AU2014234272A1 - Hydroxyalkyl starch derivatives as reactants for coupling to thiol groups - Google Patents
Hydroxyalkyl starch derivatives as reactants for coupling to thiol groups Download PDFInfo
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Abstract
The present invention relates to a hydroxyalkyl starch (HAS) derivative of formula (I), wherein X is a leaving group, preferably selected from the group consisting of Ts, Ms, Cl, Br and I; Fl is a functional group comprising the group -NR'-, with R' being H or alkyl; L1 is a spacer bridging F1 and S; L2 is a spacer bridging -NR
Description
WO 2014/147175 PCT/EP2014/055598 Hydroxyalkyl starch derivatives as reactants for coupling to thiol groups The present invention relates to a hydroxyalkyl starch derivative comprising a reactive 5 acetamide group as well as to a method for preparing the same. Further, the invention relates to the use of said hydroxyalkyl starch derivative as reactant for coupling to a thiol group of a further compound. Further, the present invention relates to a hydroxyalkyl starch derivative coupled to a thiol group of a further compound and a method for preparing the same. 10 Hydroxyalkyl starch (HAS), in particular hydroxyethyl starch (HES), is a substituted derivative of the naturally occurring carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95 % by weight, and is degraded by alpha amylases in the body. HES in particular exhibits advantageous biological properties and is 15 used as a blood volume replacement agent and in hemodilution therapy in clinics (Westphal et al., Anesthesiology, 2009, 111: 187-202). Amylopectin consists of glucose moieties, wherein in the main chain alpha-1,4-glycosidic bonds are present and at the branching sites alpha-1,6-glycosidic bonds are found. The physico-chemical properties of this molecule are mainly determined by the type of glycosidic bonds. Due to the nicked 20 alpha-1,4-glycosidic bond, helical structures with about six glucose-monomers per turn are produced. The physico-chemical as well as the biochemical properties of the polymer can be modified via substitution. The introduction of a hydroxyethyl group can be achieved via alkaline hydroxyethylation. By adapting the reaction conditions it is possible to exploit the different reactivity of the respective hydroxy group in the unsubstituted glucose monomer 25 with respect to a hydroxyethylation. Owing to this fact, the skilled person is able to influence the substitution pattern to a limited extent. It is generally accepted that the stability of polypeptides can be improved and the immune response against these polypeptides is reduced when the polypeptides are coupled to 30 polymeric molecules, i.e. when a conjugate of the polypeptide with the polymeric molecule is formed. Further, polymeric prodrugs, thus drugs coupled to polymeric compounds, were suggested to prolong the circulation lifetime in the body due to the increase in size of the drug-polymer conjugate when compared to the single drug which may prevent a quick removal of the drug by glomerular filtration through the kidneys. 35 Some ways of producing a hydroxyalkyl starch derivative for coupling to thiol groups of further compounds, such as cysteine groups of proteins are described in the art. 1 WO 2014/147175 PCT/EP2014/055598 For example, WO 02/080979 discloses a method for the preparation of hydroxyalkyl starch derivatives for coupling to thiol groups of DNA, wherein a hydroxyalkyl starch is first oxidized at its reducing end, subsequently modified with an amino group and finally reacted with succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) to 5 give a maleimide modified HAS derivative. Said HAS derivative is further coupled to a thiol group of DNA resulting in a hydroxyalkyl starch DNA conjugate. Halogenacetamide modified HAS derivatives and their coupling to further compounds are not mentioned. Similarly, WO 2005/014050 discloses a method for the preparation of hydroxyalkyl starch 10 GCS-F conjugates, wherein e.g. a HAS derivative comprising a maleimide group is disclosed. In this method, the hydroxyalkyl starch is first oxidized at its reducing end, subsequently modified with an amino group and finally reacted with N alpha(maleimidoacetoxy)succinimide ester (AMAS) to give the maleimide modified HAS derivative. Said HAS derivative is further coupled to a thiol group of GCS-F. HAS 15 derivatives modified with a reactive acetamide group and their coupling to further compounds are also not mentioned. Further, WO 2005/014050 mentiones, generally, the derivatization of an amino-modfied HAS with a monohalogen-subsituted acetic acids in the presence of an activating agent, such as EDC. 20 Halogenacetyl modified HAS molecules and their coupling to thiol groups of further compounds are further described e.g. in W02003/070772 and EP 1 398 322 Al. In the described method, HAS is modified via its oxidized reducing end with a linker compound to give the halogenacetyl modified HAS derivate. 25 Many of the above-described conjugation methods employ a type of chemistry whereby activated carboxylic acid derivatives of hydroxyalkyl starch are either formed as intermediate products or are used to introduce a functional group into the hydroxyalkyl group via a linker. However, the presence of multiple, potentially reactive, hydroxyl groups in the activated carboxylic acid derivative of the hydroxyalkyl starch can result in 30 intra- or intermolecular bond formation between hydroxyalkyl starch molecules, e.g. between the oxidized reducing end and hydroxyl groups, so potentially undesired by products may be formed, which are sometimes hard or even impossible to be purified away from the desired product. If a linker is coupled to hydroxyalkyl starch using activated carboxylic acid chemistry, a product mixture may potentially be obtained by reactions with 35 the multiple hydroxyl groups. This mixture may contain macromolecules carrying different numbers of linker molecules and/or variations in their attachment position within the macromolecules. So often a lower yield for the desired conjugation reaction between HES and the conjugation partner results and, in particular, an inhomogeneous composition potentially comprising crosslinked polymer side products is obtained. In addition, in these 2 WO 2014/147175 PCT/EP2014/055598 cases, the described oxidation of the reducing end of hydroxyalkyl starch is considered to be not completely selective, thus, also for this reason, potentially inhomogeneous products may result. Further some of the methods taught in the art, such as derivatization of amino modfied HAS with a monohalogen-subsituted acetic acids as taught in WO 2005/014050, 5 yield in derivatives which only display a low reactive group content and/or show disadvantageous reaction turnovers when converted to protein conjuagtes (see comparative examples E18 and E19). Further, in some of the ligation methods taught in the art, the resulting conjugates are disadvantageous as regards the efficiency of the method and/or in particular as regards the stability of the resulting derivatives. Further, some linking 10 strategies taught in the art, e.g. the maleimide-thio-linkage, are considered to have unpleasant side effects such as (unwanted) immunogenicity, a low stability of the thiol reactive functional group during storage and/or under reductive conditions (such as disulfide bonds) and/or in the conjugation reaction and the like. 15 Thus, there is still the need for advantageous hydroxyalkyl starch derivatives for coupling to thiol groups of further compounds, which can be formed in a highly selective manner, which highly selectively react with the further compound and/or with which a stable and biocompatible linkage to the further compound can be provided. Further, there is the need for a selective method for the preparation of such derivatives, in which possible side 20 reactions such as inter- and intramolecular crosslinking are significantly diminished or avoided and with which the derivatives can be provided in a high yield and high purity. It was thus an object of the present invention to provide hydroxyalkyl starch derivatives for coupling to thiol groups of further compounds which derivatives overcome the problems of 25 the prior art as well as a method for preparing the same, in particular which method provides the desired derivatives in high yield and with high specificity and purity. It is a further object of the present invention to provide novel, stable and biocompatible hydroxyalkyl starch derivatives comprising a protein attached to HAS via a thiol group of the protein as well as a method for preparing the same, in particular which method 30 provides the desired derivatives in high yield and with high specificity and purity. Therefore, the present invention relates to a hydroxyalkyl starch (HAS) derivative of formula (I) ORa HAS 'OH O 0 R O- C-F1-L1-S N-L2-N-J X
H
2 Rx ORc 35 wherein 3 WO 2014/147175 PCT/EP2014/055598 X is a leaving group, preferably selected from the group consisting of mesyl, tosyl, Cl, Br and I; F1 is a functional group comprising the group -NR'-, with R' being H or alkyl; LI is a spacer bridging F1 and S; 5 L2 is a spacer bridging -NR- and -NRY-; Rx and RY are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; HAS' is the remainder of the HAS molecule,R and R' are -[(CRR 2 )mO]n-H and are the 10 same or different from each other; Ra is -[(CR 1
R
2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR 1
R
2 ; n is from 0 to 6. 15 It is to be understood that if Ra is HAS", the hydroxyalkyl starch molecule has a branching site at the C6 position of the reducing end. Further, the present invention relates to a hydroxyalkyl starch (HAS) derivative of formula ORa HAS' ' OH O 0 RbO~~ C-Fl-Li-S N-L2-N 1 ZSQ
ORH
2 RX R (IV) 20 wherein -Q' is the remainder of a thiol group comprising compound Q which is linked via the group -S- of the thiol group to the group -NRY-C(=O)-CH 2 -; F1 is a functional group comprising the group -NR'-, with R' being H or alkyl; 25 LI is a spacer bridging F1 and S; L2 is a spacer bridging -NRx- and -NRY-; Rx and RY are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; HAS' is the remainder of the HAS molecule and Rb and R' are -[(CRR 2 )mO]n-H and are 30 the same or different from each other; Ra is -[(CR 1
R
2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR 1
R
2 ; n is 35 from 0 to 6. 4 WO 2014/147175 PCT/EP2014/055598 The present invention also relates to a method for the preparation of a hydroxyalkyl starch (HAS) derivative, and a hydroxyalkyl starch (HAS) derivative obtained or obtainable by said method, said method comprising 5 (i) reacting hydroxyalkyl starch (HAS) of formula (Ta) ORa HAS'-,0- 0 R b - O C *H ORc H (la) via carbon atom C* of the reducing end of the HAS with the functional group M of a crosslinking compound according to formula (TI) 10 M-L1-S-T (II) wherein M comprises the group -NHR', with R' being H or alkyl; LI is a spacer bridging M and S; T is H or a thiol protecting group PG; 15 HAS' is the remainder of the HAS molecule and Rb and R' are -[(CR 1
R
2 )mO]n-H and are the same or different from each other; Ra is -[(CRlR 2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 20 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR1R2; n is from 0 to 6, thereby obtaining a HAS derivative of formula (Ib) ORa HAS' OH RbO' C-F1-L1--S-T
H
2 ORc (Tb) 25 wherein -CH 2 -F1- is the moiety resulting from the reaction of the group M with the HAS via the carbon atom C* of the reducing end, and Fl is a functional group comprising the group -NR'-; optionally removing PG in case T is PG to give T = H; 30 (ii) reacting the HAS derivative obtained in step (i) with a crosslinking compound of formula (TTT) 5 WO 2014/147175 PCT/EP2014/055598 0 N-L2 - R X RYI) L2 is a spacer bridging -NR- and -NRY-; R and RY are, independently of each other, H or alkyl, and optionally form together 5 with the moiety N-L2-N a ring; thereby obtaining a HAS derivative of formula (I) ORa
HAS',
0 OH 0 O Rbo' C-F1-L1-S N-L2-- 'X ORcH 2 Rx (y 10 wherein X is a leaving group, preferably selected from the group consisting of mesyl, tosyl, Cl, Br and I. It has surprisingly been found that when conducting the above described method, the reaction product of the hydroxyalkyl starch derivative of formula (I) can be obtained in 15 high yields with high purity. Further, it has surprisingly been found that the derivatives according to formula (I) in which HAS is linked via its non-oxidized reducing end via a linker comprising an -NR' group and a group -S-, said group -S- being linked to an acetamide group were obtained 20 with a surprisingly high degree of selectivity for the modification of HAS via the reducing end. In other words, the reaction of hydroxyalkyl starch (HAS) of formula (Ta) with the crosslinking compound according to formula (II) showed an surprising selectivity for the reducing end of HAS. In addition, the reaction of the HAS derivative obtained in step (i) with a crosslinking compound of formula (III) was surprisingly chemoselectivFurther, the 25 derivatives according to formula (I) were surprisingly stable while at the same time being reactive and surprisingly selective towards thiol groups of further compounds. The resulting derivatives according to formula (IV) prepared using the derivatives of formula (I) were further obtained with surprisingly high yields and/or with a high 30 selectivity (see figure 4), thus they showed a high purity, and/or showed a surprisingly high stability (see e.g. figures 5-7) over a broad pH range, In particular at a physiological pH or higher the resulting derivatives according to formula (IV) showed surprisingly high stability. 6 WO 2014/147175 PCT/EP2014/055598 Further, derivatives according to formula (IV) (which may hereiunder also be referred to as "conjugates") comprising a protein as compound Q surprisingly showed a comparable activity in biological assays than than protein as such (see e.g. examples C3 to C4 hereinunder). 5 Thus, these conjugates are highly advantagoues since it is contemplated that the hydroxyalkyl starch prolongs the circulation time of the active agent in the body. Hydroxyalkyl Starch 10 Hydroxyalkyl starch is an ether derivative of optionally partially hydrolyzed native starches wherein hydroxyl groups of the starch are suitably hydroxyalkylated. As hydroxyalkyl starches, hydroxypropyl starch and hydroxyethyl starch are preferred, with hydroxyethyl starch being most preferred. 15 Starch is a well-known polysaccharide according to formula (C 6 Ho 10 0 5 )" which essentially consists of alpha-D glucose units which are coupled via glycosidic linkages. Usually, starch essentially consists of amylose and amylopectin. Amylose consists of linear chains wherein the glucose units are linked via alpha-1,4-glycosidic linkages. Amylopectin is a 20 highly branched structure with alpha-1,4-glycosidic linkages and alpha-1,6-glycosidic linkages. Native starches from which hydroxyalkyl starches can be prepared include, but are not limited to, cereal starches and potato starches. Cereal starches include, but are not limited 25 to, rice starches, wheat starches such as einkorn starches, spelt starches, soft wheat starches, emmer starches, durum wheat starches, or kamut starches, corn starches, rye starches, oat starches, barley starches, triticale starches, spelt starches, and millet starches such as sorghum starches or teff starches. Preferred native starches from which hydroxyalkyl starches are prepared have a high content of amylopectin relative to amylose. 30 The amylopectin content of these starches is, for example, at least 70 % by weight, preferably at least 75 % by weight, more preferably at least 80 % by weight, more preferably at least 85 % by weight, more preferably at least 90 % by weight such as up to 95 % by weight, up to 96 % by weight, up to 97 % by weight, up to 98 % by weight, up to 99 % by weight, or up to 100 % by weight. Native starches having an especially high 35 amylopectin content are, for example, suitable potato starches such as waxy potato starches which are preferably extracted from essentially amylose-free potatoes which are either traditionally bred (e.g. the natural variety Eliane) or genetically modified amylopectin potato varieties, and starches of waxy varieties of cereals such as waxy corn or waxy rice. 7 WO 2014/147175 PCT/EP2014/055598 A preferred hydroxyalkyl starch of the present invention has a constitution according to formula (Ta) ORa HAS'0 0 R bo. \C*H OOH ORc (1a) 5 wherein HAS' is the remainder of the HAS molecule and Rb and R' are -[(CRlR 2 )mO]n-H and are the same or different from each other; Ra is -[(CRlR 2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, 10 wherein R 1 and R 2 are the same or different from each other in the m groups CR 1
R
2 ; n is from 0 to 6. According to a preferred embodiment, R 1 , R 2 are, independently of each other, selected from the group consisting of hydrogen and a methyl group, more preferably all of R 1 , R 2 are H. 15 Integer m is of from 2 to 4, such as 2, 3 or 4, preferably m is 2. Integer n is of from 0 to 20, preferably of from 0 to 4, more preferably 0, 1, 2 or 3, most preferably 2. 20 According to a particularly preferred embodiment of the invention, the HAS derivative is a hydroxyethyl starch (HES) derivative. In this case, R 1 and R 2 are hydrogen, m is 2, n is 0 to 6, namely 0, 1, 2, 3, 4, 5, or 6, and Ra, R, R' are the same or different from each other. Preferably, Rb and R' are -[(CRlR 2 )mO]n-H and Ra is -[(CR 1
R
2 )mO]-H with HAS' being 25 the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule, with n being 0 to 6, namely 0, 1, 2, 3, 4, 5 or 6, wherein in each group Ra, R , Re, and n are the same or different from each other. 30 In formula (Ta) the reducing end of the starch molecule is shown in the non-oxidized form and the terminal saccharide unit of HAS is shown in the hemiacetal form which depending on e.g. the solvent, may be in equilibrium with the (free) aldehyde form. The abbreviation HAS' as used in the context of the present invention refers to the HAS molecule without the terminal saccharide unit at the reducing end of the HAS molecule. This is meant by the 35 term "remainder of the hydroxyalkyl starch molecule" as used herein. 8 WO 2014/147175 PCT/EP2014/055598 The term "hydroxyalkyl starch" within the meaning of the present invention is not limited to compounds where the terminal carbohydrate moiety comprises groups R, R and/or R' being -[(CRlR 2 )mO]n-H and/or HAS" as depicted, for the sake of brevity, in formula (Ta), 5 but refers to compounds in which at least one hydroxy group which is present anywhere else in the hydroxyalkyl starch, i.e. either in the terminal saccharide unit of the hydroxyalkyl starch molecule and/or in the remainder of the hydroxyalkyl starch molecule, HAS', is substituted by a group -[(CRlR 2 )mO]n-H . 10 It is to be understood that the integer m in each group -[(CR 1
R
2 )mO]n-H present in the HAS molecule may be the same or may be different. The same applies to integer n. The HAS may further contain one or more hydroxyalkyl groups, which comprise more than one hydroxyl group, in particular two or more hydroxyl groups. According to a preferred embodiment, the hydroxyalkyl groups comprised in HAS contain one hydroxy 15 group only. According to a preferred embodiment of the present invention, hydroxyalkyl starch according to above-mentioned formula (Ta) is employed. The other saccharide ring structures comprised in HAS' may be the same as or different from the explicitly described 20 saccharide ring, with the difference that they lack a reducing end. HAS, in particular HES, is mainly characterized by the molecular weight distribution, the degree of substitution and the ratio of C 2
/C
6 substitution. 25 There are two possibilities of describing the substitution degree. The degree of substitution (DS) of HAS, preferably HES, is described relatively to the portion of substituted glucose monomers with respect to all glucose moieties. The substitution pattern of HAS, preferably HES, can also be described as the molar 30 substitution (MS), wherein the number of hydroxyethyl groups per glucose moiety is counted. MS values of the respective HAS starting material, in particular the HES starting material, are given. It is assumed that the MS value is not affected during the method according to 35 the invention. HAS and in particular HES solutions are present as polydisperse compositions, wherein each molecule differs from the other with respect to the polymerization degree, the number and pattern of branching sites, and the substitution pattern. HAS and in particular HES is 9 WO 2014/147175 PCT/EP2014/055598 therefore a mixture of compounds with different molecular weight. Consequently, a particular HAS solution, and preferably a particular HES solution, is determined by the average molecular weight with the help of statistical means. In this context, M, is calculated as the arithmetic mean depending on the number of molecules and their 5 molecular weight. Alternatively, the mass distribution in HAS, and in particular HES, may be described by the weight average molecular weight M, (or Mw). The parameter Mn 10 The number average molecular weight is defined by the following equation: Mn =YIi niMi / Ii ni wherein ni is the number of hydroxyalkyl starch molecules of species i having molar mass Mi. 15 The parameter M. The weight average molecular weight is defined by the following equation: M, = Ii niMi 2 / Ii niMi 20 wherein ni is the number of hydroxyalkyl starch molecules of species i having molar mass Mi. According to the present invention, typical Mw values are preferably in the range of from 1 to 1000 kDa, more preferably of from 1 to 800 kDa, more preferably of from 1 to 700 kDa, more preferably of from 2 to 600 kDa, more preferably of from 5 to 500 kDa, 25 most preferably of from 25 to 400 kDa. The parameter MS The second parameter which is usually referred to as "MS" (molecular substitution) 30 describes the number of hydroxyalkylated sites per anhydroglucose unit of a given hydroxyalkyl starch (Sommermeyer et al., Krankenhauspharmazie 8 (8), 1987, pp 271-278, in particular page 273). The values of MS correspond to the degradability of the hydroxyalkyl starch by alpha-amylase. Generally, the higher the MS value of the hydroxyalkyl starch, the lower is its respective degradability. 35 The parameter MS can be determined according to Ying-Che Lee et al., Anal. Chem. 55, 1983, pp 334-338; or K. L. Hodges et al., Anal. Chem 51, 1979, p 2171. According to these methods, a known amount of the hydroxyalkyl starch is subjected to ether cleavage in xylene whereby adipinic acid and hydroiodic acid are added. The amount of released 10 WO 2014/147175 PCT/EP2014/055598 iodoalkane is subsequently determined via gaschromatography using toluene as an internal standard and iodoalkane calibration solutions as external standards. According to the present invention, typical MS values are in the range of from 0.1 to 3, preferably of from 0.4 to 1.3, such as 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 or 1.3. 5 The parameter C2/C6 ratio The third parameter which is referred to as "C2/C6 ratio" describes the ratio of the number of the anhydroglucose units being substituted in C2 position relative to the number of the 10 anhydroglucose units being substituted in C6 position. During the preparation of the hydroxyalkyl starch, the C2/C6 ratio can be influenced via the pH used for the hydroxyalkylation reaction. Generally, the higher the pH, the more hydroxyl groups in C6 position are hydroxyalkylated. The parameter C2/C6 ratio can be determined, for example, according to Sommermeyer et al., Krankenhauspharmazie 8 (8), 1987, pp 271-278, in 15 particular page 273. According to the present invention, typical values of the C2/C6 ratio are in the range of from 2 to 20, preferably of from 2 to 15, more preferably of from 2 to 12. 20 The reducing end According to a preferred embodiment the compound according to formula (II) is selectively reacted via carbon atom C* of the reducing end, i.e. with the reducing end of HAS. The term "selectively reacted with the reducing end" relates to processes according 25 to which preferably a least 95 %, more preferably at least 98 %, more preferably at least 99 %, more preferably at least 99.5 %, more preferably at least 99.9 % of all reacted HAS molecules are exclusively reacted via their reducing end group. According to the present invention HAS is reacted via its non-oxidized reducing end. 30 Step (i) In step (i) of the method according to the invention, the HAS according to formula (Ia) is reacted via carbon atom C* of the reducing end of the HAS with the functional group M of 35 a crosslinking compound according to formula M-L1-S-T (II), wherein a HAS derivative according to formula (Ib) is obtained, wherein -CH 2 -F1- is the moiety resulting from the reaction of the group M with the HAS via the carbon atom C* of the reducing end, and wherein F1 is a functional group comprising the group -NR'-. 11 WO 2014/147175 PCT/EP2014/055598 The functional group F1 and the functional group M M is a functional group comprising the moiety -NHR', with R' being H or alkyl. 5 Preferably R' is selected from the group consisting of H, methyl, ethyl and propyl. According to a preferred embodiment of the invention, the functional group M of the crosslinking compound according to formula (II) is selected from the group consisting of of CH 3 -NH-, CH 3
-CH
2 -NH-, CH 3
-CH
2
-CH
2 -NH-, (CH 3
)
2 -CH-NH-, H 2 N-, H 2 N-O-, H 2
N
10 NH-, H 2 N-NH-(C=G)-, H 2 N-NH-(C=G)-GG- and H 2
N-NH-SO
2 - with G being 0, S or NR m with R m being H or alkyl, preferably with G being 0; and with GG being 0, S or NRG and with RG being H or alkyl, in particular with GG being 0 or NRG with RG being H, the crosslinking compound thus preferably having one of the following structures: CH 3
-NH
Li-S-T, CH 3
-CH
2 -NH-L1-S-T, CH 3
-CH
2
-CH
2 -NH-L1-S-T, (CH 3
)
2 -CH-NH-L1-S-T, H 2
N
15 Li-S-T, H 2 N-0-L1-S-T, H 2 N-NH-L1-S-T, H 2 N-NH-(C=G)-L1-S-T, H 2 N-NH-(C=G)-G G_ Li-S-T or H 2
N-NH-SO
2 -L1-S-T, and more preferably one of the following structures:
CH
3 -NH-L1-S-T, CH 3
-CH
2 -NH-L1-S-T, CH 3
-CH
2
-CH
2 -NH-L1-S-T, (CH 3
)
2 -CH-NH-L1 S-T, H 2 N-L1-S-T, H 2 N-0-L1-S-T, H 2 N-NH-L1-S-T, H 2 N-NH-(C=0)-L1-S-T, H 2
N-NH
(C=0)-NH-L 1-S-T, H 2 N-NH-(C=0)-O-L 1-S-T or H 2
N-NH-SO
2 -L 1-S-T. 20 Thus, the present invention also relates to a method for the preparation of a hydroxyalkyl starch derivative, as described above, wherein in step (i), the hydroxyalkyl starch (HAS) of formula (Ta) is reacted via carbon atom C* of the reducing end of the HAS with the functional group M of a crosslinking compound according to formula (II) 25 M-L1-S-T (II) wherein M of the crosslinking compound according to formula (II) is selected from the group consisting of CH 3 -NH-, CH 3
-CH
2 -NH-, CH 3
-CH
2
-CH
2 -NH-, (CH 3
)
2 -CH-NH-, H 2 N-, 30 H 2 N-0-, H 2 N-NH-, H 2 N-NH-(C=G)-, H 2 N-NH-(C=G)-G G- and H 2
N-NH-SO
2 - with G being 0, S or NR m with R m being H or alkyl, preferably with G being 0; and with GG being 0, S or NRG with RG being H or alkyl, in particular with GG being 0 or NRG and with RG being H, the crosslinking compound thus preferably having one of the following structures: CH 3 -NH-L1-S-T, CH 3
-CH
2 -NH-L1-S-T, CH 3
-CH
2
-CH
2 -NH-L1-S-T, (CH 3
)
2 35 CH-NH-L1-S-T, H 2 N-L1-S-T, H 2 N-0-L1-S-T, H 2 N-NH-L1-S-T, H 2 N-NH-(C=G)-L1-S-T,
H
2 N-NH-(C=G)-G G-Li-S-T or H 2 N-NH-S0 2 -L1-S-T, more preferably one of the following structures: CH 3 -NH-L1-S-T, CH 3
-CH
2 -NH-L1-S-T, CH 3
-CH
2
-CH
2 -NH-L1-S-T,
(CH
3
)
2 -CH-NH-L1-S-T, H 2 N-L1-S-T, H 2 N-0-L1-S-T, H 2 N-NH-L1-S-T, H 2 N-NH-(C=0) 12 WO 2014/147175 PCT/EP2014/055598 Li-S-T, H 2 N-NH-(C=O)-NH-L1-S-T, H 2 N-NH-(C=O)-O-L1-S-T or H 2 N-NH-S0 2 -L1-S T. Thereby, a HAS derivative according to formula (Ib) ORa HAS 0O OH RIO' C-F1-L 1 -S-T
H
2 5 ORc (Ib) is obtained, wherein -CH 2 -F1- is the moiety resulting from the reaction of the group M with the HAS via the carbon atom C* of the reducing end, and Fl is a functional group comprising the group -NR'-, with R' being H or alkyl. Preferably F1 is -(CH 3 )-N-, -(CH 3 10 CH 2 )-N-, -(CH 3
-CH
2
-CH
2 )-N-, -((CH 3
)
