derivatives (multiPEG) as claimed in claim 1, wherein R and R" can be selected out of (CH₂),y, (CH=CH)_n-, (CH≡CH)_n-, (Ph)_n' with n' ranging from 0 to 2.

3. Multifunctional polyethylene glycol derivatives (multiPEG) as claimed in any of claims 1 and 2, wherein the PEG unit is a polydisperse PEG with a defined a m.w. higher than 500 Da.

4. Process for the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) having a branched complex structure of formula (I) X- Y^*

comprising more than one PEG unit assembled through a polyfunctional linker of formula (II)

where:

Y and Y, identical pr different, are each reactive functional groups, selected out of

OH, SH, NH₂, either free or protected by adequate protecting groups; Z is a third functional group different from Y or Y' selected out of OH, SH, NH₂ and, when "present inside the compounds of formula (I), out of -O-, -S-, -NH-;

R' can be H or CH₃; n can be equal to 0 or 1 , wherein at least one of the two R and R" is present; m can be equal to 0 or 1 ; when m = 0

X can be the functional group of the PEG unit, either free or protected by an appropriate protecting group or linked to an organic molecule; X' can be a -PEG-X unit;

W can be a H or a radical of (III)

or -Z-W can be a radical of (IV)

Y-(R)_n-C-(R")_n-r-PEG-X| -r-(R")_n- (R)_n-Y-PEG-X (IV)

1,2

Z-W 1 ,2

Z-W where W or -Z- W can have the same meaning; when m = 1

X, X', W, W or -Z- W e -Z- W can be a) H b) a -PEG-X unit, c) a radical of formula (V)

-PEG-Z-(R)_n o -PEG-Z-(R")_n (V)

d) a radical of formula (VI)

o (VI)

e) a radical of formula (VII)

(R")_n-C-(R)_n-Y -(R)_n-C-(R")_n-r

(VII)

Z-W 1 ,2 Z-W 1 ,2 f) a radical of formula (VIII)

( ")n-C-(^R)n-Y-PEG-X -(R)_n-C-(R")_n-Y'-PEG-X (VIII)

Z-W 1,2 Z-W 1 ,2

g) a radical of formula (IX)

h) a radical of formula (X)

where W or -Z-W can have the same meaning as a), b), c), d), e), f), g), and h) comprising at least the following steps:

- selective monoderivatisation with an adequate protecting group of a functional group or of a PEG unit with a defined m.w. or of the linker consisting of a polyfunctional molecule with hydrocarbon skeleton;

- activation of a residual functional group either on the PEG unit or on the linker;

- assembling by repeated condensation reactions between the activated functional group and a free functional group either of one or more mono- or multimeric PEG units and one or more linkers to obtain the desired multifunctional polyethylene glycol derivatives (multiPEG).

5. The process for the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) as claimed in claim 4 comprising the following steps: a) selective monoderivatisation with an appropriate protecting group of a PEG unit with a defined molecular weight; b) activation of the residual OH functional group of the monoprotected PEG unit; c) assembling of two monoprotected and activated PEG units obtained in steps a) and b) through a linker of formula (II) having Y and Y' identical or different, and Z equal to OH, with formation of a first multifunctional unit comprising two PEG units.

6. The process for the synthesis of multifunctional polyethylene giycol derivatives (multiPEG) as claimed in claim 5, comprising further repeated steps of: activation of the functional group of the linker of a multimeric PEG unit and assembling of the obtained multimeric PEG units through the linker of formula (II), until obtaining the desired multifunctional PEG derivatives (multiPEG) comprising several PEG units with a defined molecular weight.

7. The process for⁴ the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) as claimed in claim 4, comprising the steps of: a) activation of the OH functional groups of a starting PEG unit; "T>) introduction into the activated PEG unit of two linkers of formula (II), where Y and Y' are equal to OH; and Z is different and consists of a higher-nucleophilicity group; c) selective monoderivatisation with an appropriate protecting group of another PEG unit with a defined molecular weight; d) modification of OH groups of PEG units obtained in step b) or c) with a higher nucleophilic moiety; e) activation of the residual OH functional groups of the PEG units obtained in b) or e); f) assembling of one or more PEG units obtained in step c) on the multifunctional core obtained in step e).

8. The process for the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) as claimed in claim 7, comprising further repeated steps of: deprotection of the functional group of the multimeric PEG unit obtained and activation of the same for the introduction of one or more linkers and activation of the residual, functional groups of the linker for the assembling of one or more mono- or multimeric PEG units until obtaining the desired multifunctional derivatives' (multiPEG) comprising several PEG units with a defined molecular weight.

.

9. The process for the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) as claimed in claim 4 comprising the steps of: a) activation of the OH functional groups of a starting PEG unit; b) introduction into the activated PEG unit of two linkers of formula (II), where Y and Y' are equal to OH, and Z is different and consists of a higher-nucleophilicity group; c) selective monoderivatisation of a second linker, where Y and Y' are identical or different, and Z is equal to OH, of which at least one of the two Y or Y' is protected by an appropriate protecting group; d) activation of the OH residual functional groups on the linkers of the PEG unit obtained in step b); e) introduction into the PEG unit obtained in step d) of the linker obtained in step c); "f) selective monoderivatisation of another PEG unit with a defined molecular weight with an appropriate protecting group; g) activation of the residual functional group of the PEG unit obtained in step f); h) deprotection of the functional group of the linker of the PEG unit obtained in step e) and introduction of monoprotected and activated PEG units obtained in steps f) and g).

10. The process for the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) as claimed in claim 9 comprising further repeated steps of: introduction into the multimeric PEG unit obtained of the protected and activated linker formula (II) obtained in steps c) and d) and repetition of the condensation reaction of mono- or multimeric monoprotected and activated PEG units obtained in steps f) and g) on the linker after deprotection of same until obtaining the desired multifunctional derivatives (multiPEG) comprising several PEG units with a defined molecular weight.

