Polymer for inhibiting SARS-CoV-2 and other pathogens

Description

The present invention relates to a polymer according to the preamble of claim 1 , to a hydrogel according to the preamble of claim 6 that can be obtained from such a polymer, to various uses of such a polymer and such a hydrogel according to the preambles of claims 7 to 11 , to a pharmaceutical composition comprising such a polymer or such a hydrogel according to the preamble of claim 12, and to various manufacturing methods for such a polymer according to the preambles of claims 13 to 15.

The ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) motivates scientists to develop compounds that help against a COVID-19 infection or limit the negative consequences of such infection. In this regard, the development of vaccines [19] against corona virus was certainly a milestone. However, on a world-wide basis, the shortage of vaccines in comparison to their huge demand necessitates the development of further medicaments against SARS-CoV-2.

SARS-CoV-2 is a respiratory virus that enters a host mainly through the nasal mucosa as the first entry point. The entry of SARS-CoV-2 into the host cells is primarily facilitated by the nonspecific electrostatic interaction between the heparan sulfates proteoglycans present on the host cells and the spike proteins on surface of the viruses. [40]

Recently, Wolf et al. [41] developed a novel ex vivo model system comprising of human nasal turbinate tissues to evaluate the efficiency of the synthetic inhibitors against respiratory viruses in the first stage of infection at the nasal mucosal entry site. Using this model system, they screened a series of chemical compounds to finalize the best one that would be useful in nasal spray, but they did not reveal the chemical structure of the most active compound in their studies. In this context, synthetic sulfates would be promising ingredients in nasal spray owing to their negatively charged sulfate groups that could interact with the surface proteins of SARS- CoV-2.

Recently, Haag et al. [24] reported polysulfate inhibitors, namely, a variety of structural analogues of sulfated polyglycerol such as linear polyglycerol sulfate (LPGS) and hyperbranched polyglycerol sulfate (HPGS). They concluded the LPGS as the best inhibitors. LPGS showed SARS-CoV-2 inhibition with low half maximal inhibitory concentration (IC50 ~ 66.9 pg/mL) which was ~60 and ~20 times lower than that observed for the natural polysulfates such as Heparin or pentosan sulfate, respectively. The superior activity of LPGS was explained by its flexible chain conformation that was considered to help interacting with the virus surface proteins more effectively compared to the other analogous.

It is an object of the present invention to provide SARS-CoV-2 inhibitors that are even more effective than LPGS. Another object is to provide appropriate manufacturing methods for such inhibitors.

This object is achieved with a polymer having a structure according to general formula (I):

In this context, the residues have the following meanings:

R ³ = independently from other R ³ residues in the same molecule: -OH, -OSO ₃ ’, -O(CH ₂ ) _q SO ₃ -, -(CH ₂ ) _q SO ₃ -, -OPO ₃ ² -, -OCO(CH ₂ ) _q COO-,

-N ₃ , a sialic acid residue, a fucose residue, a galactose residue, a mannose residue, or any of the following residues: or a salt of any of these residues,

R ⁴ = independently from other R ⁴ residues in the same molecule:

R ⁵ = independently from other R ⁵ residues in the same molecule: or a salt of any of these residues,

R ⁶ = independently from other R ⁶ residues in the same molecule: or a salt of any of these residues,

R ⁷ = independently from other R ⁷ residues in the same molecule:

R ⁸ = independently from other R ⁸ residues in the same molecule:

R ⁹ = independently from other R ⁹ residues in the same molecule: a methyl residue or a phenyl residue,

R ¹⁰ = independently from other R ¹⁰ residues in the same molecule: a substituted or nonsubstituted C1-C12 alkyl residue or a substituted or non-substituted C6-C12 aryl residue,

X = independently from other X residues in the same molecule: -O or -NH, m = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to 350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, n = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to 350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, o = 0 to 50, in particular 1 to 40, in particular 2 to 30, in particular 3 to 25, in particular 4 to 20, in particular 5 to 15, in particular 6 to 14, in particular 7 to 13, in particular 8 to 12, in particular 9 to 11 , p = 0 to 50, in particular 1 to 40, in particular 2 to 30, in particular 3 to 25, in particular 4 to 20, in particular 5 to 15, in particular 6 to 14, in particular 7 to 13, in particular 8 to 12, in particular 9 to 11 , q = independently from other q indices in the same molecule: 1 to 10, in particular 2 to

9, in particular 3 to 8, in particular 4 to 7, in particular 5 to 6, r = independently from other r indices in the same molecule: 0 to 11 , in particular 1 to

10, in particular 2 to 9, in particular 3 to 8, in particular 4 to 7, in particular 5 to 6, and s = independently from other s indices in the same molecule: 1 to 12, in particular 2 to

11 , in particular 3 to 10, in particular 4 to 9, in particular 5 to 8, in particular 6 to 7.

Such a polymer, in particular when carrying a negatively charged residue like sulfate, sulfonate, phosphate and/or carboxylate (-OSOT, -O(CH2)qSC>3', -(CH2)qSO3 ^_ , -OPO3 ² ;

-OCO(CH2)qCOO'), shows excellent binding capacity against SARS-CoV-2. To give a specific example, a relatively high molecular weight (-400 KDa) methacrylate based dendronized polyglycerol sulfate (pDenGIMAS, also referred to as PGMAS or P1) corresponding to general formula (I) showed an excellent SARS-CoV-2 inhibition with a remarkably low IC50 value - 0.3 pg/mL which is 220 times lower than that observed for LPGS. Despite not having a flexible structure like LPGS, pDenGI MAS surprisingly showed better inhibition results which is contrary to the recently reported results from the inventors’ group. This indicates that the chain flexibility is apparently not the only determining parameter for the potency of viral inhibitors.

Rather, the long fibers of pDenGIMAS may play an important role in virus inhibition. On the one hand, they could cover the sulfate binding domain of the SARS-CoV-2 to interact efficiently in a polyvalent fashion and on the other hand they could provide the steric shedding to restrict the virus to bind with the active receptor angiotensin-converting enzyme 2 (ACE2) on the cell surface. Therefore, probably because of this combining effect, pDenGI MAS appears to be a significantly better SARS-CoV-2 inhibitor than LPGS.