2 -CH)-N-, -HN-, -HN-0-, -HN-NH-, -HN-NH (C=G)-, -HN-NH-(C=G)-G - and -HN-NH-S0 2 -. More preferably R' is H, thus the functional group M of the crosslinking compound according to formula (II) is preferably a functional group comprising the moiety H 2 N-, 15 more preferably M is selected from the group consisting of H 2 N-, H 2 N-O-, H 2 N-NH-, H 2
N
NH-(C=G)-, H 2 N-NH-(C=G)-GG- and H 2
N-NH-SO
2 - with G being 0, S or NR' with R" being H or alkyl, preferably with G being 0; and with GG being 0, S or NRG with RG being H or alkyl, in particular with GG being 0 or NRG and with RG being H, more preferably M is selected from the group consisting of H 2 N-, H 2 N-0-, H 2 N-NH-, H 2
N-NH
20 (C=0)-, H 2 N-NH-(C=0)-NH-, H 2 N-NH-(C=0)-O- and H 2
N-NH-SO
2 -; more preferably M is selected from the group consisting of H 2 N-, H 2 N-0-, H 2 N-NH- and H 2 N-NH-C(=0)-, more preferably M is H 2 N-, the crosslinking compound thus having the structure H 2 N-L1 S-T. 25 Consequently, F 1 is preferably selected from the group consisting of -HN-, -HN-0-, -HN NH-, -HN-NH-(C=G)-, -HN-NH-(C=G)-GG- and -HN-NH-S0 2 - with G being 0, S or NR" with R' being H or alkyl, preferably with G being 0; and with GG being 0, S or NRG with RG being H or alkyl, in particular with GG being 0 or NRG and with RG being H, more preferably F1 is selected from the group consisting of -HN-, -HN-0-, -HN-NH-, -HN-NH 30 (C=0)-, -HN-NH-(C=0)-NH-, -HN-NH-(C=0)-O- and -HN-NH-SO 2 -; more preferably F1 is selected from the group consisting of, -HN-, -HN-0-, -HN-NH- and -HN-NH-C(=0)-, more preferably Fl is -NH-. Thus, the present invention also relates to a HAS derivative, as described above, or a HAS derivative obtained or obtainable by a method as described above, wherein Fl is selected from the group consisting of -HN-, -HN-0-, -HN-NH-, 35 -HN-NH-(C=G)-, -HN-NH-(C=G)-G G- and -HN-NH-SO 2 -, more preferably F1 is -NH-. 13 WO 2014/147175 PCT/EP2014/055598 Thus, the present invention relates to a hydroxyalkyl starch (HAS) derivative, as described above, or a HAS derivative obtained or obtainable by the method as described above, wherein F1 is -NH-, the derivative thus having a structure according to the following formula: ORa HAS',, OH 0 0 RbO.~ C-N-Li-S N-L2-N 5 ORcH 2 Rx RY Further, the present invention relates to a hydroxyalkyl starch (HAS) derivative, as described above, or a HAS derivative obtained or obtainable by the method as described above, wherein F1 is -NH-, and wherein the derivative has a structure according to the 10 following formula: ORa HAS, ' OH 0 O bO~~ C-N-L1-S N-L2-N-I Q' ORcH 2 Rx Ry (IV). The spacer Li 15 As described above LI is a spacer bridging M and S or bridging F1 and S, respectively. Thus, in case the HAS derivatives described above are prepared by a method comprising the step (i), as described above, thus by reacting the reducing end of HAS with the crosslinking compound according to formula (II), LI is first linking M and S in the crosslinking compound, and, subsequently, after reaction of the crosslinking compound of 20 formula (II) with the reducing end, whereupon F1 is formed, LI is linking the thus obtained functional group F1 and S. Preferably, LI comprises, more preferably consists of, an alkyl, alkenyl, alkylaryl, arylalkyl, aryl or heteroaryl group. 25 Within the meaning of the present invention, the term "alkyl" relates to non-branched alkyl residues, branched alkyl residues, cycloalkyl residues, as well as residues comprising one or more heteroatoms or functional groups, such as, by way of example, -0-, -S-, Li -NR -, -NH-C(=0)-, -C(=0)-NH- and the like, with RL being alkyl, preferably methyl. 30 The term also encompasses alkyl groups which are further substituted by one or more suitable substituent. The term "substituted alkyl" as used in this context of the present invention preferably refers to alkyl groups being substituted in any position by one or more substituents, preferably by 1, 2, 3, 4, 5 or 6 substituents, more preferably by 1, 2, or 3 14 WO 2014/147175 PCT/EP2014/055598 substituents. If two or more substituents are present, each substituent may be the same as or may be different from the at least one other substituent. There are in general no limitations as to the substituent. 5 "Suitable substituents" in the context of spacer LI are, for example, selected from the group consisting of alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, 10 arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, amide, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, carboxymethylcarbamoyl [i.e. the group -C(=O)(-NH-CH 2 -COOH)], cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl. Preferred substituents of such organic 15 residues are, for example, alkyl groups, amide groups, hydroxyl groups, and carboxyl groups. It is to be understood that, within the meaning of the present invention, the term suitable substituents as used hereinunder and above also includes suitable salts of the respective 20 substituents. The term "alkenyl" as used in the context of the present invention refers to unsaturated alkyl groups having at least one double bond. The term also encompasses alkenyl groups which are substituted by one or more suitable substituent. 25 The term "alkynyl" refers to unsaturated alkyl groups having at least one triple bond. The term also encompasses alkynyl groups which are substituted by one or more suitable substituent. 30 Within the meaning of the present invention, the term "aryl" refers to, but is not limited to, optionally suitably substituted 5- and 6-membered single-ring aromatic groups as well as optionally suitably substituted multicyclic groups, for example bicyclic or tricyclic aryl groups. The term "aryl" thus includes, for example, optionally suitably substituted phenyl groups or optionally suitably substituted naphthyl groups. Aryl groups can also be fused or 35 bridged with alicyclic or heterocycloalkyl rings which are not aromatic so as to form a polycycle, e.g., benzodioxolyl or tetraline. The term "heteroaryl" as used within the meaning of the present invention includes optionally suitably substituted 5- and 6-membered single-ring aromatic groups as well as 15 WO 2014/147175 PCT/EP2014/055598 substituted or unsubstituted multicyclic aryl groups, for example bicyclic or tricyclic aryl groups, comprising one or more, preferably from 1 to 4, such as 1, 2, 3 or 4, heteroatoms, wherein in case the aryl residue comprises more than 1 heteroatom, the heteroatoms may be the same or different. Such heteroaryl groups including from 1 to 4 heteroatoms are, for 5 example, benzodioxolyl, pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzoimidazolyl, benzothiophenyl, methylenedioxyphenyl, napthyridinyl, quinolinyl, isoquinolinyl, indolyl, benzofuranyl, purinyl, benzofuranyl, deazapurinyl, or indolizinyl. 10 The terms "substituted aryl" and "substituted heteroaryl" as used in the context of the present invention describe moieties having suitable substituents replacing a hydrogen atom on one or more atoms, e.g. C or N, of an aryl or heteroaryl moiety. 15 Preferably, the spacer Li comprises the moiety -(C(L'L"))q- with L' and L" in each repeating unit -C(L'L")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, 20 phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, carboxymethylcarbamoyl [i.e. the group -C(=O)(-NH-CH 2 -COOH)], cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or 25 piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other, with q preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4. 30 According to a preferred embodiment of the invention, L' and L" are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), amide groups, hydroxyl groups, and carboxyl groups, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other. 35 According to a particularly preferred embodiment of the invention, L' and L" are, independently of each other, selected from the group consisting of H, amide, carboxyl and alkyl (including substituted alkyl groups, in particular including hydroxyalkyl groups), 16 WO 2014/147175 PCT/EP2014/055598 more preferably L' and L" are H or alkyl, such as H or methyl, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other. More preferably, LI has a structure selected from the group consisting of LL L" L" L** 5 and s r s with q preferably being in the range of from 1 to 20, wherein L', L", L* and L**, are independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, 10 aryloxycarbonyloxy, amide, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, carboxymethylcarbamoyl [i.e. the group -C(=O)(-NH-CH 2 15 COOH)], cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other, with q preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4, and with r 20 preferably being in the range of from 1 to 10 and with s preferably being in the range of from 1 to 10, and wherein Yi is a functional moiety selected from the group consisting of 0-, -S-, -NR" 1 -, -NH-C(=0)-, -C(=0)-NH-, 0 N IH (,,p-phenyl-C(=0)-NH-"), and wherein R"' is alkyl, preferably methyl, and wherein the groups L', L", L* and L**, in 25 each repeating unit, may be the same or may differ from each other. LI may have one or more asymmetric centers. As consequence, the linker may be employed as mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All possible stereoisomers, single isomers 30 and mixtures of isomers are included within the scope of the present invention. In case LI comprises one or more asymmetric centers, LI is preferably employed in enantiomeric or diastereomeric pure form. 17 WO 2014/147175 PCT/EP2014/055598 Preferably, L', L", L* and L** are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), amide groups, hydroxyl groups, and carboxyl groups, more preferably from H, amide including -C(=O)-NH 2 , carboxyl, hydroxyl and alkyl, in 5 particular L', L", L* and L** are, independently of each other, H or alkyl, and wherein Yi is a functional moiety as described above, preferably wherein Y is 0, -NH-C(=0)- or C(=0)-NH-. In case q is > 1, the repeating units -(C(L'L"))- may be the same or may be different from each other. In case r is > 1, the repeating units -(C(L*L**))- may be the same or may be different from each other. In case s is > 1, the repeating units -Yi 10 [C(L*L**)],- may be the same or may be different from each other. According to one preferred embodiment, LI has the structure q 15 wherein L' and L" are, independently of each other, selected from H and alkyl, wherein in each repeating unit -(C(L'L"))-, in case q is > 1, L' and L" may be the same or may be different from each other. As example, without being meant to be limiting, the following groups LI are mentioned: 20 -CH 2 -, -CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2 -,
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 CH 2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH(CH 3 )-, -CH(CH 3
)-CH
2 -, -CH 2
-CH(CH
3 )-,
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH(CH
3 )-, -CH(CH 3
)-CH
2
-CH
2 25 CH 2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3 )-,
-CH(CH
3
)-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)
CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH(CH
3 )-, -CH(CH 3
)
CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)-CH
2 CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH(CH
3
)-CH
2 -, 30 -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH(CH
3 )-, -CH 2
-C(CH
3
)
2
-CH
2 -, -CH(CH 3
)-CH(CH
3 )-,
-C(CH
3
)
2
-C(CH
3
)
2 -, -CH(CH 2
OH)-CH
2 -, -CH(CH 2
OH)-CH
2
-CH
2 -, -CH(CONH 2
)-CH
2 -,
-CH(COOH)-CH
2 -, -CH(COOH)-CH 2
-CH
2 -, -CH(COOH)-CH 2
-CH
2
-CH
2
-CH
2 -,
-CH(CONH
2
)-C(CH
3
)
2 -, -CH(CONH 2
)-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(OH)-CH
2 -, -CH 2 CH(OH)-CH(OH)-CH 2 - and -CH(COOH)-C(CH 3
)
2 -. 35 More preferably L' and L" are in each repeating unit H. 18 WO 2014/147175 PCT/EP2014/055598 According to a particularly preferred embodiment of the invention, LI is -CH 2
-CH
2 -. According to an alternative embodiment, as described above, LI has as structure according to the following formula L' L* L" L** q ,r s 5 with q preferably being in the range of from 1 to 20, with r preferably being in the range of from I to 10, with s preferably being in the range of from I to 10 and with L', L", L* and L** being as described above. 10 As regards the functional moiety Y 1 , said functional moiety is preferably selected from the group consisting of -0-, -S-, -NRY 1 -, -NH-C(=0)-, -C(=0)-NH-, p-phenyl-C(=0)-NH with R"' in particular being methyl, more preferably wherein Yi is -0-, -NH-C(=0)- or C(=0)-NH-. 15 According to one preferred embodiment of the present invention, s is 1. Thus, LI is, for example, -[C(L'L'')]q-N(CH 3 )-[C(L*L**)],-, -[,,-[C'*(L ')]q N(CH3)-[C(L*L**)]r, -[C(L'L")]q-NH-C(=0)-[C(L*L**)]r, -[C(L'L")]q-C(=0)-NH [C(L*L**)]r or -[C(L'L")]q-p-phenyl-C(=0)-NH-[C(L*L**)],-. Most preferably q is in the range of from 2 to 4. Further, r is in particular in the range of from 2 to 4. According to a 20 preferred embodiment, Yi is -0- or -C(=0)-NH-. Thus, as example, without being meant to be limiting, the following groups LI are mentioned: -CH 2
-CH
2 -0-CH 2
-CH
2 -, -CH 2
-CH
2 -0-CH 2
-CH
2 -0-CH 2
-CH
2 -, -CH 2
-CH
2 -0
CH
2
-CH
2 -0-CH 2
-CH
2 -0-CH 2
-CH
2 -, and -CH(COOH)-CH 2
-CH
2 -C(=0)NH-CH-C(=0)( 25 NH-CH 2
-COOH)-CH
2 -. According to a further preferred embodiment, s is > 1, preferably of from 2 to 10. In this case, Yi is preferably -0-, -C(=0)-NH- or -NH-C(=0)-. Thus, this embodiment includes any spacer LI derived from a peptide, thus having a peptidic backbone. In case LI is 30 derived from a peptidic crosslinking compound (II), the crosslinking compound (II) is preferably employed in enantiomeric or diastereomeric pure form, more preferably in the natural occurring stereoisomeric form. In step (i) HAS is preferably dissolved in an aqueous medium or a reaction buffer, and a 35 crosslinking compound according to formula (II) is subsequently added. 19 WO 2014/147175 PCT/EP2014/055598 Preferably, the reaction in (i) is carried out in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, acetic acid reaction buffers and mixtures of two or more thereof. Preferred reaction buffers are, e.g., sodium citrate buffer, sodium acetate buffer, sodium phosphate buffer, sodium carbonate buffer, or sodium borate buffer. 5 Preferred pH values of the reaction buffers are in the range of from 4 to 9, more preferably of from 5 to 8. The pH values given hereinunder and above refer to pH values determined via a pH sensitive glass electrode. In a preferred embodiment of the invention, the reaction is carried out in water. 10 The crosslinking compound of formula (II) is preferably used as free amine or as a salt and added as solid. Preferred are salts such as the hydrochloride, sulfate, hemisulfate, acetate or trifluoroacetate salt. In step (i) preferably an excess of the crosslinking compound of formula (II) is employed. 15 Preferably, the minimum amount of crosslinking compound (II) is one molar equivalent with respect to the amount of reducing ends to be reacted. The maximum amount is given by the solubility limit of the crosslinking compound in the particular reaction solvents. Preferably, the crosslinking compound is employed at a concentration in the range of at least 0.05 mol/l, preferably at least 0.1 mol/l, more preferably in the range of from 0.2 to 4 20 mol/l, more preferably from 0.5 to 2 mol/l, more preferably 0.9 mol/l to 1.1 mol/l, most preferably about 1 mol/l.. The reaction mixture is preferably stirred at a temperature in the range of from 5 'C to 100 'C, more preferably at a temperature in the range of from 20 'C to 90 'C, more 25 preferably in the range of from 40 'C to 80'C. During the course of the reaction, the temperature may be varied, preferably in the above-given ranges, or held essentially constant. The reaction is preferably conducted for a time in the range of from 1 to 48 h, more 30 preferably from 2 to 36 h, more preferably from 4 to 18 h. Upon reaction of M which comprises the NHR'- group with the reducing end, initially a functional group F1 * is formed, said functional group comprising the structure -CR'=N-. For example, in case M consists of an NH 2 - group, initially a functional group F 1* is 35 formed, said functional group having the structure -CH=N-. In case M is an NH 2
-NH
group, the functional group F 1* which is formed upon reaction of M with the reducing end, is a -CH=N-NH- group. In case M is an NH 2 -0- group, the functional group F1 * which is formed upon reaction of M with the reducing end, is a -CH=N-0- group. 20 WO 2014/147175 PCT/EP2014/055598 The hydroxyalkyl starch derivative obtained, i.e. hydroxyalkyl starch derivative comprising the group F1 *, said group comprising the structure -CR'=N-, preferably being selected from the group consisting of -CH=N-NH-, -CH=N- and -CH=N-O-, may be isolated from the reaction mixture by ultrafiltration or dialysis, 5 preferably ultrafiltration followed by lyophilization of the isolated hydroxyalkyl starch derivative. In case this HAS derivative which comprises the functional group F* contains a free thiol group (i.e. T = H), the ultrafiltration or dialysis is preferably carried out under neutral 10 conditions, preferably in water. Further, the hydroxyalkyl starch derivative may be precipitated from the reaction mixture, in particular by adding an alcohol, preferably 2 propanol. The obtained precipitate may be collected by filtration or centrifugation and may further be purified using conventional purification protocols, preferably ultrafiltration, dialysis or chromatographic methods, preferably size exclusion chromatography. 15 According to the invention, step (i) preferably additionally comprises the conversion of the, optionally isolated, hydroxyalkyl starch derivative obtained upon reaction of HAS with the crosslinking compound according to formula (II) prior to step (ii). In this step the functional group F1* obtained upon reaction of M with the reducing end is suitably 20 reduced, to give the functional group F1. For example, in case the linking group is a -CH=N- group, said group is reduced to give the group F1 with F1 being -CH 2 -NH-. In case, the linking group is a -CH=N-NH 2 - group, said group is reduced to give the group F1 with F1 being -CH 2
-NH-NH
2 -. In case, the linking group is a -CH=N-0- group, said group is reduced to give the group F1 with F1 being -CH 2 -NH-O-. 25 The reduction is preferably carried out in the presence of a suitable reducing agent, such as sodium borohydride, sodium cyanoborohydride, sodium triacetoxy borohydride, organic borane complex compounds such as a 4-(dimethylamino)pyridine borane complex, N ethyldiisopropylamine borane complex, N-ethylmorpholine borane complex, N 30 methylmorpholine borane complex, N-phenylmorpholine borane complex, lutidine borane complex, triethylamine borane complex, or trimethylamine borane complex, preferably sodium cyanoborohydride. Preferably, this reduction is carried out using an excess of reducing agent, so preferably a 35 minimum of one molar equivalent with respect to the amount of reducing ends in HAS is applied. More preferably, the concentration of the reducing agent used for this reaction of the present invention is in the range of from 0.001 to 3.0 mol/l, more preferably in the range of from 0.05 to 2.0 mol/l, more preferably in the range of from 0.1 to 1 mol/l, more 21 WO 2014/147175 PCT/EP2014/055598 preferably in the range of 0.3-0.6 mol/L, relating, in each case, to the volume of the reaction solution. The reduction, as described above, can either be carried out subsequently to the coupling 5 process, in which M is coupled to the reducing end, optionally after isolating the coupled product prior to the reduction, or it is possible to carry out the same reaction all in one pot, with the coupling to the reducing end and the reduction occurring concurrently. Most preferably the above mentioned one pot synthesis is carried out. Both reactions are referred to in the context of the present invention as "reductive amination". 10 Thus, the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, wherein the functional group M comprises the group HR'N-, preferably H 2 N-, more preferably consist of the group H 2 N- and the reaction according to step (i) is a reductive amination. Further, the present invention also relates to 15 a method as described above, and a conjugate obtained or obtainable by said method, wherein the functional group M is H 2 N-NH- and the reaction according to step (i) is a reductive amination. Since, the the coupling and reduction, as described above, are preferably carried out in one 20 pot, the solvent used for the reduction step is preferably also selecetd from the solvents already mentioned above in the context of step (i). Thus preferably, the reductive amination is carried out in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, acetic acid reaction buffers and mixtures of two or more thereof. Preferred pH values of are thus in the range of from 4 to 9, more preferably of 25 from 5 to 8. During the reductive amination reaction, the temperature of the reaction mixture is suitably chosen. Generally, during the reductive amination reaction, the temperature of the reaction mixture is in the range of from 5 to 100 'C such as from 20 to 90 'C or from 40 to 80 'C. 30 Preferably, during the reductive amination reaction, the temperature of the reaction mixture is in the range of from 45 to 75 'C, more preferably from 55 to 65 'C. The reductive amination reaction can be carried out for any suitable time period. Generally, the time period is in the range of from I to 48 h such as from 2 to 36 h. Preferably, the time period is in the range of from 3 to 24 h, more preferably from 6 to 21 h, more preferably from 4 to 35 18 h. Preferably, to reductive amination is carried out at a temperature of the reaction mixture in the range of from 40 to 90 'C for a time period of from 1 to 36 h, more preferably at a temperature in the range of from 45 to 80 'C for a time period of from 2 h to 24 h, more preferably at a temperature in the range of from 55 to 65 'C for a time period of from 4 to 18 h. 22 WO 2014/147175 PCT/EP2014/055598 Thus, the present invention also relates to a method, as described above, and a HAS derivative obtained or obtainable by said method, wherein the reacting according to step (i) is carried out under reductive amination conditions, preferably at a temperature in the 5 range of from 5 'C to 100 'C and in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, acetic acid, and reaction buffers and mixtures of two or more thereof. According to above-described preferred embodiment wherein the reaction of the 10 crosslinking compound (II) with HAS is carried out under reductive amination conditions, the concentration of HAS, preferably HES, in the aqueous system is preferably in the range of at least 1 weight-%, more preferably at least 10 weight-%,, more preferably in the range of from 20-40 weight-%,, most preferably around 30 weight-%, based on the total weight of the whole solution. 15 After having finished the reductive amination reaction, the reaction mixture obtained is preferably subjected to a suitable work up. Such working up may comprise one or more stages wherein preferably at least one stage comprises a purification, preferably a purification by ultrafiltration, precipitation, size exclusion chromatography, and a 20 combination of two or more of these methods, more preferably by ultrafiltration. Optionally, such working up may comprise at least one stage which comprises a pH adjustment, preferably an adjustment to a pH of at least 8, preferably at least 9, more preferably in the range of from 9 to 11. Adjusting the pH of the reaction mixture to a value of at least 8, preferably at least 9, more preferably from 9 to 11 can be realized, if carried 25 out, according to all conceivable methods. Preferably, an inorganic base, preferably an alkali metal base and/or an alkaline earth metal base, more preferably an alkali metal hydroxide and/or an alkaline earth metal hydroxide, more preferably an alkaline metal hydroxide, more preferably sodium hydroxide is added in a suitable amount. The addition of such a basic compound can be performed at the temperature of the reaction mixture of 30 the reductive amination reaction. Preferably, the reaction mixture obtained from the reductive amination reaction is cooled before the basic compound is added, preferably to a temperature in the range of from 10 to 35 'C, more preferably from 20 to 30 'C. During adding the basic compound, the mixture can be suitably stirred. The pH is to be understood as the value indicated by a pH sensitive glass electrode without correction. The preferably 35 applied ultrafiltration can be performed according to all suitable methods. Preferably, the ultrafiltration comprising at least one volume exchange with water, preferably at least five volume exchanges with water, more preferably at least 10 volume exchanges with water. According to an embodiment of the present invention, the ultrafiltration does not comprise a volume exchange with an acid. Preferably, the ultrafiltration does not comprise a volume 23 WO 2014/147175 PCT/EP2014/055598 exchange with a base. More preferably, the ultrafiltration does not comprise a volume exchange with an acid and does not comprise a volume exchange with a base. The purified mixture can be subjected directly, without any further intermediate stage, to 5 step (ii) or optionally to further reducing condition as described hereinunder. It is also possible to freeze the purified mixture and subject it to further reducing condition as described hereinunder after suitable unfreezing. 10 According to a preferred embodiment of the invention, in step (i), subsequent to the reductive amination conditions, and the optional work-up described above, the HAS derivative is subjected to further reducing conditions. As possible reducing agents in this step, complex hydrides such as borohydrides, especially 15 sodium borohydride, and thiols, especially dithiothreitol (DTT) and dithioerythritol (DTE) or phosphines such as tris-(2-carboxyethyl)phosphine (TCEP) are mentioned. The reduction is preferably carried out using borohydrides, especially sodium borohydride. Preferably, the reducing agent is used in an excess, more preferably at a concentration in the range of from 0.02 to 1.5 M, more preferably in the range of from 0.05 to 1 M, most 20 preferably in the range of from 0.1 to 0.5 M with respect to the total volume of the reaction solution. Further, this deprotection step is preferably carried out at a temperature in the range of from 0 to 80 'C, more preferably in the range of from 10 to 50 'C and especially preferably in the range of from 15 to 35'C. During the course of the reaction, the temperature may be varied, preferably in the above-given ranges, or held essentially 25 constant. Preferably, the reaction is carried out in aqueous medium. The term "aqueous medium" as used in this context of the present invention refers to a solvent or a mixture of solvents comprising water in an amount of at least 10 % per weight, preferably at least 20 % per 30 weight, more preferably at least 30 % per weight, more preferably at least 40 % per weight, more preferably at least 50 % per weight, more preferably at least 60 % per weight, more preferably at least 70 % per weight, more preferably at least 80 % per weight, even more preferably at least 90 % per weight or up to 100 % per weight, based on the weight of the solvents involved. The aqueous medium may comprise additional solvents like formamide, 35 dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), N methylpyrrolidinone (NMP), alcohols such as methanol, ethanol or isopropanol, acetonitrile, tetrahydrofurane or dioxane. The aqueous solution may also contain a transition metal chelator (disodium ethylenediaminetetraacetate, EDTA, or the like) in a 24 WO 2014/147175 PCT/EP2014/055598 concentration ranging from 0.01 to 100 mM most preferably from 0.1 to 10 mM, such as about 1 mM. The preferred solvent is water. The HAS derivative is reacted in the further reduction step preferably at a concentration in 5 the range of from 1% to 30% weight.-%, more preferably in the range of from 5% to 20% weight.-%, most preferably 10% weight.-% based on the total weight of the solution. Most preferably, in the further reducing step, sodium borohydride (NaBH 4 ) is employed as reducing agent. Preferably, the mixture obtained from adding the sodium borohydride 10 comprises the sodium borohydride preferably at a concentration in the range of from 0.05 to 1.5 mol/l, more preferably from 0.05 to 1 mol/l, more preferably from 0.1 to 0.5 mol/l. Preferably, in (i), the mixture contains the hydroxyalkyl starch and the hydroxyalkyl starch derivative at a concentration in the range of from 1 to 40 weight-%, more preferably from 5 to 30 weight-%, more preferably from 10 to 20 weight-%. Subjecting the mixture to 15 these further reducing conditions in (i) can be carried out for any suitable time period. Generally, the time period is in the range of from 10 min to 24 h. Preferably, the time period is in the range of from 0.25 to 4 h, more preferably from 1 to 3 h. The further reduction reaction using sodium boohhydrate can be carried out at every suitable temperature. Preferably, the reduction with sodium borohydrate is carried out a 20 temperature in the range of from 5 to 40 'C, more preferably from 10 to 35 'C, more preferably from 20 to 30 'C such as at room temperature. Preferably, in (i), subjecting the mixture to the further reducing conditions comprises keeping the mixture at a temperature in the range of from 10 to 35 'C for a period of from 0.25 to 4 h, more preferably at a temperature in the range of from 20 to 30 'C for a period of from 1 to 3 h. 25 According to the embodiment, wherein the HAS derivative according to formula (Ib) ORa HAS 0 OH RIO- C-F1-L1-S-T