11. The process for the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) as claimed in any of claims 4 to 10 comprising a further step of deprotection of the functional group of the , PEG unit or of the linker for the introduction of one, or more organic molecules selected out of drugs, diagnostic markers, antigens and antibodies, and contrast means.

12. The process for the synthesis of multifunctional polyethylene glycol derivatives (multiPEG) as claimed in any of claims 4 to 10 comprising a further step of deprotection of t e functional group of the PEG unit for the introduction of one or more organic molecules selected out of reagents and catalysts.

13. Multifunctional polyethylene glycol derivatives (multiPEG) having a complex branched structure comprising more than one PEG unit assembled through a polyfunctional linker of formula (II)

^* where:

Y and Y , identical or different, are each reactive functional groups, selected out of

OH, SH, NH₂; Z is a third functional group different from Y or Y' selected out of

OH, SH, NH₂

R' can be H or CH₃; obtained with the processes as claimed in any of the preceding claims from 4 to

12.

14. Multifunctional polyethylene glycol derivatives (multiPEG) as claimed in claim

13 wherein R and R" can be selected out of (CH₂)_n', (CH=CH)_n', (CH≡CH)n', (Ph)_n' with n' ranging from 0 to 2.

15. Multifunctional polyethylene glycol derivatives (multiPEG) as claimed in claim 13 and 14 wherein the PEG unit is a polydisperse PEG with a m.w. higher than 500 Da.

16. Use of multifunctional polyethylene glycol derivatives (multiPEG) having a complex branched structure of formula (I)

X- Y -(R")_n-

where:

Y and Y, identical or different, are each reactive functional groups, selected out of OH, SH, NH_2ι either free or protected by appropriate protecting groups; Z is a third functional group different from Y or Y' selected out of OH, SH, NH₂ and, when present inside the compounds of formula (I), out of -O- -S-, -NH-; R and R", identical or different, are each a hydrocarbon residue, either cyclic or acyclic, linear or branched, saturated or unsaturated aliphatic, or aromatic, comprising from 1 to 10 carbon atoms; R' can be H or CH₃; n may be equal to 0 or 1 , wherein at least one of the two R and R" is present; m can be equal to 0 or 1 ; when m = 0 X can be the functional group of the PEG unit, either free or protected by an appropriate protecting group or linked to an organic molecule; X' can be a ^PEG-X unit; W can be a H or a radical of formula (III)

-(R)_n

or -Z-W can be a radical of formula (IV)

-Y- (IV)

where W or - - can have the same meaning; when m = 1

X, X', W, W or -Z- W and -Z- W can be a) H b) a -PEG-X unit c) a radical of formula (V)

-PEG-Z-(R)_n R)_n-Y (V)

d) a radical of formula (VI) R' R'

-PEG-Z-(R)_n-C-(R")_n-Y'-PEG-X o -PEG-Z-(R")_n-C-(R)_n-Y'-PEG-X (VI)

Y-PEG-X Y'-PEG-X

e) a radical of formula (VII)

f) a radical of formula (VIII)

43) a radical of formula (IX)

-r-(R")_n- (IX)

h) a radical of formula (X)

■r-(R")_n- X)

where W or -Z-W can have the same meaning as a), b), c), d), e), f), g), and h) as carriers and stabilisers of drugs, diagnostic markers, antigens and antibodies, contrast means, bound to said derivatives on the residual functional groups of the linkers and/or of the PEG units.

17. The use of multifunctional polyethylene glycol derivatives (multiPEG) having a complex structure as claimed in claim 16' for the preparation of pharmaceutical compositions that can be used for prophylactic or therapeutic or diagnostic purposes.

18. Use of multifunctional polyethylene glycol derivatives (multiPEG) having a branched complex structure formula (I)

Y'

where:

Y and Y , identical or different, are each reactive functional groups, selected out of OH, SH, NH₂, either free or protected by adequate protecting groups; Z is a third functional group different from Y or Y' selected out of OH, SH, NH₂ and, when present inside the compounds of formula (I), out of -O- -S-, -NH-;

R' can be H or CH₃; n may be equal to 0 or 1 , wherein at least one of the two R and R" is present; m can be equal to 0 or 1 ; when m = 0

X can be the functional group of the PEG unit, either free or protected by an appropriate protecting group or linked to an organic molecule;

X' can be a -PEG-X unit;

W can be a H or a radical of formula (III)

-(R)_n-C-(R")_n-Y'-PEG-X -(R")_n-C-(R)_n-Y-PEG-X

(III)

Z-W 1 ,2 Z-W 1 ,2

-Y-(R)_n (IV)

or -Z-W can be a radical of formula (IV)

where W or-Z- W can have the same meaning; when m = 1

-PEG-Z-(R)_n R)_n-Y (V)

d) a radical of formula (VI)

PEG-Z-(R)_n-C-(R")_n-Y'-PEG-X o -PEG-Z-(R")_n-C-(R)_n-Y'-PEG-X (VI)

Y-PEG-X Y'-PEG-X

e) a radical of formula (VII)

f) a radical of formula (VIII)

(VIII)

g) a radical of formula (IX)

h) a radical of formula (X)

Y-(R" X)

where W or -Z-W can have the same meaning as a), b), c), d), e), f), g), and h) as carriers and stabilisers of reagents and catalysts for liquid phase synthesis reactions, bound to said derivatives on the residual functional groups of the linkers and/or of PEG unit^'s and soluble support in synthetic processes.

19. Use according to any of preceding claims from 16 to 18 of multifunctional polyethylehe glycol derivatives (multiPEG) as claimed in any of the claims from 13 to 15.