Besides, sulfated polymers sometimes show an anticoagulant activity that restricts their use in most clinical applications. In this context, like LPGS, pDenGIMAS also showed a remarkably (-10 times) lower anticoagulant activity than Heparin sulfates. This makes the polymers according to general formula (I) promising candidates for antiviral drugs. Easy synthesis in bulk scale, the remarkably low IC50 values for SARS-CoV-2 inhibition and negligible anticoagulant activity allow to use polymers according to general formula (I) like pDenGIMAS as the pharmaceutically active ingredient in a nasal spray. Though both acrylate [42] and methacrylate [43] based hydroxyl containing dendronized glycerol polymers have already been reported from the inventors’ group, the structure of the polymers according to general formula (I) is significantly different from the reported structures. Apart from the relatively higher molecular weight compared to the compounds reported earlier, also the manufacturing method differs from the previously published manufacturing method. The polymers according to general formula (I) can be synthesized using a bi-functional chain transfer agent that helps to introduce a 2-pyridyl disulfide at the both terminals of the polymer. This 2-pyridyl disulfide can be utilized to further grow the polymer chain from both terminals to synthesize a high molecular weight version of the polymer. This opportunity is not possible with the previously reported polymeric structures [42, 43]

In an embodiment, the (in particular pharmaceutically acceptable) salt is a sodium salt or a potassium salt. Sodium is a particularly appropriate counter ion for the negatively charged residues.

In an embodiment, X is -O if residue

In an embodiment, a degree of functionalization of the polymer lies in a range of from 10 to 100 %, in particular from 11 % to 99 %, in particular from 12 % to 98 %, in particular from 15 % to 97 %, in particular from 16 % to 96 %, in particular from 17 % to 95 %, in particular from 18 % to 94 %, in particular from 19 % to 93 %, in particular from 20 % to 90 %, in particular from 30 % to 85 %, in particular from 40 % to 70 %, in particular from 50 % to 60 %, in particular from 70 % to 98 %, in particular from 75 % to 95 %, in particular from 80 % to 90 %. The degree of functionalization is defined by R ³ residues that are different from hydroxyl groups. If all R ³ residues are different from hydroxyl groups, the degree of functionalization is 100 %. If one half of the R ³ residues are hydroxyl groups and the other half are different residues, the degree of functionalization is 50 %.

In an embodiment, R ³ is independently from other R ³ residues in the same molecule -OH, -OSOT, -O(CH ₂ ) _q SO3', -(CH ₂ ) _q SO3', -OPO3 ² ; -OCO(CH ₂ ) _q COO' or a salt of any of these residues.

In an embodiment, o and p are 0. Then, general formula (I) is simplified to general formula (XVI):

(XVI)

Examples of specific polymers covered by general formula (XVI) are summarized in the following Table 1 , wherein residue R ² is in each case Table 1 : Polymers according to formula (XVI):

Further examples of specific polymers covered by general formula (XVI) are summarized in the following Table 2, wherein residue R ² is in each case

Table 2: Polymers according to formula (XVI):

In an embodiment, m and n are equally big.

In an embodiment, a sum of m and n lies in a range of from 50 to 700, in particular from 60 to 750, in particular from 70 to 700, in particular from 80 to 650, in particular from 90 to 600, in particular from 100 to 550, in particular from 150 to 500, in particular from 200 to 450, in particular from 250 to 350.

In an embodiment, X is O.

Examples of specific polymers covered by general formula (I) are summarized in the following

Table 3, wherein X is O, residue R ¹ is -CH3, residue residue R ⁴ is Table 3: Polymers according to formula (I):

Examples of specific polymers covered by general formula (I) are summarized in the following

Table 4, wherein X is O, residue R ¹ is -CH3, residue residue R ⁴ is

Table 4: Polymers according to formula (I):

Examples of specific polymers covered by general formula (I) are summarized in the following

Table 5, wherein X is O, residue R ¹ is -CH3, residue residue R ⁴ is

Table 5: Polymers according to formula (I): Examples of specific polymers covered by general formula (I) are summarized in the following

Table 6, wherein X is O, residue R ¹ is -CH3, residue residue R ⁴ is Table 6: Polymers according to formula (I):

In an aspect, the present invention relates to a hydrogel that is made from a polymer according to the preceding explanations. Such a hydrogel can be obtained by crosslinking such a polymer by ultraviolet light (e.g., having a wavelength lying in a range of from 250 nm to 400 nm, in particular from 275 nm to 375 nm, in particular from 300 nm to 365 nm, in particular from 325 nm to 350 nm) or with a crosslinker chosen from the group consisting of a PEG-dithiol (HS- PEG-SH), a 4-arm PEG-tetrathiol, and a PEG-dicyclooctyne corresponding to the following formula:

In an embodiment, the 4-arm PEG-tetrathiol corresponds to the following general formula (XIX):

In this context, n lies in a range of from 10 to 100, in particular 20 to 90, in particular 30 to 80, in particular 40 to 70, in particular 50 to 60.

Examples of specific polymers covered by general formula (XVI) that are particularly appropriate for forming hydrogels are summarized in the following Table 7, wherein residue R ² is in each case wherein m + n is in each case 20 to 1000. Table 7 further lists appropriate crosslinkers for forming a hydrogel. Table 7: Polymers according to formula (XVI) that are particularly appropriate for forming hydrogels:

Further Examples of specific polymers covered by general formula (XVI) that are particularly appropriate for forming hydrogels are summarized in the following Table 8, wherein residue R ² is in each case wherein m + n is in each case 20 to 1000. Table 8 further lists appropriate crosslinkers for forming a hydrogel.

Table 8: Polymers according to formula (XVI) that are particularly appropriate for forming hydrogels:

In an aspect, the present invention relates to the in vivo or in vitro use of a polymer or a hydrogel according to the preceding explanations for binding viruses and/or bacteria.

In an embodiment, the viruses are chosen from the group consisting of influenza viruses (e.g., influenza A), coronaviruses (e.g., SARS-CoV-2), herpes simplex viruses (HSV, e.g., HSV-1), and respiratory syncytial viruses (RSV), and/or in that the bacteria are chosen from the group consisting of pseudomonas (e.g., P. aeruginosa) and Escherichia coli (E. coli).

In an aspect, the present invention relates to the medical use of a polymer or a hydrogel according to the preceding explanations in antiviral and/or antibacterial therapy of humans or animals.