H
2 OR (Ib) has been subjected to the further reducing step, this HAS derivative may then preferably be 30 isolated from the reaction mixture by any suitable method, such as ultrafiltration or dialysis, preferably ultrafiltration, more preferably followed by lyophilization of the isolated hydroxyalkyl starch derivative. The ultrafiltration or dialysis is preferably carried out under acidic conditions, more preferably at a pH in the range of from 2 to 6, more preferably in the range of from 3 to 5, and/or in the presence of an ion chelator. 35 25 WO 2014/147175 PCT/EP2014/055598 Preferably, the acid, if present, is selected from the group consisting of hydrochloric acid, phosphoric acid, trifluoroacetic acid, acetic acid and mixtures of two or more thereof; preferred buffers are selected from the group consisting of acetate, phosphate and citrate buffers. As ion chelators EDTA (ethylenediamine tetraacetic acid), DTPA (diethylene 5 triamine pentaacetic acid) and related compounds may be mentioned. Most preferably, an acetic acid buffer (in the range of from 0.1 mM to 1 M, more preferably in the range of from 1 to 100 mM, most preferably 10 mM) with pH 4 which more preferably comprises EDTA in the range of from 0.01 to 100 mM (most preferably 10 1 mM) is used as ultrafiltration / dialysis buffer. More preferably, after removal of the reaction impurities, the ultrafiltration / dialysis buffer is replaced by water in order to remove buffer salts from the product. Further, the hydroxyalkyl starch derivative according to formula (Ib) may be precipitated 15 from the reaction mixture, in particular by adding an alcohol, preferably 2-propanol. The obtained precipitate may be collected by filtration or centrifugation and further purified using conventional purification protocols, preferably ultrafiltration, dialysis or chromatographic methods, preferably size exclusion chromatography. 20 The group T As mentioned above, T is H or a thiol protecting group PG. Thus, the present invention also relates to a method as described above, and a conjugate 25 obtained or obtainable by said method, comprising (i) reacting hydroxyalkyl starch (HAS) of formula (Ta) ORa HAS -,00 ' R b O - C * H ORC OH (la) via carbon atom C* of the reducing end of the HAS with the functional group M of a 30 crosslinking compound according to formula (I) thereby obtaining a HAS derivative of one of the following formulas: ORa ORa HAS' 0 OH HAS' OH RBO C-F1-L1--S-H RbO C-F1-L1--S-PG
H
2 H 2 ORC or ORc 26 WO 2014/147175 PCT/EP2014/055598 According to a particularly preferred embodiment of the invention, T is H. Thus, the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, comprising 5 (i) reacting hydroxyalkyl starch (HAS) of formula (Ta) ORa HAS '-,, 0 0 ' R b O - C * H % OH ORc (la) via carbon atom C* of the reducing end of the HAS with the functional group M of a crosslinking compound according to formula (II), thereby obtaining a HAS 10 derivative of the following formula: ORa
HAS'
0 OH R bo' C-F1-L 1 -S-H
H
2 ORC By way of example, the following preferred crosslinking compounds are mentioned: H 2
N
CH
2
-CH
2 -SH, H 2
N-CH
2
-CH
2
-CH
2 -SH, H 2
N-CH
2
-CH
2
-CH
2
-CH
2 -SH, H 2
N-CH
2
-CH
2
-CH
2 15 CH 2
-CH
2 -SH, H 2
N-CH(COOH)-CH
2 -SH, H 2
N-CH(COOH)-C(CH
3
)
2 -SH, H 2
N
CH(CH
2
OH)-CH
2 -SH, H 2
N-CH(CH
2
OH)-CH
2
-CH
2 -SH, H 2 N-CH(CONH2)-C(CH 3
)
2 -SH,
H
2 N-CH(CONH2)-CH 2 -SH, H 2
N-CH(COOH)-CH
2
-CH
2 -SH, H 2
N-CH
2
-CH
2 -0-CH 2
-CH
2 SH, H 2
N-CH
2
-CH
2 -0-CH 2
-CH
2 -0-CH 2
-CH
2 -SH, H 2
N-CH
2
-CH
2 -0-CH 2
-CH
2 -0-CH 2 CH 2 -0-CH 2
-CH
2 -SH, H 2
N-CH(COOH)-CH
2
-CH
2
-C(=O)NH-CH-C(=O)(-NH-CH
2 20 COOH)-CH 2 -SH, H 2 N-0-CH 2
-CH
2 -SH, H 2 N-0-CH 2
-CH
2
-CH
2 -SH, H 2 N-0-CH 2
-CH
2 CH 2
-CH
2 -SH, H 2 N-0-CH 2
-CH
2
-CH
2
-CH
2
-CH
2 -SH, H 2 N-0-CH(COOH)-CH 2 -SH, H 2 N-0
CH(COOH)-C(CH
3
)
2 -SH, H 2 N-0-CH(CH 2
OH)-CH
2 -SH, H 2 N-0-CH(CH 2
OH)-CH
2
-CH
2 SH, H 2 N-0-CH(CONH 2
)-C(CH
3
)
2 -SH, H 2
N-NH-CH
2
-CH
2 -SH, H 2
N-NH-CH
2
-CH
2
-CH
2 SH, H 2
N-NH-CH
2
-CH
2
-CH
2
-CH
2 -SH, H 2
N-NH-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -SH, H 2
N-NH
25 CH(COOH)-CH 2 -SH, H 2
N-NH-CH(COOH)-C(CH
3
)
2 -SH, H 2
N-NH-CH(CH
2
OH)-CH
2 -SH,
H
2
N-NH-CH(CH
2
OH)-CH
2
-CH
2 -SH, H 2
N-NH-CH(CONH
2
)-C(CH
3
)
2 -SH, H 2
N-NH
C(=O)-CH
2 -SH, H 2
N-NH-C(=O)-CH
2
-CH
2 -SH, H 2
N-NH-C(=O)-CH
2
-CH
2
-CH
2 -SH, H 2
N
NH-C(=O)-CH
2
-CH
2
-CH
2
-CH
2 -SH, H 2
N-NH-C(=O)-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -SH, H 2
N
NH-C(=O)-CH(COOH)-CH
2 -SH, H 2
N-NH-C(=O)-CH(COOH)-C(CH
3
)
2 -SH, H 2
N-NH
30 C(=O)-CH(CH 2
OH)-CH
2 -SH, H 2
N-NH-C(=O)-CH(CH
2
OH)-CH
2
-CH
2 -SH and H 2
N-NH
C(=O)-CH(CONH
2
)-C(CH
3
)
2 -SH. 27 WO 2014/147175 PCT/EP2014/055598 Preferably, the crosslinking compound according to formula (II) is selected from the group consisting of H 2
N-CH
2
-CH
2 -SH, H 2
N-CH
2
-CH
2
-CH
2 -SH, H 2
N-CH
2
-CH
2
-CH
2
-CH
2 -SH,
H
2
N-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -SH, H 2
N-CH(COOH)-CH
2 -SH and H 2
N-CH(COOH)
C(CH
3
)
2 -SH, H 2
N-CH(CH
2
OH)-CH
2 -SH, H 2
N-CH(CH
2
OH)-CH
2
-CH
2 -SH, H 2
N
5 CH(CONH 2
)-C(CH
3
)
2 -SH, H 2
N-CH(CONH
2
)-CH
2 -SH, H 2
N-CH(COOH)-CH
2
-CH
2 -SH,
H
2
N-CH
2
-CH
2 -0-CH 2
-CH
2 -SH, H 2
N-CH
2
-CH
2 -0-CH 2
-CH
2 -0-CH 2
-CH
2 -SH, H 2
N-CH
2 CH 2 -0-CH 2
-CH
2 -0-CH 2
-CH
2 -0-CH 2
-CH
2 -SH, H 2
N-CH(COOH)-CH
2
-CH
2
-C(=O)NH
CH-[C(=O)(-NH-CH
2
-COOH)]-CH
2 -SH and H 2 N-0-CH 2
-CH
2 -SH. 10 Most preferably, the crosslinking compound (II) is cysteamine H 2
N-CH
2
-CH
2 -SH. According to an alternative embodiment of the invention T is PG, wherein PG may be any suitable SH protecting group known to those skilled in the art. Preferably, PG is a protecting group forming together with -S- a thioether (e.g. benzyl, allyl, triarylmethyl 15 groups, such as trityl (Trt)), a disulfide (e.g. S-sulfonates, S-tert-butyl, S-(2-aminoethyl), 2 pyridylthio). In case T is a thiol protecting group PG, step (i) further comprises a deprotection step. Thus, the present invention also relates to a method as described above, and a conjugate 20 obtained or obtainable by said method, wherein T is a thiol protecting group PG. In this case, step (i) preferably further comprises removing PG from the HAS derivative (Ib) To give T = H. Alternatively, the protecting group may be removed in situ in step (ii) and the S-H group formed in situ may then directly be reacted with the compound (III). Preferably, the protecting group PG is removed prior to step (i). 25 In case the group -S-PG is a disulfide, the crosslinking compound according to formula (II) M-L1-S-PG is preferably a symmetrical disulfide, with PG having the structure -S-L1-M or is selected from the group consisting of 2-pyridylthio, -S-SO3, -S-S02-aryl and -S-SO 2 alkyl. 30 According to a preferred embodiment of the invention, the group S-PG present in the crosslinking compound according to formula (II) is selected from the group consisting of -S-Trt, -S-S-L1-M, -S-S-tBu, -S-S-(2-pyridyl), -S-SO3, -S-S02-aryl and -S-S0 2 -alkyl, in particular the group -S-PG is -S-S-L1-M. 35 According to this embodiment, the crosslinking compound according to formula (II) is selected from the group consisting of H 2 N-L1-S-Trt, H 2 N-L1-S-S-L1-NH 2 , H 2 N-L1-S-S tBu, H 2 N-L1-S-S-(2-pyridyl), H 2 N-L1-S-SO3-, H 2 N-L1-S-SO2-aryl and H 2 N-L1-S-SO 2 28 WO 2014/147175 PCT/EP2014/055598 alkyl, most preferably the crosslinking compound according to formula (II) is H 2 N-L1-S-S Li-NH 2 . By way of example, the following particularly preferred crosslinking compounds are 5 mentioned: H 2
N-CH
2
-CH
2 -S-Trt, H 2
N-CH
2
-CH
2
-CH
2 -S-Trt, H 2
N-CH
2
-CH
2
-CH
2
-CH
2
-S
Trt, H 2
N-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -S-Trt, H 2
N-CH
2
-CH
2
-S-S-CH
2
-CH
2
-NH
2 , H 2
N-CH
2 CH 2 -S-S-tBu, H 2
N-CH
2
-CH
2
-CH
2 -S-S-tBu, H 2
N-CH
2
-CH
2
-CH
2
-CH
2 -S-S-tBu, H 2
N
CH(COOH)-CH
2 -S-Trt, H 2
N-CH(COOH)-C(CH
3
)
2 -S-Trt, H 2
N-CH(COOH)-CH
2
-S-S
CH
2
-CH(COOH)-NH
2 , H 2
N-CH(COOH)-CH
2
-CH
2
-C(=O)NH-CH-[C(=O)(-NH-CH
2 10 COOH)]-CH 2
-S-S-CH
2
-CH-[C(=O)(-NH-CH
2
-COOH)]-NH-C(=O)-CH
2
-CH
2 CH(COOH)-NH 2 . Most preferably, the crosslinking compound (II) is cystamine H 2
N-CH
2 CH 2
-S-S-CH
2
-CH
2
-NH
2 or cystine H 2
N-CH(COOH)-CH
2
-S-S-CH
2
-CH(COOH)-NH
2 , more preferably cystamine. 15 The reaction conditions used in the deprotection step are adapted to the respective protecting group used. According to a preferred embodiment of the invention, the group -S-PG is a disulfide, as described above. In this case, the deprotection step comprises the reduction of this 20 disulfide bond to give the respective thiol group. This deprotection step is preferably carried out using specific reducing agents. As possible reducing agents, complex hydrides such as borohydrides, especially sodium borohydride, and thiols, especially dithiothreitol (DTT) and dithioerythritol (DTE) or phosphines such as tris-(2-carboxyethyl)phosphine (TCEP) are mentioned. The reduction is preferably carried out using borohydrides, 25 especially sodium borohydride. Preferably, the reducing agent is used in an excess, more preferably at a concentration in the range of from 0.02 to 1.5 M, more preferably in the range of from 0.05 to 1 M, most preferably in the range of from 0.1 to 0.5 M with respect to the total volume of the reaction solution. Further, this deprotection step is preferably carried out at a temperature in the range of from 0 to 80 'C, more preferably in the range of 30 from 10 to 50 'C and especially preferably in the range of from 15 to 35'C. During the course of the reaction, the temperature may be varied, preferably in the above-given ranges, or held essentially constant. Preferably, the reaction is carried out in aqueous medium. The term "aqueous medium" as 35 used in this context of the present invention refers to a solvent or a mixture of solvents comprising water in an amount of at least 10 % per weight, preferably at least 20 % per weight, more preferably at least 30 % per weight, more preferably at least 40 % per weight, more preferably at least 50 % per weight, more preferably at least 60 % per weight, more preferably at least 70 % per weight, more preferably at least 80 % per weight, even more 29 WO 2014/147175 PCT/EP2014/055598 preferably at least 90 % per weight or up to 100 % per weight, based on the weight of the solvents involved. The aqueous medium may comprise additional solvents like formamide, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), N methylpyrrolidinone (NMP), alcohols such as methanol, ethanol or isopropanol, 5 acetonitrile, tetrahydrofurane or dioxane. The aqueous solution may also contain a transition metal chelator (disodium ethylenediaminetetraacetate, EDTA, or the like) in a concentration ranging from 0.01 to 100 mM most preferably from 0.1 to 10 mM, such as about 1 mM. The preferred solvent is water. 10 The HAS derivative is reacted in the reduction step at a concentration in the range of from 1% to 30% weight.-%, more preferably in the range of from 5% to 20% weight.-%, most preferably 10% weight.-% based on the total weight of the solution. Preferably, the retentate of the ultrafiltration step is directly used for the reduction with 15 NaBH 4 . Thus, the present invention also relates to a method, as described above, and a HAS derivative obtained or obtainable by said method, wherein the removing of the protecting group PG in step (i) is carried out at a temperature in the range of from 0 to 80 'C and in an 20 aqueous solvent system, the group S-PG being a disulfide. The pH value in this deprotection step may be adapted to the specific needs of the reactants for example by using aqueous buffer solutions. Among the preferred buffers, carbonate, phosphate, borate and acetate buffers as well as tris(hydroxymethyl)aminomethane (TRIS) may be mentioned. Preferably in case of sodium borohydride, the reaction is carried out in water at 25 a pH value in the range of 7 to 14. The deprotection step is preferably conducted for a time in the range of from from 0.25 to 24 h, more preferably of from 0.5 to 18 h; most preferably of from 0.5 to 4 h. The conditions for deprotection of thiol groups comprising trityl groups are known to those 30 skilled in the art and are e.g. described in. S. Herman et al., Bioconjugate Chemistry, 1993, 4, 402-405. For the removal of other protecting groups, literature such as P.G.M. Wuts and T.W. Greene: Protecting Groups in Organic Synthesis, Wiley, 2007, Ch. 6 (p. 647-695), is 35 referenced. Due the tendency of thiols to form disulfide bridges in solution, the reduction conditions described above with respect to the removal of the protecting group may optionally as well be applied on HAS derivatives with T = Hthus derivatives obtained upon reacting HAS 30 WO 2014/147175 PCT/EP2014/055598 with compound (II), in which T is H, or HAS derivatives obtained after removal of the protecting group PG, in particular after deprotection under non-reductive conditions. After the reaction with the crosslinking compound according to formula (II) and optionally 5 the deprotection step, at least one isolation step/and or purification step, as described above, may be carried out. The HAS derivative obtained in step (i) may e.g. be isolated from the reaction mixture by any suitable method, such as ultrafiltration or dialysis, preferably ultrafiltration followed by lyophilization of the isolated hydroxyalkyl starch derivative. 10 In case ultrafiltration or dialysis is carried out, this ultrafiltration or dialysis is preferably performed under acidic conditions, preferably at a pH in the range of from 2 to 6, more preferably in the range of from 3 to 5, and/or in the presence of an ion chelator. Preferably an acid selected from the group comprising hydrochloric acid, phosphoric acid, 15 trifluoroacetic acid and acetic acid is employed. Preferred buffers are selected from the group comprising acetate, phosphate and citrate buffers. As suitable ion chelators EDTA (ethylenediamine tetraacetic acid), DTPA (diethylene triamine pentaacetic acid) and related compounds may be mentioned. 20 Most preferably, an acetic acid buffer (preferably in the range of from 0.1 mM to 1 M, more preferably in the range of from 1 to 100 mM, most preferably 10 mM) with pH 4 preferably comprising EDTA in the range of from 0.01 to 100 mM (most preferably 1 - 10 mM) is used as ultrafiltration or dialysis buffer. After removal of the reaction impurities, the ultrafiltration / dialysis buffer is replaced by water in order to remove buffer salts from 25 the product. Further, the hydroxyalkyl starch derivative may be precipitated from the reaction mixture, in particular by adding an alcohol, preferably 2-propanol. The obtained precipitate may be collected by filtration or centrifugation and further purified using conventional purification 30 protocols, preferably ultrafiltration, dialysis or chromatographic methods, preferably size exclusion chromatography. Most preferably the obtained derivative is further lyophilized prior to step (ii) until the solvent content of the reaction product is sufficiently low according to the desired 35 specifications of the derivative. Step (ii) 31 WO 2014/147175 PCT/EP2014/055598 In step (ii) of the invention, the HAS derivative of formula (Ib) obtained in step (i) is reacted with a crosslinking compound of formula (III) N-L2--N RX RYII) 5 thereby obtaining a HAS derivative of formula (I) ORa HAS'-s, R OH O 0 R O' C-F1-L--S N--L2-IN X ORcH 2 RX RI wherein X is a leaving group, and wherein L2 is a spacer bridging -NR- and -NRY-, wherein R and RY are, independently of each other, H or alkyl, and optionally form 10 together with the moiety N-L2-N a ring. The leaving group X The term "leaving group", as used in this context of the present invention, is denoted to 15 mean a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage upon reaction with a thiol group. Examples are, inter alia, halogens or sulfonic esters. Examples for sulfonic esters are, inter alia, the mesyl and tosyl group. Within the meaning of the present invention, the groups X-CH 2 -N(Rx)- and -N(Ry)-C(=O)
CH
2 -X are referred to as "reactive acetamide groups". 20 Preferably X is selected from the group consisting of tosyl (Ts), mesyl (Ms), Cl, Br and I. More preferably X is Br or I, most preferably I. The spacer L2 25 As described above L2 is a spacer bridging -NRx- and -NRY-. According to a preferred embodiment of the invention, Rx and RY are, independently of each other, selected from the group consisting of H, methyl, ethyl, propyl or butyl or are forming a ring according to formula (V) N-L2-N /\ 30 (V). 32 WO 2014/147175 PCT/EP2014/055598 In case, R and RY are forming together with the moiety N-L2-N a ring, the ring preferably being a 5-membered to 7-membered ring. More preferably, in this case, R and RY, taken together, are -(CH 2
)
2 - or -(CH 2
)
3 -. According to this embodiment, L2 is preferably an alkyl 5 group, preferably an optionally substituted methylene, ethylene, or propylene group, more preferably -CH 2 -, -(CH 2
)
2 - or -(CH2)3-. Thus, in case R and RY are forming together with the moiety N-L2-N a ring, the moiety -NRx-L2-NR- preferably has one of the following structures: N 'N- -N' \ N- _ 10 -N N More preferably, in this case L2 is -(CH 2
)
2 - and R and RY, taken together, are -(CH2)2-. Thus the moiety -NRx-L2-NR- has the following structure: J-N N 15 According to a preferred embodiment of the invention, R and RY are, independently of each other, H or alkyl, preferably selected from the group consisting of H, methyl, ethyl, propyl or butyl, more preferably both are H, and R and RY are not forming a ring together with the moiety N-L2-N. 20 According to this preferred embodiment, L2 comprises, more preferably consists of, an alkyl, alkenyl, alkylaryl, arylalkyl, aryl or heteroaryl group. As to the terms "alkyl, alkenyl, alkylaryl, arylalkyl, aryl or heteroaryl group ", reference is made to the definition of these groups mentioned hereinabove. 25 It is thus to be understood that, e.g. the the term "alkyl" as used in the context of linker L2, also relates to non-branched alkyl residues, branched alkyl residues, cycloalkyl residues, as well as residues comprising one or more heteroatoms or functional groups, such as, by way of example, -0-, -S-, -NRL-, -NH-C(=0)-, -C(=0)-NH- and the like, with RL 2 being alkyl, 30 preferably methyl. The term also encompasses alkyl groups which are further substituted by one or more suitable substituent. The term "substituted alkyl" as used in this context of the present invention preferably refers to alkyl groups being substituted in any position by one or more substituents, preferably by 1, 2, 3, 4, 5 or 6 substituents, more preferably by 1, 2, or 3 substituents. If two or more substituents are present, each substituent may be the 35 same as or may be different from the at least one other substituent. 33 WO 2014/147175 PCT/EP2014/055598 As mentioned above, the terms "alkyl", "alkenyl", "alkylaryl", "arylalkyl", "aryl" and "heteroaryl" include the respective suitable substituted groups. "Suitable substituents" in the context of spacer L2 are, for example, selected from the 5 group consisting of alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, amide, sulfate, 10 alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, carboxymethylcarbamoyl, cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl. Preferred substituents of such organic residues are, for example, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), 15 fluorine, hydroxyl groups, keto groups, aldehyde groups and carboxyl groups. More preferably, the spacer L2 comprises the moiety -(C(L'Ltt))t- with Lt and L"t in each repeating unit -C(LtL")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, 20 arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, keto groups, aldehyde groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, amidino, nitro, imino, alkylthio, arylthio, 25 thiocarboxyl, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups Lt and L"t in each repeating unit may be the same or may differ from each other, with t preferably being in the range of from 1 to 20, more preferably in the range of from 1 30 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4. According to a preferred embodiment of the invention, Lt and Ltt are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), fluorine, tertiary amino groups, 35 hydroxyl groups, keto groups, aldehyde groups and carboxyl groups, wherein the groups Lt and L"t in each repeating unit may be the same or may differ from each other. According to a particularly preferred embodiment of the invention, Lt and Ltt are, independently of each other, selected from the group consisting of H, carboxyl and alkyl, 34 WO 2014/147175 PCT/EP2014/055598 more preferably L' and Ltt are H or alkyl, wherein the groups L' and L"t in each repeating unit may be the same or may differ from each other. More preferably, L2 has a structure selected from the group consisting of Lt Lt Lu Ltt Ltt L Uu 5 . . t . . t . . u t u u Y L L t1. .u-i L - u-i . . . -and Lt Lu - II Ltt Luu - t u v with t preferably being in the range of from 1 to 20, wherein L', L", Lu, and Luu, are 10 independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, keto groups, aldehyde groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, 15 acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, amidino, nitro, imino, alkylthio, arylthio, thiocarboxyl, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L', Ltt, Lu, and Luu in each repeating unit may be the 20 same or may differ from each other, with t preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4, and with u preferably being in the range of from 1 to 10 and with v preferably being in the range of from 1 to 10, and wherein Y 2 is a functional moiety selected from the group consisting of -O-, -S-, -NRY 2 -, -NH-C(=O)-, -C(=O)-NH-, 0 N L IH 25 - (,,p-phenyl-C(=O)-NH") and wherein RY 2 is alkyl, preferably methyl, and wherein the groups L', Ltt, Lu, and Luu in each repeating unit, may be the same or may differ from each other. More preferably Y 2 , if present, is -0-. 35 WO 2014/147175 PCT/EP2014/055598 Preferably, L', L", L', and Luu are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), hydroxyl groups, keto groups, aldehyde groups and carboxyl groups, more preferably selected from the group consisting of H, carboxyl and alkyl, in 5 particular L', L", Lu, and Luu are, independently of each other, H or alkyl, and wherein Y 2 is a functional moiety as described above, preferably wherein Y 2 is -0-. In case t is > 1, the repeating units -C(L'L")- may be the same or may be different from each other. In case u is > 1, the repeating units -C(LuLuu)- may be the same or may be different from each other. In case v is > 1, the repeating units -Y 2 -[C(LuLuu)]u- may be the same or may be different 10 from each other. According to a preferred embodiment, L2 is a symmetrical spacer. Preferably, L2 has the structure Lt -1 - ' - -I 15 . t wherein L' and L" are, independently of each other, selected from H, hydroxyl, carboxyl, -C(=0)-NH 2 , and alkyl, wherein in each repeating unit -C(L'L")-, in case t is > 1, L' and L" may be the same or may be different from each other. 20 As example, without being meant to be limiting, the following groups L2 are mentioned:
-CH
2 -, -CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2 -,
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 CH 2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 25 CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH(CH 3 )-, -CH(CH 3
)-CH
2 -, -CH 2
-CH(CH
3 )-,
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH(CH
3 )-, -CH(CH 3
)-CH
2
-CH
2 CH 2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3 )-,
-CH(CH
3
)-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)
CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH(CH
3 )-, -CH(CH 3
)
30 CH 2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)-CH
2 CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH(CH
3
)-CH
2 -,
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH(CH
3 )-, -CH 2
-CH
2
-CH(OH)-CH
2 -, -CH 2
-CH(OH)-CH(OH)
CH
2 -, -C(CH 3
)
2
-CH
2 -, -CH(CH 3
)-CH(CH
3 )-, -C(CH 3
)
2
-C(CH
3
)
2 -, -CH(COOH)-(CH 2
)
4 -,
-CH(CONH
2
)-(CH
2
)
4 -, -CH(COOH)-CH 2 -, and -CH(CONH 2
)-CH
2 -. 35 More preferably L' and L" are, independently of each other, selected from H and alkyl. More preferably L' and L" in each repeating unit are H. 36 WO 2014/147175 PCT/EP2014/055598 According to a particularly preferred embodiment of the invention, L2 is -CH 2
-CH
2 -. According to a further embodiment, L2 has the structure Lt Lu -~ -- C-C C Ltt LUU 5 . t u whererin L', Ltt, Lu, and Luu are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), hydroxyl groups, keto groups, aldehyde groups and carboxyl 10 groups, more preferably selected from the group consisting of H, carboxyl and alkyl, in particular L', Ltt, Lu, and Luu are, independently of each other, H or alkyl. In case t is > 1, the repeating units -(C(L'L"))- may be the same or different from each other. In case u is > 1, the repeating units -(C(LuLuu))- may be the same or different from each other. 15 As example, without being meant to be limiting, in this context, the following groups L2 are mentioned: -CH 2
-C-C-CH
2 -, -CH 2
-CH
2
-C-C-CH
2
-CH
2 - and -CH 2
-CH
2
-CH
2
-C-C
CH
2
-CH
2
-CH
2 -, in particular -CH 2
-C-C-CH
2 -. According to an alternative embodiment, as described above, L2 has as structure according 20 to the following formula Lt Lu ----- -2- - -- V - - t , . .uv with t preferably being in the range of from 1 to 20, with u preferably being in the range of from I to 10, with v preferably being in the range of from I to 10 and with L', Lt, Lu and 25 Luu being as described above. As regards the functional moiety Y 2 , said functional moiety is preferably selected from the group consisting of -0-, -S-, -N(CH 3 )-, -NH-C(=0)-, -C(=0)-NH-, p-phenyl-C(=0)-NH. In this case, v is preferably of from 1 to 3. Thus, according to one embodiment of the 30 present invention L2 is, for example, -[C(L'Ltt)]t-N(CH 3 )-[C(LuLuu)]u-, -[C(LtL")]t-S [C(LuLuu)]u, -[C(LL")]t-O-[C(LuLuu)]u-, -[C(LtLtt)]t-NH-C(=0)-[C(LuLuu)]u-, -[C(LtLtt)]t C(=0)-NH-[C(LuLuu)]u- or -[C(LtL")]t-p-phenyl-C(=0)-NH-[C(LuLuu)]u-. Most preferably t is in the range of from 2 to 4. Further, u is in particular in the range of from 2 to 4. 37 WO 2014/147175 PCT/EP2014/055598 According to a preferred embodiment, Y 2 is -0- or -N(CH 3 )- and u is of from 1 to 3. Preferred crosslinking compounds according to this embodiment are, for example, -CH 2 CH 2 -0-CH 2
-CH
2 -, -CH 2
-CH
2 -0-CH 2
-CH
2 -0-CH 2
-CH
2 -, -CH 2
-CH
2 -0-CH 2
-CH
2 -0-CH 2 CH 2 -0-CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2 -0-CH 2
-CH
2 -0-CH 2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 -0 5 CH 2
-CH
2
-CH
2
-CH
2 -0-CH 2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 -0-CH 2
-CH
2 -0-CH 2
-CH
2 -0-CH 2 CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2
-N(CH
3
)-CH
2
-CH
2
-CH
2 - and -CH 2
-CH
2
-N(CH
3
)-CH
2
-CH
2 -. According to a further embodiment, L2 has one of the following structures: Lt Lu Lt LU tt Luu t
.