In an aspect, the present invention relates to an antiviral and/or antibacterial method of treatment of a human or animal patient in need thereof. This method comprises a step of administering a polymer or a hydrogel according to the preceding explanations in a pharmaceutically active amount to a patient in need thereof. The administering can be done topically or systemically. In an embodiment, the administering is done topically, e.g., by applying the polymer or hydrogel in form of a nasal spray.

In an aspect, the present invention relates to the medical use of a polymer or a hydrogel according to the preceding explanations as mucus replacement, as cartilage replacement, or as synovial fluid replacement in a patient in need thereof. The rheological and viscoelastic properties of the hydrogel can be finely tuned to match the corresponding properties of mucus, cartilage, or synovial fluid. This opens a wide scope of applications of the presently claimed and described polymer and hydrogel. In addition, the respective mucus replacement, cartilage replacement, or synovial fluid replacement exhibits antiviral/antibacterial properties and can thus prevent infections and assist healing processes. In an aspect, the present invention relates to the use of a polymer or a hydrogel according to the preceding explanations as matrix for cultivating and/or expanding cells and/or organoids in vitro. Particularly appropriate cells for such cell culture/expansion technique are stem cells.

In an aspect, the present invention relates to a pharmaceutical composition comprising a polymer or a hydrogel according to the preceding explanations. An appropriate dosage form of this pharmaceutical composition is a nasal spray.

In an aspect, the present invention relates to the use of a polymer or a hydrogel according to the preceding explanations as lubricant. A polymer in which all residues R ³ are non-substituted hydroxyl groups is particularly appropriate for such use as lubricant.

In an aspect, the present invention relates to a first method for manufacturing a polymer according to the preceding explanations. This method comprises the steps explained in the following.

In a method step, a compound according to general formula (II) is reacted with a compound according to general formula (III) to obtain a compound according to general formula (IV):

In this context,

R ¹ = -CH ₃ or -H,

R ⁸ = independently from other R ⁸ residues in the same molecule:

R ⁹ = independently from other R ⁹ residues in the same molecule: a methyl residue or a phenyl residue,

R ¹⁰ = independently from other R ¹⁰ residues in the same molecule: a substituted or non-substituted C1-C12 alkyl residue or a substituted or non-substituted Ce- C12 aryl residue,

X = independently from other X residues in the same molecule: -O or -NH, m = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to

350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, n = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to 350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, and s = independently from other s indices in the same molecule: 1 to 12, in particular 2 to 11 , in particular 3 to 10, in particular 4 to 9, in particular 5 to 8, in particular 6 to 7.

This step is performed in an organic solvent. Dimethylformamide (DMF) is a particularly appropriate organic solvent. An appropriate reaction time lies in a range of from 6 hours to 24 hours, in particular from 10 hours to 20 hours, in particular from 12 hours to 18 hours, in particular from 14 hours to 16 hours. An appropriate reaction temperature lies in a range of from 20 °C to 100 °C, in particular from 30 °C to 90 °C, in particular from 40 °C to 80 °C, in particular from 50 °C to 75 °C, in particular from 60 °C to 70 °C.

In an optional preceding method step, the compound according to general formula (II) can be synthesized by a reaction between an acryloyl chloride (an prop-2-enoyl chloride) according to general formula (XVII) with a compound according to general formula (XVIII):

(XVIII)

This optional step is performed in an organic solvent. Dichloromethane (DCM) is a particularly appropriate organic solvent. The reaction is furthermore performed in the presence of a base, in particular an organic base. Triethylamine (EtsN) is a particularly appropriate organic base. An appropriate reaction time lies in a range of from 6 hours to 24 hours, in particular from 10 hours to 20 hours, in particular from 12 hours to 18 hours, in particular from 14 hours to 16 hours. An appropriate reaction temperature lies in a range of from 0 °C to 30 °C, in particular from 5 °C to 25 °C, in particular from 10 °C to 20 °C.

Afterwards, the end groups of residue R ²¹ are deprotected to obtain a compound according to wherein

In an embodiment, this deprotection step is done in an acidic aqueous solution of an alcohol. Ethanol is a particularly appropriate alcohol. Hydrochloric acid is a particularly appropriate acid. A concentration of the acid lies in a range of from 1 % to 10 % (w/v), in particular from 2 % to 9 %, in particular from 3 % to 8 %, in particular from 4 % to 7 %, in particular from 5 % to 6 %. An appropriate reaction time lies in a range of from 1 hour to 12 hours, in particular from 3 hours to 10 hours, in particular from 6 hours to 8 hours.

In an optional functionalization step, at least some hydroxyl groups of residue R ²² are substituted by at least one of the following residues: -OSOT, -O(CH2)qSC>3', -(CH2)qSO3 ^_ , -OPO3 ² ; -OCO(CH2)qCOO', -N3, a sialic acid residue, a fucose residue, a galactose residue, a mannose residue, or any of the following residues: or a salt of any of these residues.

To give an example, a sulfation may be achieved with sulfamic acid (amidosulfonic acid; H3NSO3) in an organic solvent. An appropriate organic solvent is DMF, in particular dry DMF. An appropriate reaction time lies in a range of from 12 hours to 48 hours, in particular from 20 hours to 40 hours, in particular from 24 hours to 36 hours. An appropriate reaction temperature lies in a range of from 0 °C to 30 °C, in particular from 5 °C to 25 °C, in particular from 10 °C to 20 °C.

In an embodiment, residue R ⁸ corresponds to the following residue in formula (IV): In an optional method step preceding the deprotection step, the compound according to general formula (IV) is reacted with 2 ,2'-dipyridyldisulfide to obtain a compound according to general formula (VI):

This method step is performed in an organic solvent. Dichloromethane (DCM) is a particularly appropriate organic solvent. In an embodiment, the reaction is furthermore performed in the presence of butylamine. An appropriate reaction time lies in a range of from 1 hour to 12 hours, in particular from 3 hours to 10 hours, in particular from 6 hours to 8 hours. An appropriate reaction temperature lies in a range of from 0 °C to 30 °C, in particular from 5 °C to 25 °C, in particular from 10 °C to 20 °C.

The compound according to general formula (VI) can then be deprotected according to the above explanations to obtain a compound according to general formula (V), wherein residue

R ⁸ corresponds the following residue:

In an embodiment, residue R ¹⁰ is -CH2-CH2-.