u-1 or u-1 10 wherein L', Ltt, Lu, and Luu are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), hydroxyl groups, keto groups, aldehyde groups and carboxyl groups, more preferably selected from the group consisting of H, carboxyl and alkyl, in 15 particular L, L 1 , Lu, and Luu are, independently of each other, H or alkyl, in particular H. In case t is > 1, the repeating units -(C(LtLtt))- may be the same or different from each other. In case u is > 1, the repeating units -(C(LuLuu))- may be the same or different from each other. Preferably t and u are, independently of each other 1 or 2, preferably both, t and u, are 1 or both, t and u, are 2. 20 As example, without being meant to be limiting, in this context, the following groups L2 are mentioned: 25 Thus, by way of example, the following preferred compounds according to formula (III) are mentioned 0 X A N--L2 -N R x X R L2 38 WO 2014/147175 PCT/EP2014/055598 Br H H Selected from the group consisting of: -CH 2 -, -CH 2
-CH
2 -,
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2 CH 2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2 CH 2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -,
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2 CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH(CH 3 )-,
-CH(CH
3
)-CH
2 -, -CH 2
-CH(CH
3 )-, -CH(CH 3
)-CH
2
-CH
2 -,
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH(CH
3 )-, -CH(CH 3
)-CH
2 CH 2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)
CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3 )-, -CH(CH 3
)-CH
2
-CH
2
-CH
2 CH 2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)
CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH
2 CH 2
-CH(CH
3 )-, -CH(CH 3
)-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2 CH(CH 3
)-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)-CH
2 CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH
2 CH 2
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2 CH(CH 3 )-, -CH 2
-CH
2
-CH(OH)-CH
2 -, -CH 2
-CH(OH)
CH(OH)-CH
2 -, -C(CH 3
)
2
-CH
2 -, -CH(CH 3
)-CH(CH
3 )-,
-C(CH
3
)
2
-C(CH
3
)
2 -, -CH(COOH)-(CH 2
)
4 -, -CH(CONH 2
)
(CH
2
)
4 -, -CH(COOH)-CH 2 -, -CH(CONH 2
)-CH
2 -, -CH 2
-CH
2 O-CH 2
-CH
2 -, -CH 2
-CH
2
-O-CH
2
-CH
2
-O-CH
2
-CH
2 -, -CH 2 CH 2
-O-CH
2
-CH
2
-O-CH
2
-CH
2
-O-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 O-CH 2
-CH
2
-O-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-O-CH
2
-CH
2 CH 2
-CH
2
-O-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-O-CH
2
-CH
2
-O
CH
2
-CH
2
-O-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-N(CH
3
)-CH
2 CH 2
-CH
2 - and -CH 2
-CH
2
-N(CH
3
)-CH
2
-CH
2 -, and preferably L2 is a symmetrical spacer, selected from the group consisting of -CH 2
-,-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 -, -CH 2
-CH
2 CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2 CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2 CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2 39 WO 2014/147175 PCT/EP2014/055598
CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 CH 2
-CH
2
-CH
2 -, -CH(CH 3 )-, -CH 2
-CH(CH
3
)-CH
2 -, -CH 2 CH 2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH(OH)-CH(OH)-CH
2 -,
-CH(CH
3
)-CH(CH
3 )-, -C(CH 3
)
2
-C(CH
3
)
2 -, -CH 2
-CH
2
-O-CH
2 CH 2 -, -CH 2
-CH
2
-O-CH
2
-CH
2
-O-CH
2
-CH
2 -, -CH 2
-CH
2
-O
CH
2
-CH
2
-O-CH
2
-CH
2
-O-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-O-CH
2 CH 2
-O-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-O-CH
2
-CH
2
-CH
2 CH 2
-O-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-O-CH
2
-CH
2
-O-CH
2 CH 2
-O-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-N(CH
3
)-CH
2
-CH
2 CH 2 - and -CH 2
-CH
2
-N(CH
3
)-CH
2
-CH
2 -, - and I H H See above Br CH 3
CH
3 See above I CH 3
CH
3 See above Br CH 3 - CH 3 - See above
CH
2 - CH 2 I CH 3 - CH 3 - See above
CH
2 - CH 2 Br CH 3 - CH 3 - See above
CH
2 - CH 2 CH 2 - CH 2 I CH 3 - CH 3 - See above
CH
2 - CH 2 CH 2 - CH 2 Br (CH 3
)
2 - (CH 3
)
2 - See above CH- CH I (CH 3
)
2 - (CH 3
)
2 - See above CH- CH Br -CH 2
-CH
2 - -CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2 (cycle) I -CH 2
-CH
2 - -CH 2
-CH
2 -, -CH 2
-CH
2
-CH
2 (cycle) 40 WO 2014/147175 PCT/EP2014/055598 Step (ii) according to the invention is preferably carried out in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, reaction buffers and mixtures of two or more thereof. Especially preferred solvents are DMSO and DMF. 5 The crosslinking compound of formula (III) is preferably dissolved in the reaction solvent prior to addition of the HAS derivative. Alternatively, the crosslinking compound is added to the HAS derivative solution as a solid. The reaction mixture is preferably stirred at a temperature in the range of from 10 'C to 10 50 'C, more preferably at a temperature in the range of from 20 to 40 'C, more preferably in the range of from 22 to 27 0 C. During the course of the reaction, the temperature may be varied, preferably in the above-given ranges, or held essentially constant. Thus, the present invention also relates to a method, as described above, and a HAS 15 derivative obtained or obtainable by said method, wherein the reacting according to step (ii) is carried out at a temperature in the range of from 10 'C to 50 'C and in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, reaction buffers and mixtures of two or more thereof. Preferably, the reacting is carried out under non-aqeous conditions, more preferably in a solvent selected from the group 20 consisting of DMSO, DMF, DMA, NMP and mixtures of two or more thereof. The reaction is preferably conducted for a time in the range of from 30 min to 48 h, more preferably of from 1 h to 24 h, more preferably of from 2 h to 18 h. 25 The crosslinking compound of formula (III) is preferably employed in excess (when compared to the HAS derivative (Ib)). Therefore the minimum amount is preferably twice the amount of free thiol groups in the HAS derivative (Ib). Preferably, the crosslinking compound of formula (III) is used at a concentration in the range of from 5 to 1000 mM, more preferably of from 10 to 100 mM, and more preferably of from 30 to 80 mM. The 30 maximum concentration is preferably given by the solubility of the crosslinking compound of formula (III) in the reaction solvent. The concentration of the HAS derivative (Ib) in the solvent, preferably the aqueous system, is preferably in the range of from 3 to 40 wt.-%, more preferably of from 5 to 30 wt.-%, 35 and more preferably of from 10 to 20 wt.-%, relating, in each case, to the weight of the reaction solution. Preferably, the hydroxyalkyl starch derivative (I) 41 WO 2014/147175 PCT/EP2014/055598 ORa HAS, OH O 0 R O~ C-F1-L-S N-L2N X ORcH 2 X RI obtained in step (ii), is isolated from the reaction mixture by a sequence of dilution with water, filtration and either ultrafiltration or dialysis, preferably ultrafiltration followed by 5 lyophilization, or the hydroxyalkyl starch derivative is precipitated from the reaction mixture, in particular by adding an alcohol, preferably 2-propanol or water, and separated by ultrafiltration, dialysis or chromatographic methods, preferably by size exclusion chromatography and/or lyophilization. 10 In the case of ultrafiltration, the derivative (I) is preferably purified by at least one volume exchange with aqueous acid, preferably hydroiodic acid, hydrobromic acid, hydrochloric acid, methylsulfonic acid or toluenesulfonic acid, preferably at a pH of in the range of from 1 to 6, more preferably in the range of from 2 to 4. Residual acid is preferably washed out by at least one volume exchange with water. 15 Use of the HAS derivative (I) The HAS derivative according to formula (I) is preferably used as reactant for coupling to 20 a thiol group comprising compound Q. The term "thiol group comprising compound Q" as used in the context of the present invention relates to any substance comprising a thiol group, preferably to a substance which can affect any physical or biochemical property of a biological organism including, 25 but not limited to, viruses, bacteria, fungi, plants, animals, and humans. In particular, the term "thiol group comprising compound Q" as used in the context of the present invention relates to a substance intended for diagnosis, cure, mitigation, treatment, or prevention of a disease in humans or animals, or to otherwise enhance physical or mental well-being of humans or animals. Preferred examples of such substances include, but are not limited to, 30 thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, lipids, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, cells, viruses, liposomes, microparticles, and micelles. It is to be understood that any derivatives of the aforementioned terms are included within the meaning of the present invention. The term "derivatives thereof' refers to chemically modified derivatives, 35 mutants, and functional mimetics of the aforementioned compounds. Preferably, a biologically active substance according to the present invention contains a native thiol 42 WO 2014/147175 PCT/EP2014/055598 group. However, such thiol group may also be introduced by methods well known to those skilled in the art. The term "thiol group comprising peptide" as used in the context of the present invention 5 is denoted to mean peptides comprising up to 50 natural or unnatural, D- or L-amino acids and comprising at least one thiol group. The thiol group may be part of a cysteine or may be introduced into the peptide by a chemical modification. The term "thiol comprising polypeptide" as used in the context of the present invention 10 includes all compounds having a peptidic backbone and more than 50 monomer (amino acid) units and which comprise at least one thiol group. This term thus in particular includes proteins. The term "protein" as used in the context of the present invention includes natural proteins as well as chemically modified derivatives, mutants and analogs thereof. The term "protein mutant" is denoted to mean a protein being modified with at 15 least one natural or unnatural amino acid either at the N- or at the C-terminus of the protein or in which at least one naturally-occurring amino acid within the sequence is replaced with another natural or unnatural amino acid. It has to be understood that the thiol group may be present in the wild type protein as such or may be introduced by any suitable method such as by adding (e.g. by recombinant means) a cysteine residue either at the N 20 or at the C-terminus of the polypeptide or by replacing (e.g. by recombinant means) a naturally-occurring amino acid by cysteine to give a mutant of the protein. The respective methods are known to the person skilled in the art (see Elliott, Lorenzini, Chang, Barzilay, Delorme, 1997, Mapping of the active site of recombinant human erythropoietin, Blood, 89(2), 493-502; Boissel, Lee, Presnell, Cohen, Bunn, 1993, Erythropoietin structure 25 function relationships. Mutant proteins that test a model of tertiary structure, J. Biol. Chem., 268(21), 15983-93)). Further, any group present in the protein may be chemically modified to give a chemically modified derivative of the protein, such as by addition of a suitable linker compound to the N-terminus or C-terminus or to a side chain either during the synthesis of the protein or to the existing full length protein as such. 30 Preferred examples of peptides include, but are not limited to, peptide hormones and peptide aptamers. Preferred examples of polypeptides or proteins include, but are not limited to, the following proteins, plasma proteins such as immunoglobulins, growth factors, cytokines, coagulation factors including vWF, enzymes and enzyme inhibitors, 35 albumins and binding proteins such as alternative scaffold proteins, antibody fragments and soluble receptors. The term "alternative scaffold protein" as used in the context of the present invention relates to a molecule having binding abilities similar to a given antibody wherein the molecule is based on an alternative non-antibody protein framework (see e.g. 43 WO 2014/147175 PCT/EP2014/055598 Hey, T. et al., 2005, Artificial, non-antibody binding proteins for pharmaceutical and industrial applications, Trends Biotechnol., 23(10), 514-22). As used herein, the term "oligonucleotide" or "nucleic acids" refers to polymers, such as 5 DNA and RNA, of nucleotide monomers or nucleic acid analogs thereof, including double and single-stranded deoxyribonucleotides, ribonucleotides, alpha-anomeric forms thereof, and the like. Usually the monomers are linked by phosphodiester linkages, wherein the term "phosphodiester linkage" refers to phosphodiester bonds or bonds including phosphate analogs thereof, including associated counterions, e.g., H, NH 4 +, Na+. The term 10 oligonucleotide further includes polymers comprising mixtures of deoxyribonucleotides and ribonucleotides (DNA/RNA-hybrids). Further the term includes derivatives thereof chemically modified derivative of the oligonucleotides. "Nucleoside" refers to a compound consisting of a purine, deazapurine, or pyrimidine 15 nucleobase, e.g., adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like, linked to a pentose at the 1-position. When the nucleoside base is purine or 7-deazapurine, the pentose is attached to the nucleobase at the 9-position of the purine or deazapurine, and when the nucleobase is pyrimidine, the pentose is attached to the nucleobase at the 1-position of the pyrimidine. 20 "Nucleotide" refers to a phosphate ester of a nucleoside, e.g., a triphosphate ester, wherein the most common site of esterification is the hydroxyl group attached to the C-5 position of the pentose. A nucleotide is composed of three moieties: a sugar, a phosphate, and a nucleobase (Blackburn, G. and Gait, M. Eds. "DNA and RNA structure" in Nucleic Acids 25 in Chemistry and Biology, 2nd Edition, (1996) Oxford University Press, pp. 15-81). When part of a duplex, nucleotides are also referred to as "bases" or "base pairs". The term "nucleic acid analogs" refers to analogs of nucleic acids made from monomeric nucleotide analog units, and possessing some of the qualities and properties associated 30 with nucleic acids. For example, nucleic acid analogs comprise modifications in the chemical structure of the base (e.g. C-5-propyne pyrimidine, pseudo-isocytidine and isoguanosine), the sugar (e.g. 2'-O-alkyl ribonucleotides) and/or the phosphate (e.g. 3'-N phosphoramidate). See for example Englisch, U. and Gauss, D. "Chemically modified oligonucleotides as probes and inhibitors", Angew. Chem. Int. Ed. Engl. 30:613-29 35 (1991)). Nucleotide analogs in particular include, but are not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group such as an H, OR, R, halogen, SH, SR, NH 2 , NHR, NR 2 , or CN, wherein R is 44 WO 2014/147175 PCT/EP2014/055598 an alkyl moiety. Nucleotide analogs are also meant to include nucleotides with other modified bases, or with different sugars such as 2'-methyl ribose as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, 5 and other sugars, heterocycles, or carbocycles. Nucleotide analogs are also meant to include nucleotides with non-natural linkages such as methylphosphonates and phosphorothioates. Further the term is meant to include analogs such as locked nucleic acids in which the ribose moiety of the nucleotide is modified with an extra bridge connecting the 2'-oxygen and 4'-carbon (LNA). Further, the term nucleotide also includes 10 those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide. A class of analogs where the sugar and phosphate moieties have been replaced with a 2-aminoethylglycine amide backbone polymer is peptide nucleic acid (PNA) (Nielsen, P., Egholm, M., Berg, R. and Buchardt, 0. "Sequence-selective recognition of DNA by strand displacement with a thymidine 15 substituted polyamide", Science 254:1497-1500 (1991)). Further the oligonucleotide according to the invention may comprise one or more abasic sites. By "abasic site" is meant a monomeric unit contained within an oligonucleotide chain but which does not contain a purine or pyrimidine base. 20 Preferred examples of oligonucleotides and nucleic acid analogs include, but are not limited to, thiol group comprising, ribonucleic acids, deoxyribonucleic acids, peptide nucleic acids (PNA), locked nucleic acids (LNA). The thiol group present in the oligonucleotides, nucleotides, nucleosides or nucleic acid 25 anaolgs of the invention may be attached by any method known to those skilled in the art, i.e., for example, either by introducing a thiol modified building block during the preparation of the respective compound or by chemical modification to any suitable position of the respective compound, in particular by attaching a thiol group comprising linker in 3'- or 5'-position. 30 The term "lipids" refers to a broad group of naturally occurring and unnatural molecules that include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, sterols, glycophosopholipids and others. Lipid compounds are suitable for the transport of 35 biologically active substances or molecules. Polymer conjugated lipid compounds are useful in drug delivery, for example in the form of liposomes. (Immordino et al., IN, 2006: 1 (3) 297-315). 45 WO 2014/147175 PCT/EP2014/055598 Preferably, compound Q is selected from the group consisting of, thiol group comprising, peptides, polypeptides, oligonucleotides and nucleic acid analogs, more preferably from the group consisting of peptide hormones, peptide aptamers, plasma proteins (such as immunoglobulins, growth factors, cytokines, coagulation factors including vWF), 5 enzymes, enzyme inhibitors, albumins, natural or artificial binding proteins (such as alternative scaffold proteins, antibody fragments, soluble receptors), ribonucleic acids, deoxyribonucleic acids, peptide nucleic acids (PNA), and locked nucleic acids (LNA). Thus, the present invention also relates to a hydroxyalkyl starch (HAS) derivative of formula (IV), as described above, or a salt or solvate therof, ORa HAS ' OH 0 b SQ R O- C-Fl-Li-S N-L2-N 10 ORcRx R (IV) wherein -Q' is the remainder of a thiol group comprising compound Q which is linked via the group -S- of the thiol group to the group -NRY-C(=O)-CH 2 -, and wherein Q is selected from the 15 group consisting of, thiol group comprising, peptides, polypeptides, oligonucleotides and nucleic acid analogs, more preferably from the group consisting of peptide hormones, peptide aptamers, plasma proteins (such as immunoglobulins, growth factors, cytokines, coagulation factors including vWF), enzymes, enzyme inhibitors, albumins, natural or artificial binding proteins (such as alternative scaffold proteins, antibody fragments, 20 soluble receptors), ribonucleic acids, deoxyribonucleic acids, peptide nucleic acids (PNA), and locked nucleic acids (LNA). Further, the present invention also relates to the use of such HAS derivative as a medicament. According to a preferred embodiment of the invention, compound Q is a, thiol group 25 comprising, peptide or polypeptide. Thus, the present invention also relates to a hydroxyalkyl starch (HAS) derivative of formula (IV) as described above, or a salt or solvate therof, wherein Q is a, thiol group comprising, peptide or polypeptide. Further, the present invention also relates to the use of 30 such HAS derivatives as a medicament. Particularly preferred examples of thiol group comprising, peptides and polypeptides (proteins) include, but are not limited to, the following peptides and proteins, or derivatives thereof: erythropoietin (EPO), such as recombinant human EPO (rhEPO) or an EPO 35 mimetic, colony-stimulating factors (CSF), such as G-CSF like recombinant human G-CSF (rhG-CSF), alpha-Interferon (IFN alpha), beta-Interferon (IFN beta) or gamma-Interferon 46 WO 2014/147175 PCT/EP2014/055598 (IFN gamma), such as IFN alpha, IFN beta and IFN gamma like recombinant human IFN alpha, IFN beta or IFN gamma (rhIFN alpha, rhIFN beta or rhIFN gamma), interleukines, e.g. IL-I to IL-34 such as IL-2 or IL-3 or IL-1I like recombinant human IL-2 or IL-3 (rhIL-2 or rhIL-3), serum proteins such as coagulation factors II-XIII like factor II, factor 5 III, factor V, factor VI, factor VII, factor VIla, factor VIII, such as full-length FVIII, BDD FVIII or single-chain FVIII, factor IX, factor X, factor XI, factor XII, factor XIII, von Willebrand factor (vWF), enzymes such as lipases, proteases, peptidases, hydrolases, glycosidases, isomerases, reductases, oxidases, transferases, kinases, phosphatases, serine protease inhibitors such as alpha-1-antitrypsin (A1AT), activated protein C (APC), 10 plasminogen activators such as tissue-type plasminogen activator (tPA), such as human tissue plasminogen activator (hTPA), AT III such as recombinant human AT III (rhAT III), myoglobin, albumins such as human serum albumin (HSA), growth factors, such as epidermal growth factor (EGF), thrombocyte growth factor (PDGF), fibroblast growth factors (FGF), brain-derived growth factor (BDGF), nerve growth factor (NGF), B-cell 15 growth factor (BCGF), brain-derived neurotrophic growth factor (BDNF), ciliary neurotrophic factor (CNTF), transforming growth factors such as TGF alpha or TGF beta, BMPs (bone morphogenic proteins), growth hormones such as human growth hormone (hGH) like recombinant human growth hormone (rhGH), tumor necrosis factors such as TNF alpha or TNF beta, somatostatine, somatotropine, somatomedines, hemoglobin, 20 hormones or prohormones such as insulin, gonadotropin, melanocyte-stimulating hormone (alpha-MSH), triptorelin, hypthalamic hormones such as antidiuretic hormones (ADH and oxytocin as well as releasing hormones and release-inhibiting hormones, parathyroid hormone, thyroid hormones such as thyroxine, thyrotropin, thyroliberin, calcitonin, glucagon, glucagon-like peptides (GLP-1, GLP-2 etc.), exendines such as exendin-4, leptin 25 such as recombinant human leptin (rhLeptin), vasopressin, gastrin, secretin, integrins, glycoprotein hormones (e.g. LH, FSH etc.), melanoside-stimulating hormones, lipoproteins and apo-lipoproteins such as apo-B, apo-E, apo-La, immunoglobulins such as IgG, IgE, IgM, IgA, IgD and fragments thereof, such as Fab fragments derived from immunoglobuline G molecules (Fab), di-Fabs, tri-Fabs, scFv, bis-scFv, diabodies, 30 triabodies, tetrabodies, minibodies, domain antibodies, vH domain, vL domain, murine immunoglobuline G (mIgG), shark antibodies (IgNAR) and fragments thereof, camelid immunoglobulins and fragments thereof such as vHH domain, receptor proteins, such as cell surface receptors or soluble receptors, hirudin, tissue-pathway inhibitor, plant proteins such as lectin or ricin, bee-venom, snake-venom, immunotoxins, antigen E, alpha-proteinase 35 inhibitor, ragweed allergen, melanin, oligolysine proteins, RGD proteins or optionally corresponding receptors for one of these proteins; prolactin, or an alternative scaffold protein. Particularly preferred examples of polypeptides used as alternative scaffold proteins are derivatives of Protein A, Protein G, lipocalins, CTLA-4, A domain from LDL receptor like module, ubiquitin, gamma crystallin, repeat proteins such as ankyrin repeat 47 WO 2014/147175 PCT/EP2014/055598 proteins, leucine-rich repeat proteins, tetratricopeptide repeat proteins, HEAT-like proteins, armadillo repeat protein, transferrin, beta-lactamase, C-type lectin domain, fibronectin type III domain 10 proteins, Kunitz domain, knottins such as Ecballium elaterium trypsin inhibitor II (EETI-II) and the C-terminal domain of the human Agouti-related protein 5 (AGRP), tendamistat, thioredoxin, PDZ domain, zinc finger proteins such as the plant homeodomain (PHD) finger protein, T-cell receptors, green-fluorescent protein, Fyn domain 3, Alphabodies, CH2 or CH3 domains of an antibody Fc part. According to a further particularly preferred embodiment, compound Q is selected from 10 the group consisting of insulin, glucagon, gastric inhibitory peptides, exendins, ghrelin, PYY and peptide aptamers. According to a further particularly preferred embodiment, compound Q is a growth factor or a cytokine, preferably selected from the group consisting of (EPO), such as recombinant 15 human EPO (rhEPO), colony-stimulating factors (CSF), such as G-CSF like recombinant human G-CSF (rhG-CSF), alpha-Interferon (IFN alpha), beta-Interferon (IFN beta), and gamma-Interferon (IFN gamma), such as recombinant human IFN alpha or IFN beta (rhIFN alpha or rhIFN beta), fibroblast growth factors (FGF), human growth hormone (hGH) like recombinant human growth hormone (rhGH), BMPs (bone morphogenic 20 proteins), interleukines, tumor necrosis factors such as TNF alpha and TNF beta. According to a further particularly preferred embodiment, compound Q is a protein hormone, preferably selected from the group consisting of leptins, follicle stimulating hormon (FSH) and luteinizing hormon (LH). 25 According to a further particularly preferred embodiment, compound Q is an enzyme or enzyme inhibitor, preferably selected from the group consisting of alpha-i -antitrypsin (A1AT), antithrombin such as AT III, glucocerebrosidase, acid maltase, alpha galactosidase, iduronidase, iduronate-2-sulfatase, arylsulfatase B, asparaginase, 30 phenylalanine ammonia-lyase, and L-methioninase. According to a further particularly preferred embodiment, compound Q is coagulation factor or a protein involved in hemostasis, preferably selected from the group consisting of factor II, factor III, factor V, factor VI, factor VII, factor VIla, factor VIII, factor IX, factor 35 X, factor XI, factor XII, factor XIII, von Willebrand factor, tissue factor pathway inhibitor (TFPI) and Protein C such as APC. 48 WO 2014/147175 PCT/EP2014/055598 According to a further particularly preferred embodiment, compound Q is an immunoglobulin or fragment thereof, preferably selected from the group consisting of IgG, Fab fragments, Fc fragments, scFvs and dAbs. 5 According to a further particularly preferred embodiment, compound Q is an artificial binding protein or alternative scaffold protein, preferably selected from the group consisting of ubiquitin, Protein A, lipocalins, transferrin, fibronectins and soluble receptors. 10 In another particularly preferred embodiment, compound Q is an oligonucleotide or nucleic acid, such as a DNA or RNA aptamer. It is to be understood that the term oligonucleotide or nucleic acid, such as a DNA or RNA aptamer is denoted to mean thiol comprising derivatives of these compounds. Such thiol modifications are known to those skilled in the art. 15 Thus, the present invention also relates to a hydroxyalkyl starch (HAS) derivative of formula (IV) as described above, or a salt or solvate therof, wherein compound Q is an oligonucleotide or nucleic acid, such as a DNA or RNA aptamer. Further, the present invention also relates to the use of such HAS derivatives as a medicament. 20 According to a further particularly preferred embodiment, compound Q is selected from the group consisting of ribonucleic acid, deoxyribonucleic acid, peptide nucleic acid (PNA), locked nucleic acid (LNA), antisense RNA, RNAi, siRNA, Spiegelmer, aptamer, ribozyme and phosphorothioate-modified nucleic acid. 25 Step (iii) In case the HAS derivative according to formula (I) is reacted with a thiol group comprising compound, the method according to the invention further comprises 30 (iii) reacting the HAS derivative of formula (I) via the group -NRY-C(=O)-CH 2 -X with an -SH group of a thiol group comprising compound Q, thereby forming a HAS derivative of formula (IV) ORa HAS'-, OH 0 OH RbO- C-F1-L1-S N-L2-N 8 ORcH 2 Rx R (IV) 35 wherein 49 WO 2014/147175 PCT/EP2014/055598 -Q' is the remainder of the thiol group comprising compound Q which is linked via the group -S- of the thiol group to the group -NRI-C(=O)-CH 2 -. It has surprisingly been found that when conducting the above described method, the 5 reaction product of the hydroxyalkyl starch derivative as obtained in step (ii) with the further compound can be obtained in a high yield and in a high purity. Further, the respective derivative according to formula (IV) shows advantageous properties in terms of stability. 10 The solvent is chosen depending on the nature of the compound Q to be coupled. In case Q is a polypeptide, protein or derivative thereof, the reaction is preferably carried out in a solvent selected from the group consisting of water, reaction buffers, DMSO, DMF, DMA, NMP, formamide, and mixtures of two or more thereof. 15 Preferably the reaction is carried out in an aqueous medium. The term "aqueous medium" is denoted to mean a solvent comprising water and/or at least one reaction buffer. Preferably, the the solvent comprises only minor amounts of organic solvents such as in an amount in the range of from 0 to 10 % by weight, preferably 0 to 5 % by weight, more 20 preferably 0 to 2 % by weight, most preferably less than 1 % by weight, based on the total weight of the reaction solvent. Further the reaction solvent may comprise detergents, stabilizers, antioxidants and/or reducing agents, preferably at least one antioxidant and/or at least one reducing agent to avoid oxidation of the free thiol groups. A suitable reducing agent may be TCEP, which does not contain a free thiol group and thus does not compete 25 with the Q in the conjugation reaction. A suitable antioxidant may be EDTA, which acts in an indirect manner by complexing transition metal ions, that can catalyze peroxide formation. Preferably, the reaction is carried out in the presence of TCEP and/or EDTA. According to one preferred embodiment, according to which, Q is a polypeptide, protein or 30 derivative thereof, the polypeptide, protein or derivative thereof is preferably incubated with at least one reducing agent and optionally at least one antioxidant, more preferably with DTT, DTE, beta-mercaptoethanol or TCEP, most preferably with DTT and TCEP prior to the addition to or of the HAS derivative of formula (I). Thiol-containing reducing agents should be carefully removed from the reduced protein by methods known to those 35 skilled in the art to prevent unwanted quenching of the thiol-reactive hydroxyalkyl starch derivative. The reaction mixture is preferably stirred at a temperature in the range of from 0 'C to 50 'C, more preferably at a temperature in the range of from 5 to 40 'C, more preferably in 50 WO 2014/147175 PCT/EP2014/055598 the range of from 5 to 25'C. During the course of the reaction, the temperature may be varied, preferably in the above-given ranges, or held essentially constant. Thus, the present invention also relates to a method, as described above, and a HAS 5 derivative obtained or obtainable by said method, wherein the reacting according to step (iii) is carried out at a temperature in the range of from 0 'C to 50 'C and in a solvent selected from the group consisting of water, reaction buffers, DMSO, DMF, DMA, NMP, formamide, and mixtures of two or more thereof. 10 The reaction is preferably conducted for a time in the range of from 5 min to 48 h, more preferably of from 20 min to 24 h, more preferably of from 30 min to 18 h. The molar ratio of compound Q : HAS derivative (I) is preferably in the range of from 1:0.5 to 1:100, more preferably of from 1:1 to 1:20, more preferably of from 1:1 to 1:5 and 15 more preferably of from 1:1.5 to 1:3. The concentration of the HAS derivative (I) in the solvent, preferably the aqueous system, is preferably in the range of from 0.1 to 50 wt.-%, more preferably of from 1 to 30 wt.-%, and more preferably of from 1 to 10 wt.-%, relating, in each case, to the weight of the 20 reaction solution. The term "derivatives of formula (IV)" or "conjugates" of formula (IV) as used hereinunder includes the respective pharmaceutically acceptable salts and solvates of these derivatives. 25 Preferably, the derivatives of formula (IV) described hereinunder and above are stable at a pH in range of from 4.0-8.5, preferably in the range of from 6 to 8.5. The term "stable" is denoted to mean that the percent of degradation of 1 mg of the 30 derivative according to formula (IV) in 1 mL buffer solution of a respective pH measured after 20 days of incubation at 40 'C, measured by RP-HPLC as described in example C1 is preferably less than 8 %, more preferably less than 4%, more preferably less than 3%. In the following, particularly preferred embodiments of the invention are described: 35 1. A hydroxyalkyl starch (HAS) derivative of formula (I) 51 WO 2014/147175 PCT/EP2014/055598 ORa HAS'-, OH O bX R O- C-F-L1--S N-L2--N
H
2 Rx y ORG I wherein X is a leaving group, preferably selected from the group consisting of Ts, Ms, Cl, Br 5 and I; F1 is a functional group comprising the group -NR'-, with R' being H or alkyl; LI is a spacer bridging F1 and S; L2 is a spacer bridging -NR- and -NRY-; R and RY are, independently of each other, H or alkyl, and optionally form together 10 with the moiety N-L2-N a ring; HAS' is the remainder of the HAS molecule and Rb and R' are -[(CR 1
R
2 )mO]n-H and are the same or different from each other; Ra is -[(CRlR 2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and 15 HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR1R2; n is from 0 to 6. 20 2. The HAS derivative of embodiment 1, wherein X is Br or I, preferably I. 3. A hydroxyalkyl starch (HAS) derivative of formula (IV) ORa HAS'- , OH 0 o RBO~ C-F1-L1-S N--L2-N---N_ ORcH 2 R R (IV) 25 wherein -Q' is the remainder of a thiol group comprising compound Q which is linked via the group -S- of the thiol group to the group -NRY-C(=O)-CH 2 -; F1 is a functional group comprising the group -NR'-, with R' being H or alkyl; LI is a spacer bridging F1 and S; 30 L2 is a spacer bridging -NR- and -NRY-; R and RY are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; 52 WO 2014/147175 PCT/EP2014/055598 HAS' is the remainder of the HAS molecule and Rb and R' are -[(CRR 2 )mO].-H and are the same or different from each other; Ra is -[(CR 1
R
2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 5 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR1R2; n is from 0 to 6. 4. The HAS derivative of embodiment 3, wherein Q is selected from the group 10 consisting of, thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, cells, viruses, liposomes, microparticles, and micelles. 5. The HAS derivative of any one of embodiments 1 to 4, wherein the HAS is 15 hydroxyethyl starch (HES), R1, R2, R 3 , and R 4 are hydrogen, m is 2; n is 0 to 6. 20 6. The HAS derivative of any one of embodiments 1 to 5, wherein Fl is selected from the group consisting of -(CH 3 )-N-, -(CH 3
-CH
2 )-N-, -(CH 3
-CH
2
-CH
2 )-N-, -((CH 3
)
2 CH)-N-, -HN-, -HN-O-, -HN-NH-, -HN-NH-(C=G)-, -HN-NH-(C=G)-G G- and -HN
NH-SO
2 -, wherein F1 is preferably -NH-. 25 7. The HAS derivative of any one of embodiments 1 to 5, wherein R and RY are, independently of each other selected from the group consisting of H, methyl, ethyl, propyl and butyl, or are forming a ring according to formula N-L2-N (V) 30 wherein R and RY, taken together, are -(CH 2
)
2 - or -(CH 2
)
3 -, preferably wherein Rx and RY are H. 8. The HAS derivative of any one of embodiments 1 to 7, wherein the spacer Li comprises, preferably consists of the moiety -(C(L'L"))q- with L' and L" in each 35 repeating unit -C(L'L")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, 53 WO 2014/147175 PCT/EP2014/055598 alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amine, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, 5 carboxymethylcarbamoyl [i.e. the group -C(=O)(-NH-CH 2 -COOH)], cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other, with q preferably being in the range of from 1 to 20, more preferably in the range of from 1 10 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4. 9. The HAS derivative of any one of embodiments 1 to 8, wherein the spacer Li is
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2 -, CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2 15 CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, CH 2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH(CH 3 )-, -CH(CH 3
)-CH
2 -,
-CH
2
-CH(CH
3 )-, -CH(CH 3
)-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH(CH
3 )-,
-CH(CH
3
)-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)-CH
2 -,
-CH
2
-CH
2
-CH
2
-CH(CH
3 )-, -CH(CH 3
)-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)-CH
2 20 CH 2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2 CH 2
-CH
2
-CH
2
-CH(CH
3 )-, -CH(CH 3
)-CH
2
-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(CH
3
)
CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH(CH
3
)-CH
2
-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2 CH(CH 3
)-CH
2
-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2
-CH(CH
3
)-CH
2 -, -CH 2
-CH
2
-CH
2
-CH
2 CH 2
-CH(CH
3 )-, -CH 2
-C(CH
3
)
2
-CH
2 -, -CH(CH 3
)-CH(CH
3 )-, -C(CH 3
)
2
-C(CH
3
)
2 -, 25 -CH(CH 2
OH)-CH
2 -, -CH(CH 2
OH)-CH
2
-CH
2 -, -CH(CONH 2
)-CH
2 -, -CH(COOH)
CH
2 -, -CH(COOH)-CH 2
-CH
2 -, -CH(COOH)-CH 2
-CH
2
-CH
2
-CH
2 -, -CH(CONH 2
)
C(CH
3
)
2 -, -CH(CONH 2
)-CH
2
-CH
2
-CH
2
-CH
2 -, -CH 2
-CH(OH)-CH
2 -, -CH 2
-CH(OH)
CH(OH)-CH
2 - and -CH(COOH)-C(CH 3
)
2 -, preferably -CH 2
-CH
2 -. 30 10. The HAS derivative of any one of embodiments 1 to 9, wherein the spacer L2 comprises the moiety -(C(L'L"))t- with Lt and L"t in each repeating unit -C(LtL") being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, keto groups, aldehyde 35 groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, amidino, nitro, imino, alkylthio, arylthio, thiocarboxyl, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, 54 WO 2014/147175 PCT/EP2014/055598 azido, cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L' and L"t in each repeating unit may be the same or may differ from each other, with t preferably being in the range of from 1 to 20, more preferably 5 in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4. 11. The HAS derivative of any one of embodiments 1 to 10, wherein the spacer L2 is a symmetrical spacer. 10 12. The HAS derivative of any one of embodiments 1 to 10, wherein the spacer L2 is
-CH
2
-CH
2 -. 13. A method for the preparation of a hydroxyalkyl starch derivative comprising 15 (i) reacting hydroxyalkyl starch (HAS) of formula (Ta) ORa HAS', 0- 0 H S R ' O1 C *H ORc OH (la) via carbon atom C* of the reducing end of the HAS with the functional group M of a crosslinking compound according to formula (TI) 20 M-L1-S-T (II) wherein M comprises the group -NHR', with R' H or alkyl; LI is a spacer bridging M and S; T is H or a thiol protecting group PG; 25 HAS' is the remainder of the HAS molecule and Rb and R' are -[(CR 1
R
2 )mO]n H and are the same or different from each other; Ra is -(CR 1
R
2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having 30 from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR R2; n is from 0 to 6, thereby obtaining a HAS derivative of formula (Ib) ORa HAS 0 OH RbO' C-F1-L 1 -S-T
H
2 ORC (Tb) 55 WO 2014/147175 PCT/EP2014/055598 wherein -CH 2 -F 1- is the moiety resulting from the reaction of the group M with the HAS via the carbon atom C* of the reducing end, and Fl is a functional group comprising the group -NR'-; optionally removing PG in case T is PG to 5 give T = H; (ii) reacting the HAS derivative obtained in step (i) with a crosslinking compound of formula (III) 0 N-L2--N RX RYII) 10 L2 is a spacer bridging -NR- and -NRY-; R and RY are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; 15 thereby obtaining a HAS derivative of formula (I) ORa HAS, OH O RbO- C-F1-L 1 -S N-L2-- X ORcH 2 RX R wherein X is a leaving group, preferably selected from the group consisting of Ts. Ms, Cl, Br and I. 20 14. The method of embodiment 13, wherein T is a thiol protecting group PG, and wherein step (i) further comprises removing PG from the HAS derivative (Ib). 15. The method of embodiment 14, wherein PG has the structure -S-L1-M or -Trt 25 (Trityl), preferably S-Li -M. 16. The method of any one of embodiments 13 to 15, wherein HAS is hydroxyethyl starch (HES), R1, R2, R3, and R 4 are hydrogen, 30 m is 2 n is 0 to 6. 17. The method of any one of embodiments 13 to 16, wherein X is Br or I, preferably I. 56 WO 2014/147175 PCT/EP2014/055598 18. The method of any one of claims 13 to 17, wherein M is selected from the group consisting of CH 3 -NH-, CH 3
-CH
2 -NH-, CH 3
-CH
2
-CH
2 -NH-, (CH 3
)
2 -CH-NH-, H 2 N-,
H
2 N-O-, H 2 N-NH-, H 2 N-NH-(C=G)-, H 2 N-NH-(C=G)-GG- and H 2
N-NH-SO
2 -, M preferably being H 2 N-. 5 19. The method of any one of claims 13 to 18, wherein R and RY are, independently of each other selected from the group consisting of H, methyl, ethyl, propyl and butyl, or are forming a ring according to formula N-L2-N (V) 10 wherein R and RY, taken together, are -(CH 2
)
2 - or -(CH 2
)
3 -, preferably wherein R and RY are H. 20. The method of any one of embodiments 13 to 19, wherein the reacting according to 15 step (i) is carried out under reductive amination conditions, preferably at a temperature 5 'C to 100 'C and in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, water, formamide, buffer and reaction buffers and mixtures of two or more thereof, in particular using NaCNBH 4 . 20 21. The method according to embodiment 20, wherein subsequent to the reductive amination conditions, the HAS derivative is purified, preferably by ultrafiltration, and optionally subjected to further reducing conditions prior to step (ii), in particular by employing NaBH 4 . 25 22. The method of any one of embodiments 13 to 21, wherein the spacer Li comprises, preferably consists of the moiety -(C(L'L"))q- with L' and L" in each repeating unit -C(L'L")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, 30 alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amine, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, carboxymethylcarbamoyl [i.e. the group -C(=0)(-NH-CH 2 -COOH)], cycloalkyl such 35 as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other, with q 57 WO 2014/147175 PCT/EP2014/055598 preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4. 23. The method of any one of embodiments 13 to 22, wherein the spacer Li is -CH 2 5 CH 2 -. 24. The method of embodiment 14 or 15, wherein PG has the structure -S-L1-M and wherein the removing of the protecting group PG according to step (i) is carried out at a temperature in the range of from 0 to 80 'C and in an aqueous solvent system. 10 25. The method of any one of embodiments 13 to 24, wherein the spacer L2 comprises the moiety -(C(L'L"))t- with Lt and L"t in each repeating unit -C(LtL")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, 15 alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, keto groups, aldehyde groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, amidino, nitro, imino, alkylthio, arylthio, 20 thiocarboxyl, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups Lt and L"t in each repeating unit may be the same or may differ from each other, with t preferably being in the range of from 1 to 20, more preferably 25 in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4. 26. The method of any one of embodiments 13 to 25, wherein the spacer L2 is a symmetrical spacer, preferably -CH 2
-CH
2 -. 30 27. The method of any one of embodiments 13 to 26, wherein the reacting according to step (ii) is carried out at a temperature in the range of from 10 'C to 50 'C and preferably in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, reaction buffers and mixtures of two or more thereof, more 35 preferably in DMF and/or DMSO, 28. The method of any one of embodiments 13 to 27, wherein the reacting according to step (ii) is preferably carried out in non aqueous medium, in particular in a solvent 58 WO 2014/147175 PCT/EP2014/055598 selected from the group consisting of DMSO, DMF, DMA, NMP and a mixture of two or more thereof. 29. The method of any one of embodiments 13 to 28, further comprising 5 (iii) reacting the HAS derivative of formula (I) via the group -NRY-C(=0)-CH 2 -X with an -SH group of a thiol group comprising compound Q, thereby forming a HAS derivative of formula (IV) ORa HAS '-, OH 0 0 R bo~~ C-F1-L1--S N--L2--- -- S ORc RX y (IV) 10 wherein -Q' is the remainder of the thiol group comprising compound Q which is linked via the group -S- of the thiol group to the group -NRY-C(=0)-CH 2 -. 30. The method of embodiment 29, wherein Q is selected from the group consisting of, 15 thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, cells, viruses, liposomes, microparticles, and micelles. 31. The method of embodiment 29, wherein Q is selected from the group consisting of, 20 thiol group comprising, peptides, polypeptides, oligonucleotides and nucleic acid analogs, more preferably from the group consisting of peptide hormones, peptide aptamers, plasma proteins (such as immunoglobulins, growth factors, cytokines, coagulation factors including vWF), enzymes, enzyme inhibitors, albumins, natural or artificial binding proteins (such as alternative scaffold proteins, antibody 25 fragments, soluble receptors), ribonucleic acids, deoxyribonucleic acids, peptide nucleic acids (PNA), and locked nucleic acids (LNA). 32. The method of embodiments 18 to 31, wherein Q is a peptide or polypeptide, and wherein the reacting according to step (iii) is carried out at a temperature in the range 30 of from 5 0 C to 50 0 C and in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, water, formamide, reaction buffers and mixtures of two or more thereof. 33. A HAS derivative obtainable or obtained by a method according to any one of 35 embodiments 13 to 28. 59 WO 2014/147175 PCT/EP2014/055598 34. A HAS derivative, or a salt or solvate therof, obtainable or obtained by a method according to any one of embodiments 29 to 33. 35. A HAS derivative according to embodiment 34, wherein the derivative is at least 5 stable at a pH in range of from 4.0 to 8.5, preferably in the range of from 6 to 8.5. 36. A HAS derivative according to embodiment 34 or 35, wherein Q is a, thiol group comprising, peptide or polypeptide. 10 37. A HAS derivative according to embodiment 34 or 35, wherein Q is a, thiol group comprising, oligonucleotide or nucleic acid, such as a modified DNA or RNA aptamer. 38. Use of a HAS derivative according to any one of embodiments 1, 2, 5 to 11, or 33 as 15 reactant for coupling to a thiol group of a thiol group comprising compound Q. 39. A HAS derivative, or a salt or solvate therof, according to any one of embodiments 34 to 37 for use as a medicament. 20 Figures: Figure 1: Cation exchange chromatography of an L7-HES-Ubi conjugate The chromatographic separation of the L7-HES-Ubi coupling reaction according to 25 example C1 is monitored by UV spectroscopy at 220 nm (continuous line, y = absorbance in AU) as function of the elution volume (x = elution volume in ml). The percentage of eluent B (broken line) and the conductivity (dotted line, 100% eluent B = 86 mS/cm) are shown. Chromatographic conditions were as follows: 30 Chromatography system: Akta Purifier 100 (GE Healthcare) Column: Hi Trap SP HP 1 ml (GE Healthcare) Eluent A: 20 mM acetate, pH 4.0 Eluent B: 20 mM acetate, pH 4.0, 1 M NaCl Operating conditions: flow rate 1.5 ml/min, 25 'C 35 Gradient: equilibration, 5 CV, 0% B; sample load; wash, 5 CV, 0% B; elution conjugate, 5 CV, 40% B; elution free Ubi, 40 CV, 40-65% B; regeneration, 5 CV, 100% B; reequilibration, 5 CV, 0% B. Sample load: conjugate 20fold diluted in eluent A and adjusted to pH 4.0 60 WO 2014/147175 PCT/EP2014/055598 Non-reacted HES derivative is found in the flow-through. The conjugation to the HES derivative weakens the interaction of the protein with the column material resulting in a decrease of elution time for the L7-HES-Ubi conjugate (c) as compared to the unmodified Ubi (u). 