In an embodiment, residue R ¹⁰ is one of the following residues: In an aspect, the present invention relates to a second method for manufacturing a polymer according to the preceding explanations. This method comprises the steps explained in the following. In a method step, a compound according to general formula (II) and a compound according to general formula (VII) are reacted with a compound according to general formula (III) to obtain

In this context, R ¹ = -CH ₃ or -H,

R ⁸ = independently from other R ⁸ residues in the same molecule:

R ⁹ = independently from other R ⁹ residues in the same molecule: a methyl residue or a phenyl residue,

R ¹⁰ = independently from other R ¹⁰ residues in the same molecule: a substituted or non-substituted C1-C12 alkyl residue or a substituted or non-substituted Ce- C12 aryl residue,

X = independently from other X residues in the same molecule: -O or -NH, m = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to

350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, n = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to 350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, o = 0 to 50, in particular 1 to 40, in particular 2 to 30, in particular 3 to 25, in particular 4 to 20, in particular 5 to 15, in particular 6 to 14, in particular 7 to 13, in particular 8 to 12, in particular 9 to 11 , and p = 0 to 50, in particular 1 to 40, in particular 2 to 30, in particular 3 to 25, in particular 4 to 20, in particular 5 to 15, in particular 6 to 14, in particular 7 to 13, in particular 8 to 12, in particular 9 to 11.

This step is performed in an organic solvent. DMF is a particularly appropriate organic solvent. An appropriate reaction time lies in a range of from 6 hours to 24 hours, in particular from 10 hours to 20 hours, in particular from 12 hours to 18 hours, in particular from 14 hours to 16 hours. An appropriate reaction temperature lies in a range of from 20 °C to 100 °C, in particular from 30 °C to 90 °C, in particular from 40 °C to 80 °C, in particular from 50 °C to 75 °C, in particular from 60 °C to 70 °C. In another method step, the compound according to general formula (VIII) is reacted with a compound according to general formula (IX) to obtain a compound according to general formula (X):

In this context, or a salt of any of these residues.

This method step is performed in an organic solvent. DCM is a particularly appropriate organic solvent. An appropriate reaction time lies in a range of from 1 hour to 12 hours, in particular from 3 hours to 10 hours, in particular from 6 hours to 8 hours. An appropriate reaction temperature lies in a range of from 0 °C to 30 °C, in particular from 5 °C to 25 °C, in particular from 10 °C to 20 °C.

Afterwards, the end groups of residue R ²¹ are deprotected to obtain a compound according to general formula (XI):

In an embodiment, this deprotection step is done in an acidic aqueous solution of an alcohol. Ethanol is a particularly appropriate alcohol. Hydrochloric acid is a particularly appropriate acid. A concentration of the acid lies in a range of from 1 % to 10 % (w/v), in particular from 2 % to 9 %, in particular from 3 % to 8 %, in particular from 4 % to 7 %, in particular from 5 % to 6 %. An appropriate reaction time lies in a range of from 1 hour to 12 hours, in particular from 3 hours to 10 hours, in particular from 6 hours to 8 hours.

In an optional functionalization step, at least some hydroxyl groups of residue R ²² are substituted by at least one of the following residues: -OSOT, -O(CH2)qSC>3', -(CH2)qSO3 ^_ ,

-OPO ₃ ² -, -OCO(CH2)qCOO', -N3, a sialic acid residue, a fucose residue, a galactose residue, a mannose residue, or any of the following residues: or a salt of any of these residues.

In an aspect, the present invention relates to a third method for manufacturing a polymer according to the preceding explanations. This method can be denoted as statistical copolymer synthesis and comprises the steps explained in the following.

In a method step, a compound according to general formula (II) and a compound according to general formula (XII) are reacted with a compound according to general formula (III) to obtain a compound according to general formula (XIII):

In this context,

R ¹ = -CH ₃ or -H,

R ⁷ = independently from other R ⁷ residues in the same molecule: independently from other R ⁸ residues in the same molecule:

R ⁹ = independently from other R ⁹ residues in the same molecule: a methyl residue or a phenyl residue,

R ¹⁰ = independently from other R ¹⁰ residues in the same molecule: a substituted or non-substituted C1-C12 alkyl residue or a substituted or non-substituted Ce-

C12 aryl residue,

X = independently from other X residues in the same molecule: -O or -NH, m = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to

350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, n = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to 350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, o = 0 to 50, in particular 1 to 40, in particular 2 to 30, in particular 3 to 25, in particular 4 to 20, in particular 5 to 15, in particular 6 to 14, in particular 7 to

13, in particular 8 to 12, in particular 9 to 11, p = 0 to 50, in particular 1 to 40, in particular 2 to 30, in particular 3 to 25, in particular 4 to 20, in particular 5 to 15, in particular 6 to 14, in particular 7 to 13, in particular 8 to 12, in particular 9 to 11, r = independently from other r indices in the same molecule: 0 to 11 , in particular

1 to 10, in particular 2 to 9, in particular 3 to 8, in particular 4 to 7, in particular 5 to 6, and s = independently from other s indices in the same molecule: 1 to 12, in particular

2 to 11 , in particular 3 to 10, in particular 4 to 9, in particular 5 to 8, in particular 6 to 7.

This step is performed in an organic solvent. DMF is a particularly appropriate organic solvent. An appropriate reaction time lies in a range of from 6 hours to 24 hours, in particular from 10 hours to 20 hours, in particular from 12 hours to 18 hours, in particular from 14 hours to 16 hours. An appropriate reaction temperature lies in a range of from 20 °C to 100 °C, in particular from 30 °C to 90 °C, in particular from 40 °C to 80 °C, in particular from 50 °C to 75 °C, in particular from 60 °C to 70 °C.

In a further method step, the end groups of residue R ²¹ are deprotected to obtain a compound according to general formula (XIV):

In this context, In an embodiment, this deprotection step is done in an acidic aqueous solution of an alcohol. Ethanol is a particularly appropriate alcohol. Hydrochloric acid is a particularly appropriate acid. A concentration of the acid lies in a range of from 1 % to 10 % (w/v), in particular from 2 % to 9 %, in particular from 3 % to 8 %, in particular from 4 % to 7 %, in particular from 5 % to 6 %. An appropriate reaction time lies in a range of from 1 hour to 12 hours, in particular from 3 hours to 10 hours, in particular from 6 hours to 8 hours.

In an optional functionalization step, at least some hydroxyl groups of residue R ²² are substituted by at least one of the following residues: -OSOT, -O(CH2)qSC>3', -(CH2)qSO3 ^_ ,

-OPO3 ² ; -OCO(CH2)qCOO', -N3, a sialic acid residue, a fucose residue, a galactose residue, a mannose residue, or any of the following residues: or a salt of any of these residues.

In an embodiment, residue R ⁸ corresponds to the following residue in formula (XIII):

In an optional method step preceding the deprotection step, the compound according to general formula (XIII) is reacted with 2,2'-dipyridyldisulfide to obtain a compound according to general formula (XV):

The compound according to general formula (XV) can then be deprotected according to the above explanations to obtain a compound according to general formula (XIV), wherein residue

R ⁸ corresponds the following residue: This method step is performed in an organic solvent. DCM is a particularly appropriate organic solvent. In an embodiment, the reaction is furthermore performed in the presence of butylamine. An appropriate reaction time lies in a range of from 1 hour to 12 hours, in particular from 3 hours to 10 hours, in particular from 6 hours to 8 hours. An appropriate reaction temperature lies in a range of from 0 °C to 30 °C, in particular from 5 °C to 25 °C, in particular from 10 °C to 20 °C.

In an aspect, the present invention relates to a fourth method for manufacturing a polymer according to the preceding explanations. This method can be denoted as block copolymer synthesis and comprises the steps explained in the following.

In a method step, a compound according to general formula (IV) is reacted with a compound according to general formula (XII) to obtain a compound according to general formula (XIII):

In this context,

R ¹ = -CH ₃ or -H,

R ⁷ = independently from other R ⁷ residues in the same molecule:

R ⁸ = independently from other R ⁸ residues in the same molecule:

R ⁹ = independently from other R ⁹ residues in the same molecule: a methyl residue or a phenyl residue,

R ¹⁰ = independently from other R ¹⁰ residues in the same molecule: a substituted or non-substituted C1-C12 alkyl residue or a substituted or non-substituted Ce- C12 aryl residue,

X = independently from other X residues in the same molecule: -O or -NH, m = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to 350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, n = 10 to 500, in particular 20 to 450, in particular 30 to 400, in particular 40 to 350, in particular 50 to 300, in particular 60 to 250, in particular 70 to 200, in particular 80 to 150, in particular 90 to 100, o = 0 to 50, in particular 1 to 40, in particular 2 to 30, in particular 3 to 25, in particular 4 to 20, in particular 5 to 15, in particular 6 to 14, in particular 7 to 13, in particular 8 to 12, in particular 9 to 11 , p = 0 to 50, in particular 1 to 40, in particular 2 to 30, in particular 3 to 25, in particular 4 to 20, in particular 5 to 15, in particular 6 to 14, in particular 7 to 13, in particular 8 to 12, in particular 9 to 11 , r = independently from other r indices in the same molecule: 0 to 11 , in particular

1 to 10, in particular 2 to 9, in particular 3 to 8, in particular 4 to 7, in particular 5 to 6, and s = independently from other s indices in the same molecule: 1 to 12, in particular

2 to 11 , in particular 3 to 10, in particular 4 to 9, in particular 5 to 8, in particular 6 to 7.

This step is performed in an organic solvent. DMF is a particularly appropriate organic solvent. An appropriate reaction time lies in a range of from 6 hours to 24 hours, in particular from 10 hours to 20 hours, in particular from 12 hours to 18 hours, in particular from 14 hours to 16 hours. An appropriate reaction temperature lies in a range of from 20 °C to 100 °C, in particular from 30 °C to 90 °C, in particular from 40 °C to 80 °C, in particular from 50 °C to 75 °C, in particular from 60 °C to 70 °C.

Afterwards, a deprotection step like in the third manufacturing method and optionally also a functionalization step like in the third manufacturing method are performed. Reference is made to the explanations given above with respect to these method steps.

The resulting polymer is a block copolymer, in which a plurality of building blocks carrying the index o are arranged adjacent to each other, followed by a block containing a plurality of adjacent building blocks carrying the index m, followed by residue R ¹⁰ and the chemical groups flanking residue R ¹⁰ , followed by a block containing a plurality of adjacent building blocks carrying the index n, and this finally followed by a block containing a plurality of adjacent building blocks carrying the index p. Since the building blocks carrying the indices o and p can be designed such that they have a significantly higher hydrophobicity than the building blocks carrying the indices m and n, it is possible to construe a block copolymer according to the scheme A-B-A, in which A refers to hydrophobic blocks located at the periphery of the block copolymer (made up by the building blocks carrying the indices o and p), and B denotes a hydrophilic central portion of the block copolymer (made up by the building blocks carrying the indices m and n).

In an embodiment, a ratio between blocks carrying the indices m and n and blocks carrying the indices o and p lies in a range of from 1 :1 to 10:1 , in particular from 2:1 to 9:1 , in particular from 3:1 to 8:1 , in particular from 4:1 to 7:1 , in particular from 5:1 to 6:1.

In an embodiment of the block copolymer synthesis, X is -O and residue

Then, it is possible to manufacture an N-hydroxysuccinimide (NHS) containing block copolymer. It is also possible to further react the NHS group, as explained above with respect to the second manufacturing method.

In an embodiment of any of the precedingly explained manufacturing methods, a functionalization of the polymer (i.e. , of at least some of the hydroxyl groups of residue R ²² of the resulting compound) with a sugar is carried out. This sugar functionalization is done, in an embodiment, by reacting a compound according to general structure (XI) or (XIV) with methanesulfonyl chloride (mesyl chloride; MsCI), an organic base, in particular triethylamine, and sodium azide (NaNs). This reaction is done in an organic solvent, wherein DMF is a particular appropriate organic solvent. This reaction leads to a substitution of at least some of the hydroxyl groups of residue R ²² by azide residues. The degree of substitution (and thus the degree of functionalization) can be adjusted, e.g., by choosing an appropriate reaction time.

Subsequently, an ethynylated sugar, in particular an ethynylated sialic acid, an ethynylated mannose, an ethynylated fucose, or an ethynylated galactose, is reacted with the azide- functionalized polymer. This results in a sugar functionalization of the polymer by click chemistry.