5 Figure 2: RP-HPLC analysis of the coupling reaction of L7-HES to Ubi Figure 2 shows a section of the RP-HPLC analysis of an L7-HES-Ubi conjugate that was prepared according to example Cl. The separation is monitored by UV spectroscopy at 220 nm (continuous line, y = absorbance in mAU) as function of time (x = retention time 10 in min). The section shows a part of the gradient from 28.2% to 34.0% of eluent B (broken line). Chromatographic conditions were as follows: Chromatography system: Ultimate 3000 HPLC System (Dionex) Column: Jupiter C18, 300 A, 5 gin, 4.6 x 150 mm (Phenomenex) 15 Guard column: C18 guard cartridges (Phenomenex) in a SecurityGuardTM Cartridge System (Phenomenex) Eluent A: 0.1% trifluoroacetic acid in water Eluent B: 0.l1% trifluoroacetic acid in acetonitrile Operating conditions: flow rate 1 ml/min, 25 'C 20 Gradient: 0-1.0 min, 2% eluent B; 1-3 min, 2-25% eluent B; 3-15 min, 25-35% eluent B; 15-15.5 min, 35-95% eluent B; 15.5-23 min, 95% eluent B; 23-24 min, 95-2% eluent B; 24-27 min, 2% Sample load: 10 gg of reaction mixture, diluted in demineralized water to a final protein concentration of 0.1 g/l 25 The L7-HES-Ubi conjugate (c) elutes with a retention time of 11.99 min and is separated from the free Ubi (u) eluting at 13.92 min. Figure 3: Stress stability study of an L7-HES-Ubi conjugate analyzed by SEC Figure 3 shows a section of an SEC analysis of an L7-HES-Ubi conjugate prepared 30 according to example Cl and incubated for 20 days at pH 7.0 and 40 'C. The chromatographic separation is monitored by UV spectroscopy at 220 nm (y = absorbance in mAU) as function of time (x = retention time in min). Chromatographic conditions were as follows: Chromatography system: Ultimate 3000 HPLC System (Dionex) 35 Column: Superose 12 10/300 GL (GE Healthcare) Eluent: 1x PBS (5 mM sodium phosphate, 1.7 mM potassium phosphate, 150 mM sodium chloride, pH 7.4, Lonza) Operating conditions: flow rate 0.5 ml/min, 25 'C, run time 30 min 61 WO 2014/147175 PCT/EP2014/055598 Sample load: 10 gg of reaction mixture, diluted in elution buffer to a final protein concentration of 0.33 g/l The L7-HES-Ubi conjugate (c) elutes with a retention time of 10.09 min and is separated from the free Ubi (u) eluting at 14.73 min. 5 Figure 4: Test for unwanted side-reactions with thiol-free proteins Figure 4 shows the coupling to the Cys free Ubi variant (W-Ubi) shown as black bars and IFN-a (contains no single Cys) shown as white bars to different HES derivatives. X-axis: 1: L3-HES, 2: L5-HES, 3(#): L7-HES, 4: LI-HES, 5: Ll-HES, 6: L13-HES, 7: mi 10 PEGI, 8: mi-PEG2, 9: mi-HES (For the respective linker structures, see Table 1) y-axis: yield of conjugated product. The detection of conjugated product is an indication for unwanted non-specific reactions with functional groups other than thiol groups of cysteines. The amount of protein conjugate was determined using RP-HPLC (see Table 7). (#) = according to the invention. 15 The results shown in figure 4 cleary demonstrate that derivatives according to the invention (see entry 3) show a superior selectivity for the thiol group comprising compound when compared to the other tested derivatives. 20 Figure 5: Stress stability of Ubi conjugates depending on the linker The stress stability of various conjugates was determined by RP-HPLC or SEC (see Example C1) after 20 days incubation and is shown as percent of conjugate degradation (y) at pH 4.0 (black bars), pH 7.0 (white bars) and pH 8.0 (broken bars) and at 40 'C. The 25 numbers of the bars displayed in figure 5 correspond to the respective linker L having the same number as the bar (see Table 1). Stars indicate linkers or conditions that were not investigated for stress stability analysis. This figure clearly demonstrates the high stability of the tested Ubi comprising conjugates 30 according to the invention, in particular at physiologically pH or higher pH ranges. Figure 6: Stress stability of MEP conjugates depending on the linker The stress stability of various conjugates was determined by RP-HPLC or SEC (see 35 Example E11) after 20 days incubation and is shown as percent of conjugate degradation (y) at pH 4.0 (black bars), pH 7.0 (white bars) and pH 8.0 (broken bars) and at 40 'C. The numbers of the bars displayed in figure 6 correspond to the respective linker L having the same number as the bar (see Table 1). Stars indicate linkers or conditions that were not investigated for stress stability analysis. 62 WO 2014/147175 PCT/EP2014/055598 This figure clearly demonstrates the high stability of the measured MEP comprising conjugates according to the invention, in particular at physiologically pH or higher pH ranges. 5 Figure 7: Average decomposition rate for conjugates with 3 linkers attached to HES molecules of differing size and different target molecules The linker structures LI, L7 and LI (see Table 1) were attached to thiol-modified HES molecules of different size (Mw ~30, 100 and 250 kDa). Conjugates with MEP, Ubi and 10 HSA (only largest HES) were prepared and subjected to stress stability as described in examples E1I and C6. The average degradation rate in % after 20 days for all conjugates of the respective linker tested is shown in the figure. Figure 8: RP-HPLC analysis of Example E22 comparative example E21 15 Figure 8 shows a representative section of the RP-HPLC analysis of HES-Ubi conjugation reactions conducted according to table 10. The separation is monitored by UV spectroscopy at 220 nm (continuous line, y = absorbance in mAU) as function of time (x = retention time in min). The section shows a part of the gradient from 28.2% to 34.0% of 20 eluent B (broken line). Chromatographic conditions were as follows: Chromatography system: Ultimate 3000 HPLC System (Dionex) Column: Jupiter C18, 300 A, 5 gin, 4.6 x 150 mm (Phenomenex) Guard column: C18 guard cartridges (Phenomenex) in a SecurityGuard Cartridge System 25 (Phenomenex) Eluent A: 0.l1% trifluoroacetic acid in water Eluent B: 0.l1% trifluoroacetic acid in acetonitrile Operating conditions: flow rate 1 ml/min, 25 'C Gradient: 0-0.5 min, 5% eluent B ; 0.5-0.6 min, 5-25% eluent B; 0.6-6.1 min, 25-35% 30 eluent B; 6.1-6.2 min, 35-95% eluent B; 6.2-8.2 min, 95% eluent B; 8.2-8.3 min, 95-5% eluent B; 8.3-10.3 min, 5% Sample load: 10 gg of reaction mixture, diluted in demineralized water to a final protein concentration of 0.1 g/l The HES-Ubi conjugate eluted as a broad peak with a retention time between 4.5 and 5.5 35 min, multiple by-products of the conjugation between Ubi and presumably linker-derived impurities were found at 5.5 to 6 min, while free Ubi protein was eluting at 6.2 min. Three samples are shown in stacked mode, in the upper chromatogram (solid line) L7-HES (with HES derivative E20) reaction mixtures are investigated. In the middle and lower sample analysis the results for the HES derivatives according to Example E18 (dashed 63 WO 2014/147175 PCT/EP2014/055598 line) and example E19 (dotted line) are presented, with significantly more reaction by products showing up. X: retention time in min; Y: arbitrary absorption units at 220 nm 5 The following examples are intended to illustrate the present invention without limiting it. Examples The following abbreviations were used: 10 AlAT al-antitrypsin Ac-R6-NH 2 Ac-Arg-Ser-Cys-Arg-Trp-Arg-NH 2 , NeoMPS, France AU arbitrary unit CV column volume DMF N,N-dimethylformamide 15 DMSO dimethylsulfoxide DTT dithiothreitol EDTA ethylenediaminetetraacetic acid disodium salt dihydrate HES hydroxyethyl starch HSA human serum albumin 20 IFN-a Interferon a2b IL 1RA interleukin-1 receptor antagonist mi-PEGI maleimide PEG (Jenkem, ZZ102P046) mi-PEG2 maleimide PEG (NOF, ME-200MAOB) MWCO molecular weight cut-off 25 Ms Mesyl, methanesulfonyl none n.d. not determined PET 4-pyridineethanethiol hydrochloride, TCI Pierce today Thermo Fisher Scientific 30 RGC reactive group content: defined as percentage of HES molecules modified with a certain functional group with respect to all HES molecules (based on M,, of the respective HES derivative) R6R H-Arg-Ser-Cys-Arg-Trp-Arg-OH, NeoMPS, France RP-HPLC reversed phase high performance liquid chromatography 35 SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC size exclusion chromatography SlyD sensitive to lysis protein D TCI Tokyo Chemical Industry TFA trifluoroacetic acid 64 WO 2014/147175 PCT/EP2014/055598 TFE 2,2,2-trifluoroethanol THF tetrahydrofurane TRC Toronto Research Chemicals Ts Tosyl, p-toluenesulfonyl 5 Ubi Ubiquitin F45W S57C W-Ubi Ubiquitin F45W All chemicals were obtained from Sigma-Aldrich, Taufkirchen, Germany unless otherwise noted. 10 I: Homobifunctional linker compounds The structures of bifunctional linker compounds used in the following examples are given in table 1. 15 Example El: Synthesis of linker (Li)
C
16
H
20
N
2 0 2
S
4 Mo Wt 400,61 N HS, O O SH DIEA O OS N 2-[2-(2-Mercapto-ethoxy)- 2-{2-[2-(2-[Pyrid-2-yF}-disulfanyl-ethoxy} ethoxy]-ethanethiol ethoxy]-ethy!disulfanyl}-pyridine A 11 three-neck flask was equipped with pressure exchange, magnetic stirring bar and dropping funnel. The flask was loaded with 15.1 g of 2,2'-dithiodipyridine, 500 ml of 20 methanol and 50 ptl of N,N-diisopropylethylamine under inert atmosphere. A solution of 2 [2-(2-mercapto-ethoxy)-ethoxy]-ethanethiol in methanol (2 g/120 ml) was added drop-wise over a period of 30 min. 1 h after the ending of the first addition a second portion of the 2 [2-(2-mercapto-ethoxy)-ethoxy]-ethanethiol (0.5 g in 30 ml methanol) was added drop wise and the reaction mixture was stirred at room temperature. 25 After complete conversion of the disulfide, the solvent was removed at room temperature under reduced pressure. The residual oil (18.4 g) was purified by flash chromatography on silica (mobile phase: hexane/ethyl acetate 2:1 (v/v)). The crude product was further purified by a second chromatography on silica (mobile phase: hexane/ethyl acetate 1:1 30 (v/v)). Li was obtained in 49% yield (2.7 g). Example E2: Synthesis of linker (L6) 65 WO 2014/147175 PCT/EP2014/055598 0 20 0 _O O^,,H 2 N N + H 2 N O NNH2 0 00 A 250 ml Schlenk flask was charged with maleimidopropionic acid-N-hydroxysuccinimide ester (ABCR, Karlsruhe, Germany, 1.24 g; 4.66 mmol) and dry dichloromethane (100 ml). To the resulting suspension triethylamine (650 gl; 4.66 mmol) was added followed by 2,2' 5 (ethylenedioxy)bis(ethylamine) (341 gl; 2.33 mmol) at room temperature. The resulting solution was stirred for 24 h. It was washed with 25 ml of a saturated sodium bicarbonate solution followed by 25 ml of brine. The organic phase was dried over sodium sulfate, filtered and evaporated to yield L6 (600 mg; 1.33 mmol; 57.10%) as a pink powder. 10 Example E3: Synthesis of linker (L8) H0 H2N N I 0 0 H4 A 500 ml round bottom flask was charged with 1,6-diaminohexane (600 mg; 5.16 mmol) and 200 ml of sodium hydroxide (2M). A solution of iodoacetyl chloride (ABCR, Karlsruhe, Germany, 986 pil; 10.99 pimol) in 50 ml of 1,2-dichloroethane was added. The 15 mixture was stirred for 10 min at room temperature followed by filtration. The residue was crystallized from ethanol to yield L8 (1.28 g; 2.83 mmol; 54.8%) as a white solid. Example E4: Synthesis of linker (L9) 0) 0 HN'O NH, + 2 1-AI ' N 1 H H 20 A 50 ml three-neck flask was equipped with two dropping funnels and an internal thermometer. The reactor was charged with 2-(2-aminoethoxy)ethylamine (296 pil; 2.79 mmol) and chloroform (5 ml). The solution was cooled to -5 'C in an acetone dry ice bath and solutions of iodoacetyl chloride (ABCR, Karlsruhe, Germany, 500 pil; 5.58 mmol) in 5 ml of chloroform and potassium carbonate (780 mg; 5.64 mmol) in 3 ml of water were 25 added simultaneous over a period of 30 min keeping the internal temperature below 5 'C. The mixture was stirred for 5 h at room temperature followed by addition of 20 ml of chloroform and 10 ml of water. After separation, the organic phase was washed with 10 ml of chloroform. The organic extracts were dried over sodium sulfate, followed by filtration and evaporation. The crude product was crystallized from toluene to yield L9 (240 mg; 30 545 ptmol; 19.6%) as a pale yellow solid. 66 WO 2014/147175 PCT/EP2014/055598 Example E5: Synthesis of linker (L13) 0 0 Diazom ethane CI C HBr BrBr 0 0 A solution of diazomethane in diethyl ether (0.245 mol/l) was prepared from DIAZALD* 5 as described in T.H. Black, Aldrichimica Acta, 1983, 16, 3-10. A 11 one-neck flask was equipped with a magnetic stirring bar. An outside cooling (ice/water) was prepared. The flask was loaded with the diazomethane solution (500 ml). A dropping funnel with pressure exchange was installed. A solution of freshly distilled 10 hexanedioyl-dichloride (4.58 g) in 30 ml diethyl ether was added drop-wise under slight development of gas. The funnel was washed with 20 ml diethyl ether and the mixture was stirred for 1 h at 0 'C. An aqueous HBr-solution (9.5 ml, 62% (w/w)) was poured to this reaction mixture in one portion under a strong development of gas. A precipitate appeared. The temperature was allowed to warm to room temperature and the resulting mixture was 15 stirred overnight. Diethyl ether was added (6 1) to dissolve the precipitate. The resulting solution was washed three times with 200 ml of water. The organic phase was dried with sodium sulfate and filtered. Activated charcoal (2.4 g) was added; the mixture was stirred for 30 min and filtered over kieselguhr. Again the organic phase was dried with sodium sulfate and filtered. Then 8 0
-
9 0 % of the solvent were evaporated under reduced pressure at 20 room temperature. The precipitate was collected by filtration, washed with cold diethyl ether and dried in vacuo. L13 was obtained in 47% yield (3.5 g) and used without further purification. Example E6: Synthesis of linker (L14) 0 0 Na I, Acetone 25 0 0 A 25 ml two-neck flask was equipped with a magnetic stirring bar under inert atmosphere. The flask was loaded with sodium iodide (1.1 g), evacuated and refilled with nitrogen. Under inert atmosphere acetone (7 ml) was added and the mixture was stirred until the salt dissolved. In a nitrogen flow L13 (1 g) was added and the resulting mixture was stirred at 30 room temperature for 3 h. Then a further portion of sodium iodide (0.1 g) was added. The reaction was monitored by GC. The solvent was removed at room temperature under reduced pressure and dichloromethane (100 ml) was added. The residual solid was filtrated and washed with 67 WO 2014/147175 PCT/EP2014/055598 dichloromethane (5 ml). The combined organic phases were washed with 20 ml of an aqueous solution of sodium hydrogensulfite/sodium sulfite and twice with 10 ml of water, dried with sodium sulfate and filtered. Approximately 75% of the solvent were evaporated under reduced pressure at room temperature. The precipitate was collected by filtration and 5 dried in vacuo. L14 was obtained in 81 % yield (1.06 g) as a white to off-white solid. II: Synthesis of thiol modified hydroxyethyl starch (thiol-modified HES) General Procedure E7: Preparation of thiol-modified HES 10 10 g HES (M, between 10 kDa and 700 kDa) were dissolved in 23 ml sodium acetate buffer (pH 5.0 and c = 1 mol/l) by vigorous stirring and heating (up to 60 C). To the clear solution, the crosslinking compound (II) (concentration varied from 0.18 to 2 mol/l, see Table 2) and NaCNBH 3 (concentration according to Table 2) were added. The reaction mixture was stirred at 60 'C for 16 to 24 h, diluted with water to 100 ml, neutralized with 15 diluted sodium hydroxide solution, purified by ultrafiltration with a membrane (e.g. MWCO 10 kDa) against 2 1 ultrapure water and concentrated to approximately 100 ml. 1 g sodium borohydride was added and the reaction mixture was stirred at 25 'C for 18 h. In case of cysteamine as crosslinking compound (II), the reaction mixture was stirred for 2 h. The pH was adjusted to 4.0 with IM aqueous HCl solution and the thiol-modified HES 20 was purified by ultrafiltration (e.g. membrane MWCO 10 kDa) against 1.5 1 of a 10 mmol/l sodium acetate buffer pH 4.0, containing 1 mmol/l EDTA and subsequently with 500 ml ultrapure water and lyophilized. The RGC of thiol-modified HES was determined with Ellman's Reagent as described in Instructions Ellman's Reagent, Pierce Biotechnology, Inc. 7/2004, USA. 25 For determination of M, and M,, see Example E1O. III: Synthesis of hydroxyethyl starch linker derivatives On the one hand, for a given linker structure, thiol-modified HES was varied with respect 30 to the mean molecular weight, and with respect to its molar substitution. On the other hand, the chemical nature of the linker was varied, for a given thiol-modified HES starting material. The reaction details are summarized in Tables 3 and 4. Example E8: General synthesis procedure 35 The amounts Al of thiol-modified HES X (weight average molecular weight Mw and molecular substitution MS see Table 2) were dissolved in the appropriate volume Vi of solvent Si by stirring at room temperature. To the clear solution, the indicated amount A2 of linker L was added dissolved in a given volume V2 of solvent S2. The reaction mixture 68 WO 2014/147175 PCT/EP2014/055598 was incubated for time t at 21 'C at a mixing rate of 750 rpm (see Table 3). All experiments with iodine containing linkers were performed in the dark. Work-up procedure 1 (for samples with < 1 g thiol-modified HES) 5 For work-up the solution was poured into seven-fold excess volumes of 2-propanol and centrifuged at room temperature for 15 min at 7000*g. The supernatant was discarded and the precipitate dissolved in ultrapure water to a final concentration of 5% (w/v). The product was purified by size exclusion chromatography using ultrapure water as 10 eluent, a guard column HiTrapTM Desalting 1*5 ml and a separation column HiPrepTM 26/10 Desalting 53 ml (both GE Healthcare). In each run 5 ml samples were injected. The chromatographic procedure was monitored by UV spectroscopy at the wavelength of 210 nm and 5 ml-fractions were collected. After use the column was equilibrated with 5 column volumes of 0.5M acetic acid and 5 column volumes of ultrapure water. The 15 fraction containing the HES-derivative were pooled and lyophilized. Work-up procedure 2 (for samples with > 1 g thiol-modified HES) All samples with more than 1 g thiol-modified HES were diluted to a final concentration of maximum 10% (v/v) DMF or DMSO and filtered. The HES-linker derivative was purified 20 by ultrafiltration with a membrane (e.g. MWCO 10 kDa) against 20 times its own volume of ultrapure water and lyophilized. IV: Analysis of the hydroxyethyl starch linker derivatives 25 Example E9: Determination of reactive group content of HES-linker derivatives General synthesis procedure Between 4 and 25 mg HES-linker derivative was dissolved either in 0.21M phosphate buffer pH 8.5 containing 5 mM EDTA (linker L7-L19) or PBS-buffer pH 6.5 containing 5 mM EDTA (linker L1-L6). One equivalent of thiol T (referred to M" of the HES species; 30 see Table 3) was dissolved in 5 mM EDTA solution pH 6.0 and added to the HES-linker derivative solution. The final concentration of HES-linker derivative in the reaction mixture was 15% (w/v). The reaction mixture was incubated at 21 'C for 2 h. The samples were analyzed by RP-HPLC/UV (conditions see below in Example Ellc) at the UV maximum of T (for peptides 280 nm and for PET 254 nm). The RGC was evaluated by 35 comparison of the relative peak area of the conjugate to the sum of all other products. The reaction conditions for the various target molecules were not optimized. Example E10: General procedure for the determination of the mean molecular weight Mw 69 WO 2014/147175 PCT/EP2014/055598 M, and M,, were determined as described in WO 2012/004007 Al, Example 1.9. Example Eli: Stability studies a) General synthesis procedure: Synthesis of HES-linker-(2-mercaptoethyl)pyrazine 5 conjugates Between 150 and 200 mg HES-linker derivative (defined by the linker and the HES species) was dissolved in 0.1M borate buffer pH 8 containing 5 mM EDTA to a concentration of 20% (w/v). (2-Mercaptoethyl)pyrazine (20 equivalents referred to M,, of the HES species) was dissolved in the same volume of DMF and added to the HES-linker 10 derivative solution. The reaction mixture was incubated at 21 'C for 16-24 h. After incubation the samples were purified by first precipitation and subsequently desalting and isolated by freeze drying as described above in example E8. The conjugates were analyzed by RP-HPLC (for conditions see below). The reaction conditions for the various target molecules were not optimized. 15 b) Evaluation of the stability of HES-linker-(2-mercaptoethyl)pyrazine conjugates in aqueous buffer solutions of different pH The conjugates were dissolved in buffer and diluted to a concentration of 20 mg/ml. The stability study was performed in a) 0. 1M sodium acetate buffer pH = 4.0, b) 0. 1M sodium 20 phosphate buffer pH = 7.0 or c) 0.1M sodium borate buffer pH = 8.0. The samples were incubated for up to 32 d at 40 'C. Samples were taken after 0, 1, 5, 10 and 20 d. They were analyzed by RP-HPLC/UV at 266 nm (for conditions see below). The decay was evaluated by comparison of the relative peak area of the conjugate to the sum of all decomposition products. 25 c) RP-HPLC analysis of HES-linker-(2-mercaptoethyl)pyrazine conjugates The product was analyzed by RP-HPLC using ultrapure water with 0.1% TFA (Uvasol*, for spectroscopy, Merck, Code No. 1.08262.0100) as eluent A and acetonitrile (Uvasol*, Reag. Ph. Eur., gradient grade for liquid chromatography, Code No. 1.00030.2500) with 30 0.1% TFA as eluent B. The analysis was performed with a pre-column SecurityGuardTM Cartridge system, Widepore C18, ODS, 4 mm L * 3.0 mm ID (Phenomenex, Code No. AJO-4321) and a separation column Reprosil Gold 300, C18, 5g, 150 * 4.6 mm (Dr. Maisch, Code No. r35.9g.s1546, SN: 120109 251501). In each run 100 gl samples were injected. The samples were analyzed with the following gradient with a flow of 2 ml/min: 35 0-2.5 min 2% B 2.5-10 min 2-60% B 10-12.6 min 98% B 12.7-15.3 min 2% B 70 WO 2014/147175 PCT/EP2014/055598 Example E12: Synthesis of linker (L12) diamino- H Br ,tha 0 BrN N B BrBr Br etane Hr 0 A solution of diaminoethane (226.9 ml; 3.38 mol) in 3.39 1 of trichloromethane was cooled to an internal temperature of 0 'C and solutions of a) bromoacetylbromide (880.8 ml; 5 10.14 mol) in 4.07 1 of trichloromethane and b) K 2
CO
3 (1.12 kg; 8.08 mol) in 4.07 1 of water were added simultaneously drop-wise with vigorous stirring over a period of 4 hours while keeping the internal temperature below 5 'C. After the addition was completed, the mixture was stirred over night at room temperature. The resulting suspension was heated to reflux and the phases were separated hot. The aqueous layer was extracted with 2 1 of hot 10 trichloromethane and the extract was combined with the organic phase of the reaction. The organic extracts contained large amounts of a solid A, which was filtered off. The filtrate was dried over sodium sulfate and the solvent was evaporated after filtration at a bath temperature of 60 'C to yield 130 g of crude product, which was crystallized from 850 ml of hot ethanol to yield the first batch of E12 as an off-white solid. The solid A (1.1 kg 15 before drying) was crystallized from 3.25 1 of hot ethanol to yield another batch of E12. Both batches were combined after drying at 10- 3 mbar at room temperature to yield 516.30 g of E12 (1.71 mol; 50.5%). Example E13: Synthesis of linker (L16) HO CO 2 Me MsCI MsO CO 2 Me OH MsO CO 2 H 0 SOC1 2 diamino- O H MsO COCI ethane , Ms N OMs 20 0 a) Mesyloxy acetic acid chloride Methyl glycolate (5.0 g) was dissolved in THF (40 ml), triethylamine (9.48 ml) was added and the reaction mixture was cooled to 0 'C. A solution of mesyl chloride (7.9 g) in THF (15 ml) was added drop-wise. The reaction was stirred for 30 minutes at 0 'C and over 25 night at room temperature. The mixture was filtered to remove the triethylamine salts, the solid was washed twice with THF and the solvent was removed in vacuo. The residue was dissolved in 100 ml of dichloromethane, washed twice with 80 ml of HCl (0.1N), 100 ml of brine, dried over sodium sulfate and filtered. The solvent was removed in vacuo and the remaining oil (5 g) was re-dissolved in THF (200 ml). The reaction mixture was cooled to 30 0 'C, a solution of LiOH*H 2 0 (4.9 g) in 75 ml water was added drop-wise and the reaction 71 WO 2014/147175 PCT/EP2014/055598 was stirred vigorously for 10 minutes at 0 'C and 30 minutes at room temperature. Water (100 ml) was added, THF was removed at reduced pressure and the remaining aqueous solution was extracted twice with diethyl ether (200 ml each). The aqueous phase was acidified to pH 2-3 with IN HCl and extracted three times with 200 ml of diethyl ether. 5 The combined organic phases were dried with sodium sulfate, filtered and the solvent was removed in vacuo yielding 1.8 g of a white solid (210% yield based on methyl glycolate). Mesyloxy acetic acid (1.54 g) and SOCl 2 (11.0 ml) were heated to reflux for 3 hours. SOCl 2 was removed in vacuo without heating. The rest of SOCl 2 was removed by three cycles of adding 50 ml of dichloromethane followed by a removal of the solvent in vacuo. 10 Mesyloxy acetic acid chloride was obtained as brownish oil (724 mg, 42% yield) and was used without further purification. b) L16 To a solution of 1,2-ethylene diamine (107 mg) in dichloromethane (70 ml) triethylamine 15 (0.74 ml) was added and the reaction mixture was cooled to 0 'C. A solution of mesyloxy acetic acid chloride (675 mg) in dichloromethane (10 ml) was added drop-wise and the mixture was stirred at 0 'C for 45 minutes. L16 precipitated in the course of the reaction and was collected by filtration. It was washed 4 times with dichloromethane, dried in vacuo and used without further purification. L16 was obtained in 910% yield (m = 535 mg). 20 Example E14: Synthesis of linker (L17) TsCI LiOH HO CO 2 Me Tso CO 2 Me Ts CO 2 H 0 SOCl 2 diamino- H TsO COCl ethane , TsO N OTs 0 a) p-Tosyloxy acetic acid chloride Methyl glycolate (1.5 g) was dissolved in THF (25 ml), triethylamine (2.6 ml) was added 25 and the reaction mixture was cooled to 0 'C. A solution of tosyl chloride (3.49 g) in THF (8 ml) was added drop-wise. The reaction was stirred for 30 minutes at 0 'C and over night at room temperature. The mixture was filtered to remove the triethylamine salts, the solid was washed twice with THF and the solvent was removed in vacuo. The residue was dissolved in 50 ml of dichloromethane, washed twice with 40 ml of HCl (0. IN), 50 ml of 30 brine, dried over sodium sulfate and filtered. The solvent was removed in vavuo and the remaining oil (2.2 g) was re-dissolved in THF (60 ml). The reaction mixture was cooled to 0 'C, a solution of LiOH*H 2 0 (687 mg) in 20 ml water was added drop-wise and the 72 WO 2014/147175 PCT/EP2014/055598 reaction was stirred vigorously for 10 minutes at 0 'C and 30 minutes at room temperature. Water (40 ml) was added, THF was removed at reduced pressure and the remaining aqueous solution was extracted twice with diethyl ether (100 ml each). The aqueous phase was acidified to pH 2-3 with IN HCl and extracted three times with 100 ml of diethyl 5 ether. The combined organic phases were dried with sodium sulfate, filtered and the solvent was removed in vacuo yielding 850 mg of a white solid (22% yield based on methyl glycolate). Tosyloxy acetic acid (850 mg) and SOCl 2 (8.5 ml) were heated to reflux for 2 hours. SOCl 2 was removed in vacuo without heating. The rest of SOCl 2 was removed by three cycles of 10 adding 50 ml of dichloromethane followed by a removal of the solvent in vacuo. p Tosyloxy acetic acid chloride was obtained as brownish oil (0.8 g, 87% yield) and was used without further purification. b) L17 15 To a solution of 1,2-ethylene diamine (54 mg) in dichloromethane (20 ml) triethylamine (0.35 mL) was added and the reaction mixture was cooled to 0 'C. A solution of tosyloxy acetic acid chloride (517 mg) in dichloromethane (10 ml) was added drop-wise and the mixture was stirred at 0 'C for 30 minutes. The mixture was filtered and the precipitate was washed twice with dichloromethane. The combined organic fractions were washed 20 with 40 ml of aqueous NaHCO 3 (0.5% w/v), with 40 ml HCl (0.1N) and with 50 ml brine, dried over sodium sulfate and filtered. The solvent was removed in vacuo, yielding solid L17 in 91 % yield (0.4 g). Example E15: Synthesis of linker (L18) HO CO 2 Me TsC IN TsO CO 2 Me LOH TsO CO 2 H 0 SOC1 2 1,4-diamino- H TsO COCI butane - Ts N OTs H 25 0 p-Tosyloxy acetic acid chloride was synthesized as described in Example El4a. To a solution of 1,4-diaminobutane (48 mg) in dichloromethane (8 ml) triethylamine (0.224 ml) was added and the reaction mixture was cooled to 0 'C. A solution of tosyloxy 30 acetic acid chloride (300 mg) in dichloromethane (4 ml) was added drop-wise and the mixture was stirred at 0 'C for 30 minutes. The mixture was filtered and the precipitate was washed twice with dichloromethane. The combined organic fractions were washed with 40 ml of aqueous NaHCO 3 (0.5% w/v), with 40 ml HCl (0.1N) and with 50 ml brine, 73 WO 2014/147175 PCT/EP2014/055598 dried over sodium sulfate and filtered. The solvent was removed in vacuo, yielding solid L17 in 72% yield (0.2 g). Example E16: Synthesis of linker (L19) 5 Nal ciN N c a - N N o _ CI 0_ I A suspension of 1,1'-piperazine-1,4-diylbis(2-chloroethanone) (490 mg; 2.05 mmol) in 40 mL 2-butanone was heated at reflux for 10 min. Sodium iodide (880 mg; 5.87 mmol) was added under nitrogen followed by reflux for further 30 min. The mixture was centrifuged 10 down after cooling to room temperature. The liquid phase was evaporated and the remaining residue was taken up in dichloromethane (40 mL) and water (2 mL). The organic layer was dried over sodium sulfate, followed by filtration and evaporation to yield 100 mg 1,1'- piperazine-1,4-diylbis(2-iodethanone) (237 pimol; 11.6 %) as a yellow solid. 15 V. Conjugation of the HES derivatives to proteins Example C1: Conjugation of thiol-reactive HES and Ubiquitin Ubiquitin (Ubi, pdb code: 1UBQ) was selected as model protein for testing reactivity and 20 stability of various thiol-reactive HES derivatives. For this purpose the protein variant Ubi F45W S57C (with a C-terminal His6 tag, manufactured by Scil Proteins, Halle, Germany) was used allowing site-specific conjugation to the single cysteine residue introduced on position 57 and detection by UV spectroscopy by the tryptophan residue introduced on position 45. 25 To avoid dimerization of the protein and to get a high yield of conjugate, Ubi had to be reduced with DTT before starting the coupling procedure. DTT is used in a 50fold molar excess and the reduction takes place for 1 hour at 37 'C. Afterwards the DTT was removed by cation exchange procedure on a 1 ml HiTrap SP HP (GE Healthcare). DTT elutes from 30 the column in the flow-through, afterwards Ubi was eluted by a step gradient. For small-scale conjugation reactions, a 10% or 40% (w/v) stock solution of the thiol reactive HES derivative (defined by the linker and the HES species) was prepared. The appropriate amount of HES derivative was weighed into the reaction tubes and dissolved in 35 reaction buffer until a clear solution appeared. The protein solution and the HES derivatives were combined in a specified ratio (Table 5) and mixed thoroughly. For large 74 WO 2014/147175 PCT/EP2014/055598 scale conjugation reactions, the appropriate amount of thiol-reactive HES derivative (see Table 5) was weighed directly in a 15 ml Falcon tube, dissolved in reaction buffer (see Table 5) as described above and mixed thoroughly with the appropriate protein solution. The reaction mixtures were analyzed by RP-HPLC (example see Figure 2), SEC (example 5 see Figure 3) and SDS-PAGE. The chromatogram monitored at a wavelength of 220 nm or 280 nm was integrated and the yield of the conjugation reaction was calculated from the peak areas of the conjugate and the non-modified protein. The coupling procedure was optimized for different pH conditions and various HES : target ratios (Table 5). 10 The preparation of the HES-Ubi conjugates was performed by a cation exchange chromatography. All chromatographic steps were performed at room temperature using an Akta Purifier 100 system (GE Healthcare) and monitored by UV spectroscopy at a wavelength of 220 nm and 280 nm and by conductivity measurements. For the preparation of the HES-Ubi conjugates a HiTrap SP HP 1 ml column (GE Healthcare) was used. 15 Eluents were exchanged to eluent A (20 mM acetate, pH 4.0) and eluent B (20 mM acetate, pH 4.0, 1 M NaCl); the column was equilibrated with 10 CV eluent A. The reaction mixture was diluted approximately 20fold using buffer A and loaded onto the column using the sample pump. The flow-through was collected in 50 ml Falcon tubes. Unbound sample was washed out with five CV eluent A and the conjugate was eluted with a flow 20 rate of 1.5 ml/min and a segmented salt gradient (Figure 1). Fractions containing HES-Ubi conjugates free of unmodified Ubi were combined and concentrated in a suitable centrifugal concentrator (e.g. Amicon Ultra 4, 10 kDa MWCO) and sterile-filtered using a 0.22 gm syringe filter with low protein binding (Pall Acrodisc). The concentration was determined by UV-spectroscopy. 25 Example C2: Conjugation of thiol-reactive HES and ILIRA (Kineret@) Coupling reactions of IL1RA (pdb code: lIRA) with HES derivatives were performed as described in example C1 (examples listed in Table 5). The reaction mixtures were analyzed by RP-HPLC (as described in example C1) with a Jupiter C18 column (300 A, 5 gm, 4.6 x 30 150 mm, Phenomenex) with a segmented gradient (0-0.2 min: 2% B, 0.2-0.8 min: 2-30% B, 0.8-5.8 min: 30-40% B, 5.8-6.5 min: 98% B, 6.5-7.0 min: 98% B, 7.0-9.0 min: 2% B) and a flow rate of 1 ml/min or by SEC (as described in example C1) with a flow rate of 0.5 ml/min within 60 min on a Superose 6 10/300 GL (GE Healthcare) column. 35 Preparations of conjugates for stability tests were performed in a similar way as described in example Cl by an anion exchange chromatography with two HiTrap Q HP 1 ml columns (GE Healthcare) under following conditions: flow rate 1 ml/min, eluent A: 10 mM Tris/HCl, pH 8.0, eluent B: 10 mM Tris/HCl, 0.25 M NaCl, pH 8.0, loaded sample fold diluted and a segmented gradient (5 CV: 0% B, 6 CV: 25% B, 11 CV: 25-36% B, 5 75 WO 2014/147175 PCT/EP2014/055598 CV: 100% B, 5 CV: 0% B). Fractions containing the protein conjugate were combined and concentrated in a centrifugal concentrator. Conjugates of L7-HES with IL1RA were tested for binding affinity to its natural binding 5 partner using SPR on a BlAcore system. Interleukin 1 receptor type I (R&D Systems) was immobilized on the chip surface and the kinetic binding parameters were determined for the conjugate in comparison to the unmodified protein (chip: CM3, immobilization: 950 RU, flow rate 30 gl/min, association 180 s, dissociation 900 s, regeneration: 6 s with 5 mM glycine buffer, pH 2.0). The curves were analyzed by assuming a 1:1 binding 10 stoichiometry. The L7-HES-IL1RA conjugate retained an excellent binding affinity with a KD value of 180 pM in comparison to 90 pM for the unmodified IL1RA and 187 pM for IL1RA that is conjugated to HES via N-terminal coupling. The conjugate shows an association rate ka of 2.69- 10 5 M-Is- 1 that is comparable to unmodified IL1RA (ka = 5.88- 105 M- 1 s-) and IL1RA that is conjugated to HES via N-terminal coupling (ka = 15 2.63 -105 M-Is-1). Example C3: Conjugation of thiol-reactive HES and A1AT A1AT (pdb code: 1KCT) contains a single cysteine that is partially capped and therefore had to be reduced before starting the coupling procedure by addition of a 10fold molar 20 excess of DTT for one hour at 37 'C. After reduction, the DTT was removed by buffer exchange in a centrifugal concentrator. Coupling reactions of A1AT with HES derivatives were performed as described in example Cl (examples listed in Table 5). The reaction mixtures were analyzed by RP 25 HPLC with a Jupiter C18 column (300 A, 5 gm, 4.6 x 150 mm) as described in example C1 with a segmented gradient (0-4 min: 5% B, 4-8 min: 5-46% B, 8-17 min: 46-51 % B, 17-22 min: 98% B, 22-25 min: 5% B) and a flow rate of 1 ml/min. Preparations of conjugates for stability tests were performed in a similar way as described 30 in example C1 by an anion exchange chromatography. The chromatographic steps were performed with a HiTrap Q HP 1 ml column and the following conditions: flow rate 1 ml/min, eluent A: 20 mM Tris/HCl, pH 8.0, eluent B: 20 mM Tris/HCl, 1 M NaCl, pH 8.0, loaded sample 10fold diluted and a segmented gradient (30 CV: 0% B, 42 CV: 0 21 % B, 5 CV: 21-100% B, 5 CV: 100% B, 5 CV: 0% B). Fractions containing the protein 35 conjugate were combined and concentrated in a centrifugal concentrator. The activity of the L7-HES-A1AT conjugate was analyzed in an elastase inhibition assay as described in Beatty et al. (1980, JBC, 255, 9, 3931-3934) and compared to unmodified 76 WO 2014/147175 PCT/EP2014/055598 AlAT. The L7-HES-A1AT conjugate shows 101% of the elastase inhibition activity compared to unmodified A1AT. Example C4: Conjugation of thiol-reactive HES and SlyD 5 SlyD D101C (Zoldak and Schmid, 2011, JMB, 406(1), 176-94; pdb code: 2K81) contains a single additional cysteine that had to be reduced by addition of a 10fold molar excess of DTT for one hour at 37 'C. After reduction, the DTT was removed by buffer exchange in a centrifugal concentrator. 10 A coupling reaction of SlyD with a HES derivative was performed as described in example Cl (see Table 5). The reaction mixture was analyzed by RP-HPLC with a Jupiter C18 column (300 A, 5 gm, 4.6 x 150 mm) as described in example Cl with a segmented gradient (0-1 min: 2-30% B, 1-6 min: 30-50% B, 6-10 min: 98% B, 10-13 min: 2% B) and a flow rate of 2 ml/min or by SEC (as described in example C1) with a flow rate of 15 0.5 ml/min within 60 min on a Superose 12 10/300 GL (GE Healthcare) column. Preparations of conjugates for stability tests were performed in a similar way as described in example Cl by an anion exchange chromatography with a Q Ceramic HyperD F 1 ml column (PALL) and the following conditions: flow rate 1 ml/min, eluent A: 20 mM 20 Tris/HCl, pH 8.0, eluent B: 20 mM Tris/HCl, 1 M NaCl, pH 8.0, loaded sample 10fold diluted and a segmented gradient (5 CV: 0% B, 20 CV: 0-40% B, 5 CV: 100% B, 5 CV: 0% B). Fractions containing the protein conjugate were combined and concentrated in a centrifugal concentrator. 25 Prolylisomerases like SlyD catalyze the trans to cis isomerization of peptidyl prolyl bonds. The prolyl isomerase activities of SlyD and the L7-HES-SlyD conjugate were measured by a prolyl isomerase activity assay according to Zoldak et al. (2009, Biochemistry, 48, 10423-10436). The rate constant of the cis to trans isomerization of the prolyl bond was determined using Origin 8.1. The L7-HES-SlyD conjugate shows an equal activity for 30 prolyl isomerization as unmodified SlyD. The catalytic activity of unmodified SlyD is 5.6-106 M-1s-1 and the activity of the L7-HES-SlyD conjugate is 7.3.106 M- s- (131% of the activity of unconjugated SlyD). The higher activity of the conjugate compared to unmodified SlyD is within the error of the assay. 35 Example C5: Conjugation of thiol-reactive HES and HAS Coupling reactions of HSA (pdb code: 1E7H) with HES derivatives were performed as described in example C1, examples are shown in Table 5. The reaction mixtures were 77 WO 2014/147175 PCT/EP2014/055598 analyzed by SEC (as described in example C1) with a Superose 6 10/300 GL column with a flow rate of 0.5 ml/min within 60 min as described in example C1. Preparations of conjugates for stability tests were performed in a similar way as described in example C1 by anion exchange chromatography with a HiTrap Q HP 5 ml column under 5 following conditions: flow rate 1 ml/min, eluent A: 20 mM Tris/HCl, pH 8.0, eluent B: 20 mM Tris/HCl, 1 M NaCl, pH 8.0, loaded sample 10fold diluted and a segmented gradient (5 CV: 0% B, 40 CV: 0-30% B, 5 CV: 100% B, 5 CV: 0% B). Fractions containing the protein conjugate were combined and concentrated in a centrifugal concentrator. Stability studies were conducted as described in example C6. 10 Example C6: General description for coupling procedure The amount of the target molecule indicated in Table 5 was transferred into the appropriate reaction buffer. The indicated amount of HES derivative (defined by the linker and the HES species) was dissolved in reaction buffer and mixed with the target substance 15 solution. HES : target describes the molar ratio of reactive groups of HES to protein concentration (target). The coupling reactions were performed in 0.1 M phosphate with 5 mM EDTA or 0.1 M Tris/HCl depending on the required pH for two hours at 20 or 25 'C (Ubi and A1AT) or over night at 5 'C (all other targets). 20 Stability tests/Analysis of the specificity of the coupling to a thiol group of a protein For stability tests at pH 4.0, 5.5, 7.0 and 8.0, the conjugates were diluted into final buffer conditions of 20 mM acetate (pH 4.0 and 5.5) or 10 mM phosphate (pH 7.0 and 8.0), 0.5 mM ETDA, 154 mM NaCl to a final concentration of 1 mg/ml. The conjugates were stored at 40 'C for 20 days and analyzed by RP-HPLC (as described in example C1), SEC 25 (as described in example C1) or SDS-PAGE. The results are given in Tables 6 & 8; see also Figures 5-7. For specificity testing HES species with a molecular weight between 60 and 90 kDa (see Table 2) and PEG with a molecular weight of approximately 20 kDa were used. HES : 30 target/ PEG : target describes the molar ratio of reactive groups of HES/PEG to protein concentration (target). The coupling reactions were performed in 0.1 M phosphate, pH 8.0 at 20 'C for 72 hours. The Ubiquitin variant (W-Ubi) contains no additional single cysteine. IFN-a2b lacks a 35 single cysteine for site-specific conjugation. The specificity was analyzed by SDS Page and RP-HPLC (see Table 7 and Figure 4). As may be taken from table 7 as well as figure 4, linker L7 shows a high selectivity towards thiol groups, thus no conjugate formation with W-Ubi has been observed. 78 WO 2014/147175 PCT/EP2014/055598 VI. Preparation of HES derivatives with monohalogen-subsituted acetic acid linker compounds 5 Example E17 Preparation of amino-modified HES as described W02005/014050A2 Example 3 (p.137), (not according to the invention) Oxo-HES 50/0.7 Lot 502(Mw = 40.7 kDa, Mn = 17.7 kDa, PD 2.3) was prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim. 3.0 g (0.17 mmol) of oxo-HES 50/0.7 were dissolved in 10 mL anhydrous dimethyl 10 sulfoxide (DMSO, VWR, Lot 08A100140) and added dropwise under argon to a solution of 2.1 mL (21.2 mmol) 1,4-diaminobutane in 3.7 mL anhydrous dimethyl sulfoxide and stirred at 40'C for 19 h. The reaction mixture was poured in a mixture of 120 mL ethanol and 120 mL acetone. The resulting precipitate was separated by centrifugation, washed with a mixture of ethanol and acetone and re-dissolved in 20 mL Milli-Q water. The 15 solution was dialyzed for 4 days against water (SnakeSkin dialysis tubing 3.5 kD cut off, Thermo Fisher, Lot JE123986) and subsequently lyophilized. The yield was 20.9 % (0.632 g) amino-oxo-HES 50/0.7 (Mw = 30.3 kDa, Mn = 15.9 kDa, PD = 1.9). Example E18: Preparation of iodoacetamide-HES from iodoacetic acid - Linker "IA EDC" (not according to the invention) 20 0.2 g (0.013 mmol) amino-oxo-HES 50/0.7 was dissolved in 1 mL Milli-Q water. 13.5 mg (0.07 mmol) iodoacetic acid (Fluka, Lot BCBC8636V) was dissolved in 85 gL Milli-Q water and the pH was adjusted to 4.7 with 0.01 mol/L NaOH. The iodoacetic acid solution and and 5.0 mg (0.03 mg) 1-ethyl-3-(3-dimethyl amino propyl) carbodiimid (Sigma, Lot 55H08001) were added to the HES solution. With 0.01 mol/L NaOH and HCl respectively 25 the pH of the solution was maintained at 4.7 during the whole reaction time of 4h. The product was purified according to work-up procedure 1 and subsequently lyophilized. The yield was 81.5 % (0.163 mg). The experimental details and results were summarized in table 9. Example E19 Preparation of iodoacetamide-HES from iodoacetic acid chloride 30 Linker "IA-Cl" (not according to the invention) 0.2 g (0.013 mmol) amino-oxo-HES 50/0.7 was dissolved in 1 mL anhydrous dimethyl formamide (DMF, acros dry, Lot 1367244). 103 mg (0.5 mmol) iodoacetic acid chloride (Alfa Aesar, Lot 10153766) was added under argon. After 4h at room temperature the iodocetamide-HES was purified according to work-up procedure 1 and subsequently 35 lyophilized. The yield was 74.7 % (0.148 mg). The experimental details and results were summarized in table 9. 79 WO 2014/147175 PCT/EP2014/055598 Example E20: Preparation of L7-HES 10/1.0 with specificity control reactions Thiol-derivatized HES 10/1.0 (X22) was obtained according to example 7 under reaction conditions listed in table 2. L7-HES10/1.0 was synthesized according to example E8 and 5 analyzed as described in example E9. Specific reaction conditions are given in Table 9. To assess the nonspecific reaction of the Linker L7 with unmodified HES control reactions with unmodified HES were conducted using identical conditions. The analysis of the reactive group content (see table 9) showed that under the reaction conditions according to the invention no side reactivity of unmodified HES with the linker 10 L7 was observed (comparison of entry 1 and 2). In comparison to the HES derivatives according to comparative examples E18 and E19 the reactive group content of the L7-HES 10/1.0 according to example E20 was 2-4fold higher. The molecular weight Mn of all HES derivatives tested was in a comparable range, the Mw 15 of the NH2-oxHES based reagents was higher due to a higher polydispersity of the starting material. VII. Conjugation of the HES derivatives according to VI, Examples E18 to E20 to proteins 20 Example E21: Preparation of Ubi conjugates with HES derivatives formed upon reaction with IA/EDC or IA-Cl The coupling of the HEA derivatives according to Examples E18 to E19 was carried as 25 described in example C6. Further detailes are given in table 10. Example E22: Preparation of Ubi conjugates with L7-HES The coupling of the HES derivative according to example E20 was carried as described in 30 example C6. Further detailes are given in table 10. Although the coupling reactions were conducted with a normalized reactive group content, the HES derivatives according to example E18 and E19 (examples not according to the invention) resulted in significantly lower reaction turnovers into protein conjugate 35 compared to L7-HES described in example E20 (according to the invention). 80 WO 2014/147175 PCT/EP2014/055598 oinjcnujs0 0 -ou jonpoid olquiljqo ............ ................................ .. .... Q CJ/2 #[ioul~ 44g .. . .. . .. . ................. ....................... 0...... ..........