In an aspect, the present invention relates to a method for manufacturing a hydrogel from any of the precedingly explained polymers. Such hydrogel is formed by allowing a reaction between any of the above-explained polymers with a crosslinker chosen from the group consisting of a PEG-dithiol, a 4-arm PEG-tetrathiol, and a PEG-dicyclooctyne corresponding to the following formula: Alternatively, ultraviolet light may be used as cross-linking initiator. The formed hydrogel comprises a porous network with a heterogeneous pore size lying in a range of from 500 nm to several micrometers. Since the hydrogel formation is, amongst others, due to disulfide linkages between individual polymer molecules and between polymer molecules and crosslinker molecules, the hydrogel can be degraded by the addition of glutathione (GSH). Depending on the GSH concentration, a complete degradation of the polymer-based hydrogel is possible. To give an example, in the presence of 10 mM GSH, a complete degradation of the hydrogel occurs within 72 hours.

All embodiments of the polymer can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the various uses, to the hydrogel, to the pharmaceutical composition, and to the different manufacturing methods. Likewise, all embodiments of the uses can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the polymer, to the hydrogel, to the pharmaceutical composition, and to the different manufacturing methods. Furthermore, all embodiments of the hydrogel can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the polymer, to the various uses, to the pharmaceutical composition, and to the different manufacturing methods. All embodiments of the pharmaceutical composition can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the polymer, to the various uses, to the hydrogel, and to the different manufacturing methods. Finally, all embodiments of the different manufacturing methods can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the polymer, to the various uses, to the hydrogel, to the pharmaceutical composition, and to the respective other manufacturing method.

Further details of aspects of the present invention will be explained in the following making reference to exemplary embodiments and accompanying Figures. In the Figures:

Figure 1 shows the structure of polymers P1 , P1a, P1 b, P1c, P2, and P3;

Figures 2A to 2C show the dose dependent inhibition capability of different polymers against a SARS-CoV-2 infection of cells;

Figures 3A to 3D show the results of cytotoxicity measurements of different polymers with respect to different cell types;

Figure 4 shows the anticoagulant activity of different polymers; Figures 5A and 5B show the inhibition capability of different polymers against a HSV-1 infection of cells;

Figure 6 show the results of cytotoxicity measurements of polymer P22 with respect to different cell types;

Figure 7 shows the anticoagulant activity of polymer P22;

Figure 8 shows the rheological properties of a first P22-based hydrogel;

Figure 9 shows the rheological properties of a second P22-based hydrogel;

Figure 10 shows the rheological properties of a third P22-based hydrogel;

Figure 11 shows a plot illustrating the degradability of a P 22-based hydrogel;

Figure 12 illustrates the dependency of the rheological properties of P22a-based hydrogels from the concentration of P22a;

Figure 13 shows the rheological properties of a first P22a-based hydrogel in an intact and in a degraded state;

Figures 14A to 14D illustrate the rheological properties of the first P22a-based hydrogel and its self-healing properties;

Figure 15 illustrates the virus-binding properties of different P22a-based hydrogels with respect to HSV-1 ;

Figure 16 illustrates the ability of polymer P22a to inhibit SARS-CoV2 omicron variant; and .

Figure 17 illustrates the ability of various polymers to inhibit respiratory syndrome virus (RSV);

To synthesize different polymers having a structure according to general formula (I), a synthesized bifunctional chain transfer agent (CTA; corresponding to general formula (III)) was reacted with a monomer M1 (OGMA) corresponding to general formula (II) to produce different molecular weight polymers (POGMA) in gram scale. The terminal dithiocarbonate groups of POGMA were converted to 2-pyridyl disulfide (PDS) and UV/Vis analysis confirmed the transformation. Quantification [27] of PDS groups indicated that they are at the both chain terminals confirming polymerization grown from the both initiating sites. PDS groups at terminals could help POGMA for further polymerization in presence of di-thiol via thiol-disulfide exchange reaction. [28]

Afterwards, acetonide groups were deprotected to generate hydroxyl functionalities in residue R ²¹ . By decoration of sulfates groups [29] at the side chain, the final polymers (P1, P2 and P3) were obtained (cf. Figure 1 ; P1 : m + n = 580; P2: m + n = 180; P3: m + n = 60). The abbreviation “Ph” denotes a phenyl residue. The degree of sulfation was quantified by elemental analysis. For simplification reasons only, all hydroxyl groups of the molecule shown in Figure 1 are depicted in their sulfated state. The amount of substituted hydroxyl groups in the real polymers depends on the degree of functionalization.

With this synthetic route, the inventors have successfully synthesized polymer (P1) with highest molecular weight of ~ 430 kDa. It will be referred to in the following also as PGMAS_400 kDa or P1_400 kDa. Likewise, polymer P2 will be referred to in the following also as PGMAS_120 kDa, PGMAS_100 kDa, P2_120 kDa or P2_100 kDa, and polymer P3 as PGMAS_40 kDa or P3_40 kDa.

Table 9: Characterization and half-maximal inhibitory concentration (IC50) of polymers against SARS-CoV-2 (in pg/mL and in pM). ^{a 1} H NMR determined molecular weight; DOF stands for degree of functionalization (either sulfates or carboxylate); Heparin was used as control. Overall negative charge of the polymers owing to sulfate groups was confirmed by very high negative value of zeta potential (cf. Table 9). To check the morphology of P1 in aqueous solution, cryo-electron microscopy (cryo-EM) was performed. In accordance with the inventors’ hypothesis, worm-like defined fibers having a length between approximately 150 nm and 215 nm were observed. Tomography of the sample was measured to calculate accurate length and width of the fibers. Tomography analysis estimated (selecting 10 random fibers) a length of the fibers lying in a range of from ~ 155-211 nm and their width was ~ 2 nm matching with the theoretical average length ~ 180 nm and width ~ 2 nm of P1 (average repeat unit of P1 = 600) calculated based on the atomic distance in the polymer chain. This indicates that P1 forms single chain fibers [30] in aqueous solution. The observed size distribution of fibers can be explained by considering polydispersity (D = 1.5) in sample P1.

To validate whether the polymeric structural construct might be the key parameter for single chain fiber morphology, the inventors have checked cryo-EM morphology of P3, which is 10 times shorter in length than P1. If the hypothesis of single chain fiber with this structural construct was correct, one would expect to get single chain fibers with shortest synthesized polymer P3 as well. Interestingly, it was found that an aqueous solution of P3 showed a fiber structure with a length of ~ 18 nm matching with its theoretical extended polymer chain length. It was even more interesting that owing to the same repeat unit for polymer P1 and P3, the fibers from them showed comparable width (~ 2 nm) further confirming single chain polymer fibers. Therefore, it was concluded that the methacrylate backbone introduces a rigidity in the polymer chain that is further supported by the electrostatic repulsion among the sulfated groups. This enhances the possibilities for further chain extension to attain a mucin-like fiber structure.

To evaluate SARS-CoV-2 inhibition activities of synthetic fibers, the inventors investigated the fibers on wild-type (WT) SARS-CoV-2 variant and determined their activities by plaque reduction assay for infection against Vero-E6 and other cell lines. The morphologies of the cells were recorded after incubation with SARS-CoV-2 in presence or absence of polymer fibers. It was very clear from the results that in presence of P1 most of the cells remained healthy, whereas without polymers the cells were infected by the viruses. The negligible number of infected cells in presence of P1 confirmed its SARS-CoV-2 inhibition activity.

The analysis of dose-dependent virus inhibition curves (Figure 2A) suggested that activity of the synthetic fibers was dependent on the molecular weight, more precisely on the length of the polymer fibers (cf. Table 9). The natural polysulfate heparin was used as control. P1 showed remarkably low half-maximal inhibition concentration (C = 2.26 ±0.82 pg/mL, 5.6 nM) in the nanomolar range (cf. Table 9) which is so far the best reported IC50 value for sulfated inhibitors targeting the receptor binding domain (RBD) of SARS-CoV-2 via electrostatic interaction. Moreover, the IC50 values in Table 9 indicate that P1 is -100 times more active than P3, even though P1 is only 10 times longer than P3. Hence, SARS-CoV-2 inhibition activity appears to be not linearly proportional to the length of fibers. This is further supported when comparing the inhibition activity of P1 to the inhibition activity of P2 (the inhibition activity of P1 is - 20 times higher than that of P2, whereas its length is only 3 times bigger than that of P2), indicating that some other parameters may also play a role for inhibition in addition to the length of fibers.

This anomaly could be explained by considering the fact that long chain polymer fibers not only interact with the RBD of SARS-CoV-2 via polyvalent interaction but also provide a steric shielding [31 ] that could prevent SARS-CoV-2 to bind to the active receptor ACE-2 for infection. On the other hand, owing to their relatively shorter fibers, P2 and P3 interact with the RBD via a less effective polyvalent fashion and probably could not provide enough steric shielding to prevent viral attachment to ACE-2. Therefore, the combined effect might be the reason for remarkable low IC50 value for SARS-CoV-2 inhibition of P1.

To understand the impact of negatively charged polymers on SARS-CoV-2 inhibition [32], the inventors synthesized derivatives of P1 , the most potent inhibitor from the initial studies, by varying the degree of sulfation (P1a, P1b and P1c) and quantified them by elemental analysis. The inventors found that a -10 % sulfated polymer (P1c) showed negligible SARS-CoV-2 inhibition activity (Figure 2B) (IC50 = 2404 ± 1869 pg/mL). With increasing degree of sulfation, the zeta potential becomes more negative (Table 9). At the same time, SARS-CoV-2 inhibition activity increased (Figure 2B). This further confirmed that electrostatic interaction with the RBD of SARS-CoV-2 is responsible for the infection inhibition upon first entry of SARS-CoV-2 into a host cell.

The inventors furthermore synthesized a carboxylated derivative (P1d) of polymer P1. A plaque reduction assay of P1d showed excellent inhibition with IC50 values -37.1 pg/mL (Figure 2C). This confirmed that carboxylate functional groups are also active in SARS-CoV-2 inhibition. However, the sulfated version of P1 appeared to be more active than its carboxylated version (P1d). This could be due to the more electronegative character of the sulfated groups in comparison to the same number of carboxylate groups as observed in zeta potential measurement. The synthesized polymers, regardless of being functionalized with sulfate groups or carboxylate groups, appeared as highly cell viable in different cell lines (Vero-E6, 16HBE14o and A549) (Figures 3A to 3C for the sulfated polymers and Figure 3D for the carboxylated polymer).

The inherent anticoagulant activity restricts many sulfated compounds from direct applications. In this context, the synthesized functionalized polymers showed negligible anticoagulant activity in comparison to heparin (Figure 4). The anticoagulant activity increased with decreasing length of polymer chain [35], The carboxylated version of P1 appeared as less anticoagulant than its sulfated version.

Polymers P1, P1a, P1 b, P1c, P2 and P3 were also tested on other sulfate binding respiratory viruses, for example Herpes simplex virus-1 (HSV-1), and also showed here their high inhibition activity [34] (Figures 5A and 5B) in comparison to the negative control heparin. The experimental results are summarized in Table 10. Hence, the synthetic polymers appeared as broad-spectrum novel respiratory virus inhibitors.

Table 10: Inhibition activity of different polymers against HSV-1.

Polymer P22 (cf. Table 5) turned out to be particularly appropriate for hydrogel formation. This polymer has a molecular weight of approximately 200 kDa, a degree of polymerization of 300 and a block ratio between o and p blocks on the one hand and m and n blocks on the other hand (cf. general formula (I)) of approximately 1 :9. Due to its suitability for hydrogel formation, P22’s SARS-CoV-2 inhibition capacity, as well as its biocompatibility with respect to cytotoxicity and coagulation properties was also tested. P22 turned out to have an IC50 value of SARS-CoV-2 inhibition of 3.5 pg/ml. The results of the experiments with respect to cytotoxicity of P22 are depicted in Figure 6, and the results of the experiments with respect to the coagulation properties of P22 are depicted in Figure 7. Obviously, P22 shows only a very low cytotoxicity and has low anticoagulant properties, at least in concentrations of less than 200 pg/ml.

Polymer P22 was then used to form a hydrogel together with HS-PEG-SH (having a molecular weight of approximately 5.0 kDa). The polymer concentration was kept constant at approximately 2.0 weight percent (wt %) at different PEG:polymer ratios. The storage modulus and the loss modulus at 1.0 Hz were determined. The results are depicted in Figure 8 and summarized in the following Table 11. For better comparison, Figure 8 also illustrates the storage modulus and the loss modulus of mucus of a healthy individual.