WO 2014/147175 PCT/EP2014/055598 0 0 0 \0 =Z 0 Z = z 0z 0 Z= l 0 00') 1 00 -ou iolpoid II olqaiuwiqo Ln IC 82 WO 2014/147175 PCT/EP2014/055598 55 0 0 o 0 0 550 0 o 0 \ 00 ou ionpoid III0 olqLuiIqo 4.1 4. 4.1 4.) 4.1 ........ ............... ............ cjo/2 W')j~c~ j * -cjncjocjocj ............. ........ .............. CA CAH .......... . ................. ............. ........... .............. .... ................... ].. A Cl 83 WO 2014/147175 PCT/EP2014/055598 01 0 zmz = z 0 ou ionpoid olquiljqo C.) C.) cjlnoolo cjo cjl Xjilcj CA 84 WO 2014/147175 PCT/EP2014/055598 Table 2: Thiol-modified HES species (example E7) 80 1 1.0 126 X cystamine *2HCl 0.6 0.6 65 126 2 1.0 83 X2 cystamine *2HCl 0.6 0.6 65 84 3 1.0 248 X3 cystamine *2HCl 1.433 0.6 73 239 4 1.0 54 X4 cystamine *2HCl 0.6 0.6 59 55 1.0 126 cystamine 2HCl 0.6 0.6 125 6 1.3 84 X6 cystamine *2HCl 0.18 0.6 54 84 7 1.0 78 X7 icystamine *2HCl 1.433 0.6 70 84 8 1.3 84 X8 cystamine *2HCl 0.18 0.6 53 84 . .- ...... ..... -...... ... .... 9 1.3 84 X9 cystamine *2HCl 0.18 0.6 50 85 10 1.0 93 X10 cystamine *2HCl 0.6 0.6 64 92 1 1 1.0 80 X11 cystamine *2HCl 0.6 0.6 61 80 12 1.0 91 X12 cysteamine *HCl 1.0 0.3 83 92 13 1.0 83 X13 cystamine *2HCl 0.6 0.6 67 84 14 1.0 88 X14 cystamine *2HCl 2.0 0.6 67 95 15 1.0 78 X15 cystamine *2HCl 0.6 0.6 64 78 16 1.0 88 X16 cysteine 2.0 0.6 53 94 17 1.0 93 X17 penicillamine 1.0 0.6 67 93 18 1.0 29 X18 cysteamine * HCl 2.0 0.6 80 29 19 1. 247 X19 cysteamine *HCl 2.0 0.6 74 246 20 1.0 86 X20 cysteamine * HCl 2.0 0.6 76 85 21 1. 88 X21 cysteamine *HCl 2.0 0.6 76 88 22 1.0 10.9 X22 cystamine *2HCl 1.43 0.6 60 11.2 85 WO 2014/147175 PCT/EP2014/055598 1L101141z 1%~u] 7t 7t7 Sw" oinpood dn-Nfjom lCCllCllCll [q]141 : Cll Cl [IIW]ZS JUOAOoGwnloA :ZA I I I I I I I I I JUOAJOS :ZSI I I I [I]IS JUOAJOowuIIoA IA C 00C JUOAJOS :IS [bo] SIuOj ?Ainbo :a C)0 C) n 7t [2] junomiIo3[uli1:ZV 200 [I ] POUIOwUTI?4 :I0Vl _ S3H pGIH OUI-jJ:I C C 4o M -Juo7 86 WO 2014/147175 PCT/EP2014/055598 .. .. ... .. .. . . . . . . . .. . . . . . . . . . . . . . . . .. .. ... .. .. ... .. .. ..... ..... .. . .. . .. . . . . . . . . . . . .. . .. . . . . . . ........ .... ... ...... ....... .... 0...0...0. .. .... ... ... ... ... ... ... ... ... ... .. .. .................... l .. ... ... .. .... ...... . ... .. . ... . . ... .. ... .............. I .. .. .. .... .. ....I.. ..... ....... .......... .. 00........... 00 .... 00...00. .. ... .. .. .. .. ... ... ... ... ... ... ... ... .. ~ * ~ * 87 WO 2014/147175 PCT/EP2014/055598 110114 Gnpooodd dn-Nfjom ci - i LIW] ZS UOAjOS o 6 - ci IUOAIOS :ZS Iw] IS IUOAjOS oUIIlOA JA~ 00 IUOAjOSf JS [b ] SJU OILAib o :a . 0........ [2wi] junou Jnu!1 :zv6 juIowuTI SIH PG~OUI-jO11J TV o ~~ xsIods SIH POUIP~w-101!W 88 WO 2014/147175 PCT/EP2014/055598 .. .. .. .. ... .. .. .. .. .. .. ... AI....... ............ .. . . . . . . . .. ... I I .. .. ... ....... .. ...... . ...) .. . ) .) ....... 00 ...... ....00 . . 00.. ....... ............ 0 0....... .... 00......0.0 Cl~~ ~ - -1 , 0 0 0 0 0 0 I I II I I t It I I 00 Cl Cl rn -~ oCl .. ...... .... *....... oc CA~ 0n I ~ 89 WO 2014/147175 PCT/EP2014/055598 Table 5: Examples of coupling reactions: variation of linker molecules, target substances, buffer conditions and HES : target ratios thiol- HES linker modified target pH HES: [% w/v] target HES yield L # HES target [mg] [mg] [%] species X Li X18 Ubi 7.0 1:1 1.5 2.5 25.4 94 Li X19 Ubi 7.0 1:1 6.0 2.5 105.3 92 Li X20 Ubi 7.0 1:1 2.5 2.5 40.5 96 L3 X8 Ubi 7.0 5:1 13.9 0.05 3.83 41 L5 X6 Ubi 7.0 5:1 15.1 0.05 4.37 43 L7 X2 Ubi 7.0 1.5:1 2.8 0.05 0.93 44 L7 X2 Ubi 7.5 1.5:1 2.8 0.05 0.93 68 L7 X2 Ubi 8.0 1.5:1 2.8 0.05 0.93 80 L7 X2 Ubi 8.5 1.5:1 2.8 0.05 0.93 89 L7 X2 Ubi 9.0 1.5:1 2.8 0.05 0.93 78 L7 X2 Ubi 8.5 1:1 2.1 0.05 0.62 69 L7 X2 Ubi 8.5 2:1 3.5 0.05 1.23 82 L7 X2 Ubi 8.5 3:1 4.5 0.05 1.85 86 L7 X2 Ubi 8.5 3:1 13.2 5.8 243.5 99 L7 X18 Ubi 8.5 2:1 1.3 2.5 19.8 92 L7 X19 Ubi 8.5 2:1 7.6 2.5 140.1 87 L7 X20 Ubi 8.5 2:1 3.3 2.5 55.0 96 L8 X10 Ubi 7.0 1.5:1 2.6 0.05 0.92 36 L8 X10 Ubi 7.5 1.5:1 2.6 0.05 0.92 60 L8 X10 Ubi 8.0 1.5:1 2.6 0.05 0.92 82 L8 X10 Ubi 8.5 1.5:1 2.6 0.05 0.92 78 L8 X10 Ubi 9.0 1.5:1 2.6 0.05 0.92 76 L8 X10 Ubi 8.5 1:1 1.9 0.05 0.61 65 L8 X10 Ubi 8.5 2:1 3.2 0.05 1.23 82 L8 X10 Ubi 8.5 3:1 4.1 0.05 1.84 87 L9 X10 Ubi 7.0 1.5:1 3.7 0.05 1.53 47 L9 X10 Ubi 7.5 1.5:1 3.7 0.05 1.53 71 L9 X10 Ubi 8.0 1.5:1 3.7 0.05 1.53 82 L9 X10 Ubi 8.5 1.5:1 3.7 0.05 1.53 82 L9 X10 Ubi 9.0 1.5:1 3.7 0.05 1.53 87 L9 X10 Ubi 8.5 1:1 2.8 0.05 1.02 71 90 WO 2014/147175 PCT/EP2014/055598 L9 X1O Ubi 8.5 2:1 4.4 0.05 2.04 86 L9 X1O Ubi 8.5 3:1 5.4 0.05 3.05 87 LI X7 Ubi 7.0 1.5:1 2.6 0.05 0.92 18 LI X7 Ubi 7.5 1.5:1 2.6 0.05 0.92 27 LI X7 Ubi 8.0 1.5:1 2.6 0.05 0.92 33 LI X7 Ubi 8.5 1.5:1 2.6 0.05 0.92 30 LI X7 Ubi 9.0 1.5:1 2.6 0.05 0.92 35 LI X7 Ubi 8.5 1:1 1.9 0.05 0.61 24 LI X7 Ubi 8.5 2:1 3.2 0.05 1.23 30 LI X7 Ubi 8.5 3:1 4.1 0.05 1.84 33 LI X18 Ubi 8.5 2:1 1.7 2.5 27.6 93 LI X19 Ubi 8.5 2:1 7.9 2.5 147.6 94 LI X20 Ubi 8.5 2:1 2.8 2.5 46.1 91 L11 X1i Ubi 7.0 1:1 7.3 0.05 1.40 39 L11 X1i Ubi 7.5 1:1 7.3 0.05 1.40 46 L11 X1i Ubi 8.0 1:1 7.3 0.05 1.40 34 L11 X1i Ubi 8.5 1:1 7.3 0.05 1.40 24 L11 Xii Ubi 8.5 3:1 17 0.05 4.21 81 Lii Xii Ubi 8.5 5:1 23 0.05 7.02 94 L12 X13 Ubi 7.0 1.5:1 2.9 0.05 1.06 29 L12 X13 Ubi 7.5 1.5:1 2.9 0.05 1.06 48 L12 X13 Ubi 8.0 1.5:1 2.9 0.05 1.06 63 L12 X13 Ubi 8.5 1.5:1 2.9 0.05 1.06 60 L12 X13 Ubi 9.0 1.5:1 2.9 0.05 1.06 64 L12 X13 Ubi 8.5 1:1 2.1 0.05 0.70 49 L12 X13 Ubi 8.5 2:1 3.5 0.05 1.41 65 L12 X13 Ubi 8.5 3:1 4.5 0.05 2.11 70 L13 X2 Ubi 6.5 1.5:1 3.0 0.05 1.16 75 L13 X2 Ubi 7.0 1.5:1 3.0 0.05 1.16 78 L13 X2 Ubi 7.5 1.5:1 3.0 0.05 1.16 82 L13 X2 Ubi 8.0 1.5:1 3.0 0.05 1.16 81 L13 X2 Ubi 8.5 1.5:1 3.0 0.05 1.16 82 L13 X2 Ubi 8.5 1:1 2.2 0.05 0.77 87 L13 X2 Ubi 8.5 2:1 3.7 0.05 1.54 82 L13 X2 Ubi 8.5 3:1 4.7 0.05 2.31 84 L14 X9 Ubi 7.0 1.5:1 3.5 0.05 1.40 55 L14 X9 Ubi 7.5 1.5:1 3.5 0.05 1.40 74 L14 X9 Ubi 8.0 1.5:1 3.5 0.05 1.40 81 91 WO 2014/147175 PCT/EP2014/055598 L14 X9 Ubi 8.5 1.5:1 3.5 0.05 1.40 82 L14 X9 Ubi 9.0 1.5:1 3.5 0.05 1.40 84 L14 X9 Ubi 8.5 1:1 2.6 0.05 0.93 79 L14 X9 Ubi 8.5 2:1 4.2 0.05 1.86 80 L14 X9 Ubi 8.5 3:1 5.2 0.05 2.79 81 L15 X14 Ubi 7.0 1.5:1 3.8 0.05 1.61 18 L15 X14 Ubi 7.5 1.5:1 3.8 0.05 1.61 21 L15 X14 Ubi 8.0 1.5:1 3.8 0.05 1.61 29 L15 X14 Ubi 8.5 1.5:1 3.8 0.05 1.61 26 L15 X14 Ubi 9.0 1.5:1 3.8 0.05 1.61 39 L15 X14 Ubi 8.5 1:1 2.9 0.05 1.07 18 L15 X14 Ubi 8.5 2:1 4.5 0.05 2.14 33 L15 X14 Ubi 8.5 3:1 5.6 0.05 3.22 42 L16 X7 Ubi 7.0 1.5:1 3.9 0.05 1.67 57 L16 X7 Ubi 7.5 1.5:1 3.9 0.05 1.67 60 L16 X7 Ubi 8.0 1.5:1 3.9 0.05 1.67 62 L16 X7 Ubi 8.5 1.5:1 3.9 0.05 1.67 60 L16 X7 Ubi 9.0 1.5:1 3.9 0.05 1.67 63 L16 X7 Ubi 8.5 1:1 3.0 0.05 1.11 51 L16 X7 Ubi 8.5 2:1 4.6 0.05 2.22 69 L16 X7 Ubi 8.5 3:1 5.7 0.05 3.34 74 L17 X15 Ubi 7.0 1.5:1 3.2 0.05 1.24 31 L17 X15 Ubi 7.5 1.5:1 3.2 0.05 1.24 38 L17 X15 Ubi 8.0 1.5:1 3.2 0.05 1.24 44 L17 X15 Ubi 8.5 1.5:1 3.2 0.05 1.24 43 L17 X15 Ubi 9.0 1.5:1 3.2 0.05 1.24 51 L17 X15 Ubi 9.5 1.5:1 3.2 0.05 1.24 64 L17 X15 Ubi 8.5 1:1 2.4 0.05 0.82 33 L17 X15 Ubi 8.5 2:1 3.8 0.05 1.65 53 L17 X15 Ubi 8.5 3:1 4.9 0.05 2.48 68 L18 X15 Ubi 7.0 1:1 2.9 0.05 0.85 42 L18 X15 Ubi 8.0 1:1 1.8 0.05 0.81 45 L18 X15 Ubi 8.5 1:1 1.6 0.05 0.85 41 L18 X15 Ubi 8.5 3:1 3.7 0.05 2.55 81 L18 X15 Ubi 8.5 5:1 5.0 0.05 4.26 88 L19 X21 Ubi 8.5 1.5:1 9.8 3 232 95 L7 X2 IL1RA 8.5 10:1 4.6 15 964 29 L13 X2 IL1RA 8.5 5:1 9.7 10 486 47 92 WO 2014/147175 PCT/EP2014/055598 L7 X2 AlAT 8.0 5:1 28.7 3 40 35 LI X7 AlAT 8.0 5:1 3.7 0.05 0.53 35 L11 X2 AlAT 8.0 5:1 2.9 0.1 1.33 11 L13 X2 AlAT 8.0 5:1 30.7 3 43 44 Li X19 HSA 7.0 3:1 15 5.0 92.2 88 L7 X19 HSA 8.5 10:1 15 5.0 205.3 44 Li X19 HSA 8.5 10:1 15 5.0 215.2 31 L7 X2 SlyD 8.5 3:1 5.6 0.05 3.82 98 93 WO 2014/147175 PCT/EP2014/055598 Table 6: Stability data of conjugates with various target proteins and linker molecules linker thiol-modified conjugate degradation after 20 days (%) L # HES species X target pH 4.0 pH 5.5 pH 7.0 pH 8.0 L2 X7 Ubi 0.7 n.d. 24.1 29.0 L4 X9 Ubi 2.1 n.d. 15.2 17.4 L7 X2 Ubi 1.3 n.d. 2.1 2.6 L8 X10 Ubi 1.4 n.d. 3.1 3.1 L9 X10 Ubi 1.9 n.d. 4.0 n.d. LI X7 Ubi 1.5 n.d. 14.9 17.1 L11 X1l Ubi 2.2 n.d. 7.4 10.9 L13 X2 Ubi 5.1 n.d. 5.5 11.4 L14 X9 Ubi 3.4 n.d. 5.8 10.6 L7 X2 ILIRA 1.2 1.6 1.8 1.4 L13 X2 ILIRA 2.4 5.2 10.0 28.0 L7 X2 AlAT 1.7 n.d. 5.9 n.d. L13 X2 AlAT n.d. 23.0 22.3 n.d. L7 X2 SlyD n.d. 1.3 3.4 8.7 Li X20 HSA 1.0 1.2 4.8 7.8 L7 X20 HSA 1.1 1.8 3.2 4.4 LI X20 HSA 0.7 5.9 20.7 25.5 Li X6 MEP 5.2 n.d. 24.3 25.2 L2 X7 MEP 1.4 n.d. 17.3 18.2 L3 X8 MEP 5.3 n.d. 6.8 7.8 L4 X9 MEP 2.5 n.d. 3.8 5.9 L5 X6 MEP 2.3 n.d. 10.5 12.5 L6 X9 MEP 10.7 n.d. 9.4 10.2 L7 X2 MEP 0.2 n.d. 0.6 0.2 L8 X10 MEP 0.0 n.d. 0.2 0.3 L9 X10 MEP 0.4 n.d. 2.1 1.6 LI X7 MEP 4.0 n.d. 15.4 15.9 L11 X11 MEP 5.8 n.d. 2.4 6.0 L12 X13 MEP 0.6 n.d. 1.9 1.8 L13 X2 MEP 2.5 n.d. 4.9 7.1 Conjugates prepared by using linker L15, L16 and L17 were not tested since their structure corresponds to compounds prepared using linkers L7 and L12. 94 WO 2014/147175 PCT/EP2014/055598 Table 7: Specificity of the conjugation reaction depending on the derivative thiol linker modified HES HES/PEG: HES/PEG target HES/PEG d L # species target target [% w/v] [mg] [mg] d X/PEG L3 X8 W-Ubi 10:1 24.8 0.07 10.0 3 L5 X6 W-Ubi 10:1 32.5 0.07 13.8 6 L7 X2 W-Ubi 10:1 20.6 0.07 8.0 0 LI X7 W-Ubi 10:1 20.5 0.07 8.0 39 L11 X1l W-Ubi 10:1 23.4 0.07 9.3 41 L13 X2 W-Ubi 10:1 25.0 0.07 10.0 8 mi PEGi W-Ubi 10:1 4.1 0.07 1.5 12 mi PEG2 W-Ubi 10:1 4.7 0.07 1.7 11 L3 X8 IFN-a 10:1 10.0 0.07 5.0 0 L5 X6 IFN-a 10:1 10.0 0.07 6.9 0 L7 X2 IFN-a 10:1 10.0 0.07 4.0 0 LI X7 IFN-a 10:1 10.0 0.07 4.0 6 Lii X11 IFN-a 10:1 10.0 0.07 4.7 6 L13 X2 IFN-a 10:1 10.0 0.07 5.0 0 mi PEGi IFN-a 10:1 10.0 0.07 0.7 13 mi PEG2 IFN-a 10:1 10.0 0.07 0.8 2 95 WO 2014/147175 PCT/EP2014/055598 Table 8: Further stability data of conjugates with Ubi and MEP and linker L1, L7 and L10 linker thiol-modified target conjugate degradation after 20 days (%) L # HES species X pH 4.0 pH 5.5 pH 7.0 pH 8.0 Li X18 Ubi 0.5 0.4 15.4 23.4 L7 X18 Ubi 2.6 3.9 4.3 3.9 Li X18 Ubi 0.8 2.0 9.6 10.8 Li X19 Ubi 0.3 0.5 7.1 15.7 L7 X19 Ubi 0.9 0.5 1.2 1.5 Li X19 Ubi 1.6 3.1 17.4 19.2 Li X20 Ubi 0.4 0.2 4.3 10.8 L7 X20 Ubi 1.2 1.4 1.8 2.1 Li X20 Ubi 1.1 2.8 16.5 17.6 thiol-modified linker* HES species target conjugate degradation after 20 days (%) L# X Li X18 MEP 13.9 n.d. 17.5 17.8 L7 X18 MEP 1.5 n.d. 4.0 4.4 Li X18 MEP 0.9 n.d. 8.0 7.2 Li X19 MEP 0.5 n.d. 4.5 5.4 L7 X19 MEP 0.0 n.d. 1.2 0.0 Li X19 MEP 0.7 n.d. 14.4 14.8 Li X20 MEP 2.8 n.d. 12.9 14.3 L7 X20 MEP 0.3 n.d. 0.8 1.1 Li X20 MEP 2.2 n.d. 18.2 16.9 Stability studies performed in buffers as described in example C6 96 WO 2014/147175 PCT/EP2014/055598 HL H H H oJnpood dn-liom- - [pn] ZS IUOAJ0SownlJ0A : JUOAJ0S :ZS [jIn] IS JU3Aj0S oIflfj0A :jIA JUOAJ0S :JS QCc [bo] SjU~j13Alb3 7t [5w] junourn jo;ujj :zv C)4 X sflooll SH P39J1POW xv - x ... ........... ............. ...... ............. ....... # T1Jli .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . .. I. . . . SW -- I 7t .. ....... .... ............ . ............ .......... ;~ -~ *97 WO 2014/147175 PCT/EP2014/055598 Table 10: Examples of coupling reactions E21 and E22 (according to example C6) liner modified HES: HES target HES yield L # HES target pH target [% w/v] [mg] [mg] by C species X b E L7 X22 Ubi 8.5 1:1 0.213 0.1 0.23 79 L7 X22 Ubi 8.5 2:1 0.426 0.1 0.46 99 L7 X22 Ubi 8.5 3:1 0.639 0.1 0.69 99 L7 X22 Ubi 8.5 5:1 1.065 0.1 1.14 99 IA/ NH2 EDC oxHES Ubi 8.5 1:1 1.32 0.05 0.71 49 (Examp (Example le E18) E17) IA/ NH2 EDC oxHES Ubi 8.5 2:1 2.64 0.05 1.43 77 (Examp (Example le E18) E17) IA/ NH2 EDC oxHES Ubi 8.5 3:1 3.96 0.05 2.14 81 (Examp (Example le E18) E17) IA/ NH2 EDC oxHES Ubi 8.5 5:1 6.6 0.05 3.57 80 (Examp (Example le E18) E17) IA-Cl NH2 (Examp oxHES Ubi 8.5 1:1 0.755 0.05 0.41 23 le E19) (Example E17) IA-Cl NH2 (Examp oxHES Ubi 8.5 2:1 1.51 0.05 0.81 39 le E19) (Example E17) IA-Cl NH2 (Examp oxHES Ubi 8.5 3:1 2.265 0.05 1.22 52 le E19) (Example E17) IA-Cl NH2 (Examp oxHES Ubi 8.5 5:1 3.775 0.05 2.03 70 le E19) (Example E17) 98
Claims (21)
1. A hydroxyalkyl starch (HAS) derivative of formula (I) ORa HAS', 0 OH O 0 R O- C-F1-L1-S N-L2-N x H 2 RX RY 5 ORG I wherein X is a leaving group, preferably selected from the group consisting of Ts, Ms, Cl, Br and I; 10 Fl is a functional group comprising the group -NR'-, with R' being H or alkyl; LI is a spacer bridging F1 and S; L2 is a spacer bridging -NR- and -NRY-; R and RY are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; 15 HAS' is the remainder of the HAS molecule and Rb and R' are -[(CR 1 R 2 )mO]n-H and are the same or different from each other; Ra is -[(CRlR 2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 20 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR1R2; n is from 0 to 6.
2. The HAS derivative of claim 1, wherein X is Br or I, preferably I. 25
3. A hydroxyalkyl starch (HAS) derivative of formula (IV) ORa HAS'-, OHO R bO' C-Fl-Li-S N-L2-N 1 K SQ H 2 I I ORc Rx R (IV) wherein -Q' is the remainder of a thiol group comprising compound Q which is linked via the 30 group -S- of the thiol group to the group -NRY-C(=O)-CH 2 -; F1 is a functional group comprising the group -NR'-, with R' being H or alkyl; LI is a spacer bridging F1 and S; 99 WO 2014/147175 PCT/EP2014/055598 L2 is a spacer bridging -NR- and -NRY-; R and RY are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; HAS' is the remainder of the HAS molecule and Rb and R' are -[(CRR 2 )mO]n-H and 5 are the same or different from each other; Ra is -[(CRR 2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups 10 CR 1 R 2 ; n is from 0 to 6.
4. The HAS derivative of claim 3, wherein Q is selected from the group consisting of, thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, 15 cells, viruses, liposomes, microparticles, and micelles.
5. The HAS derivative of any one of claims 1 to 4, wherein the HAS is hydroxyethyl starch (HES), R1, R2, R 3 , and R 4 are hydrogen, 20 m is 2; n is 0 to 4.
6. The HAS derivative of any one of claims 1 to 5, wherein Fl is selected from the group consisting of -(CH 3 )N-, -(CH 3 -CH 2 )-N-, -(CH 3 -CH 2 -CH 2 )-N-, -((CH 3 ) 2 -CH) 25 N-, -IN-, -HN-O-, -HN-NH-, -HN-NH-(C=G)-, -HN-NH-(C=G)-GG- and -HN-NH SO 2 -, wherein F1 is preferably -NH-.
7. The HAS derivative of any one of claims 1 to 6, wherein R and RY are, independently of each other selected from the group consisting of H, methyl, ethyl, 30 propyl and butyl, or are forming a ring according to formula (V) N-L2-N (V) wherein R and RY, taken together, are -(CH 2 ) 2 - or -(CH 2 ) 3 -, preferably wherein Rx and RY are H. 35 100 WO 2014/147175 PCT/EP2014/055598
8. The HAS derivative of any one of claims 1 to 7, wherein the spacer Li comprises, preferably consists of the moiety -(C(L'L"))q- with L' and L" in each repeating unit -C(L'L")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, 5 arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, 10 carboxymethylcarbamoyl, cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other, with q preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of 15 from 2 to 6, more preferably, 2, 3 or 4, preferably wherein LI is -CH 2 -CH 2 -.
9. The HAS derivative of any one of claims 1 to 8, wherein the spacer L2 comprises the moiety -(C(L'Ltt))t- with Lt and L"t in each repeating unit -C(LtL")- being, 20 independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, keto groups, aldehyde groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, 25 phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, amidino, nitro, imino, alkylthio, arylthio, thiocarboxyl, sulfate, alkylsulfinyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, cycloalkyl such as e.g. cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, 30 wherein the groups Lt and L"t in each repeating unit may be the same or may differ from each other, with t preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4, preferably wherein the spacer L2 is a symmetrical spacer, 35 more preferably wherein the spacer L2 is -CH 2 -CH 2 -.
10. A method for the preparation of a hydroxyalkyl starch derivative comprising (i) reacting hydroxyalkyl starch (HAS) of formula (Ia) 101 WO 2014/147175 PCT/EP2014/055598 ORa HAS%, 0 H RbO C*H ORc H (la) via carbon atom C* of the reducing end of the HAS with the functional group M of a crosslinking compound according to formula (II) 5 M-L1-S-T (II) wherein M comprises the group -NHR', with R' being H or alkyl; LI is a spacer bridging M and S; 10 T is H or a thiol protecting group PG; HAS' is the remainder of the HAS molecule and Rb and R' are -[(CR 1 R 2 )mO]n H and are the same or different from each other; Ra is -(CR 1 R 2 )mO]-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or Ra is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch 15 molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR 1 R 2 ; n is from 0 to 6; thereby obtaining a HAS derivative of formula (Ib) ORa HAS' 0 OH RO' C-F1-L1--S-T H 2 20 ORc (Ib) wherein -CH 2 -F 1- is the moiety resulting from the reaction of the group M with the HAS via the carbon atom C* of the reducing end, and Fl is a functional group comprising the group -NR'-; optionally removing PG in case t is PG to 25 give T = H; (ii) reacting the HAS derivative obtained in step (i) with a crosslinking compound of formula (III) 0 N-L2-N RX R(II) 30 102 WO 2014/147175 PCT/EP2014/055598 wherein L2 is a spacer bridging -NR- and -NRY-; R and RY are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; 5 thereby obtaining a HAS derivative of formula (I) ORa HAS OH RbO' C-F1-L 1 -S -L2-N X ORcH 2 RX R( wherein X is a leaving group, preferably selected from the group consisting of 10 Ts, Ms, Cl, Br and I.
11. The method of claim 10, wherein T is a thiol protecting group PG, and wherein step (i) further comprises removing PG from the HAS derivative (Ib), preferably wherein PG has the structure -S-L1-M or -Trt (Trityl), preferably -S-L1-M. 15
12. The method of claim 10 or 11, wherein X is Br or I, preferably I.
13. The method of any one of claims 10 to 12, wherein M is selected from the group consisting of H 2 N-, H 2 N-NH-, H 2 N-NH-C(=O)- and H 2 N-O-, M preferably being 20 H 2 N-.
14. The method of any one of claims 10 to 13, wherein the reacting according to step (i) is carried out under reductive amination conditions, preferably at a temperature in the range of from 5 0 C to 100 0 C and in a solvent selected from the group consisting of 25 DMSO, DMF, DMA, NMP, formamide, water, acetic acid, and reaction buffers and mixtures of two or more thereof.
15. The method of claim 11, wherein PG has the structure -S-L1-M and wherein the removing of the protecting group PG according to step (i) is carried out at a 30 temperature in the range of from 0 0 C to 80 0 C and in an aqueous solvent system.
16. The method of any one of claims 10 to 15, wherein the reacting according to step (ii) is carried out at a temperature in the range of from 10 0 C to 50 0 C and in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, 35 reaction buffers and mixtures of two or more thereof, preferably in DMSO and/or DMF. 103 WO 2014/147175 PCT/EP2014/055598
17. The method of any one of claims 10 to 16, further comprising (iii) reacting the HAS derivative of formula (I) via the group -NRY-C(=O)-CH 2 -X with an -SH group of a thiol group comprising compound Q, thereby forming a 5 HAS derivative of formula (IV) ORa HAS'-, OH 0 0 R O- C-F1-L1-S N-L2-N ORc Rx y (IV) wherein -Q' is the remainder of the thiol group comprising compound Q which is linked via 10 the group -S- of the thiol group to the group -NRY-C(=O)-CH 2 -, preferably wherein Q is selected from the group consisting of, thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, cells, viruses, liposomes, microparticles, and micelles. 15
18. The method of claim 17, wherein Q is a peptide or polypeptide and wherein the reacting according to step (iii) is carried out at a temperature in the range of from 0 0 C to 50 0 C and in a solvent selected from the group consisting of water, reaction buffers, DMSO, DMF, DMA, NMP, formamide, and mixtures of two or more 20 thereof.
19. A HAS derivative obtainable or obtained by a method according to any one of claims 10 to 16. 25
20. A HAS derivative obtainable or obtained by a method according to any one of claims 17 to 19.
21. Use of a HAS derivative as claimed in any one of claims 1, 2, 5 to 9, or 20 as reactant for coupling to a thiol group of a thiol group comprising compound Q. 104
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP13160266.6 | 2013-03-20 | ||
| EP13160266 | 2013-03-20 | ||
| PCT/EP2014/055598 WO2014147175A1 (en) | 2013-03-20 | 2014-03-20 | Hydroxyalkyl starch derivatives as reactants for coupling to thiol groups |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2014234272A1 true AU2014234272A1 (en) | 2015-10-08 |
Family
ID=47913167
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2014234272A Abandoned AU2014234272A1 (en) | 2013-03-20 | 2014-03-20 | Hydroxyalkyl starch derivatives as reactants for coupling to thiol groups |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP2976366A1 (en) |
| AU (1) | AU2014234272A1 (en) |
| CA (1) | CA2907371A1 (en) |
| WO (1) | WO2014147175A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015132724A1 (en) | 2014-03-05 | 2015-09-11 | Pfizer Inc. | Improved muteins of clotting factor viii |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1681303B1 (en) * | 2002-09-11 | 2013-09-04 | Fresenius Kabi Deutschland GmbH | HASylated polypeptides, especially HASylated erythropoietin |
| BR0314107A (en) * | 2002-09-11 | 2005-07-19 | Fresenius Kabi De Gmbh | Production method of hydroxyalkyl starch derivatives |
| EP1660134B1 (en) * | 2003-08-08 | 2010-12-29 | Fresenius Kabi Deutschland GmbH | Conjugates of hydroxyalkyl starch and g-csf |
| EP2070950A1 (en) * | 2007-12-14 | 2009-06-17 | Fresenius Kabi Deutschland GmbH | Hydroxyalkyl starch derivatives and process for their preparation |
| EP2591008A1 (en) * | 2010-07-09 | 2013-05-15 | Fresenius Kabi Deutschland GmbH | Conjugates comprising hydroxyalkyl starch and a cytotoxic agent and process for their preparation |
-
2014
- 2014-03-20 WO PCT/EP2014/055598 patent/WO2014147175A1/en active Application Filing
- 2014-03-20 AU AU2014234272A patent/AU2014234272A1/en not_active Abandoned
- 2014-03-20 EP EP14711748.5A patent/EP2976366A1/en not_active Withdrawn
- 2014-03-20 CA CA2907371A patent/CA2907371A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO2014147175A1 (en) | 2014-09-25 |
| EP2976366A1 (en) | 2016-01-27 |
| CA2907371A1 (en) | 2014-09-25 |
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