Table 11 : Composition and physical properties of different hydrogels made from polymer P22 and HS-PEG-SH.

Under this experimental setup, a concentration of 2 wt % polymer is very well suited to produce mucus mimetic hydrogels.

Polymer P22 was furthermore used to form a hydrogel together with a 4-arm PEG-tetrathiol corresponding to the following general formula (XIX) (having a molecular weight of approximately 5.0 kDa), wherein n lies in a range of from 10 to 100, in particular 20 to 90, in particular 30 to 80, in particular 40 to 70, in particular 50 to 60:

The polymer concentration was varied between 2 and 4 weight percent (wt %) at a constant PEG:polymer ratio of 1:1. The storage modulus and the loss modulus at 1.0 Hz were determined. The results are depicted in Figure 9 and summarized in the following Table 12. For better comparison, Figure 9 also illustrates the storage modulus and the loss modulus of mucus of a healthy individual.

Table 12: Composition and physical properties of different hydrogels made from polymer P22 and a 4-arm PEG-tetrathiol corresponding to the following general formula (XIX).

Under this experimental setup, a concentration of 2 to 3 wt % polymer is very well suited to produce mucus mimetic hydrogels.

Polymer P22 was furthermore used to form a hydrogel together with a 4-arm PEG-tetrathiol corresponding to general formula (XIX) (having a molecular weight of approximately 10.0 kDa), wherein n lies in a range of from 10 to 100, in particular 20 to 90, in particular 30 to 80, in particular 40 to 70, in particular 50 to 60. The polymer concentration was kept at 2 weight percent (wt %) at a constant PEG:polymer ratio of 1 :1. Due to the higher molecular weight of the crosslinker in comparison to the previously explained embodiment, the amount of PEG was twice as high as in the previously explained embodiment to achieve the same PEG:polymer ratio. The storage modulus and the loss modulus at 1.0 Hz were determined. The results are depicted in Figure 10 and summarized in the following Table 13. For better comparison, Figure 10 also illustrates the storage modulus and the loss modulus of mucus of a healthy individual.

Table 13: Composition and physical properties of different hydrogels made from polymer P22 and a 4-arm PEG-tetrathiol corresponding to the following general formula (XIX).

Under this experimental setup, a concentration of 1 .5 to 2 wt % polymer is very well suited to produce mucus mimetic hydrogels.

In addition, the hydrogel degradability was tested. The structure of the hydrogels is determined to a relevant extent by disulfide bridges. The storage modulus of the formed hydrogels was tested as explained above. Afterwards, glutathione was added in a concentration of 10 mM to the hydrogels. After 72 hours, the modulus was tested again under identical conditions. As can be seen from Figure 11 , the modulus significantly decreased after the glutathione treatment, indicating that the hydrogels were fully degraded by glutathione due to breakup of the disulfide bridges. This clearly indicates that the hydrogels are easily degradable. In this context, the degradation is dependent on glutathione concentration.

In summary, a methodology for gram-scale synthesis of methacrylate-based functionalized dendronized polyglycerol polymers was developed. These polymers appeared as highly biocompatible and less anticoagulant, formed thread-like single chain fibers and showed excellent inhibition against respiratory viruses such as SARS-CoV-2 and HSV-1. They could be easily used for forming degradable hydrogels together with a crosslinker.

Polymer P22a, which has a chemical structure being very similar to the structure of polymer P22, is likewise particularly appropriate to form hydrogels. A reversible addition-fragmentation chain-transfer (RAFT) polymerization was used to synthesize a copolymer as first intermediate product. P22a comprises sulfated dendronized methacrylate oligo-glycerol units (M1) and 2-pyridyl disulfide (PDS) functionalized methacrylate units (M2). For the synthesis of the first intermediate product, a ratio of M1 :M2 was chosen such that the ratio between total monomers (M1+M2) and the chain transfer agent (CTA) was 650:1. The polymerization was stopped at approximately 85 % conversion of the monomers. The contribution of the individual monomers M1 and M2 in the first intermediate product was calculated from an ¹ H NMR analysis and confirmed that the incorporation of M2 was approximately 10 % in terms of the repeat unit. Considering the 85 % conversion of both monomers, the molecular weight of the first intermediate product was estimated to be 200 kDa that matched with the SEC-determined molecular weight and confirmed controlled radical polymerization. Then ammonolysis was done and the terminal dithiocarbonate groups were converted to 2-pyridyl disulfides to get a second intermediate product, i.e., a copolymer in which PDS groups are randomly distributed throughout the polymer chain and also at the chain end. The acetonide groups of the second intermediate product were then deprotected to get a third intermediate product. Subsequently, a further sulfation of the terminal hydroxyl groups was performed to obtain P22a.

An ¹ H NMR spectrum of the third intermediate product confirmed the deprotection of acetonide groups. The observed shift of adjacent methylene proton peaks in an ¹ H NMR spectrum of P22a in comparison to the third intermediate product further confirmed addition of electronegative sulfates groups. Total sulfur content in P22a was quantified by elemental analysis, confirming > 90 % sulfation of P22a. The molecular weight of P22a was determined from ¹ H NMR and was around 400 kDa that roughly matched with the SEC-determined molecular weight (/W _w = 300 kDa). The overall electronegative charge of P22a was confirmed from a negative zeta potential value owing to the high degree of sulfation. The morphology of P22a in aqueous solution was investigated by cryo-electron microscopy (cryo-EM). Interestingly, P22a showed a thread-like elongated structure. Since cryo-EM only provided projection images of the fibers spatially embedded in vitreous ice, cryo-electron tomography (cryo-ET) was used to determine the 3D structure and length of the fibers. Tomogram analysis of 10 randomly selected fibers revealed a fiber length of 150.5 nm (n = 10, SD = 42.4 nm). This was consistent with the theoretical average length of approximately 175 nm of P22a (average number of repeat units = 570) calculated based on the atomic distance in the polymer chain. This observation indicates that P22a forms single-chain fibers in aqueous solution. The observed length distribution of fibers is plausible considering the polydispersity (£> = 1.4) of P22a. PDS groups were randomly distributed throughout the polymer chain. A cell viability assay confirmed that P22a is biocompatible in various cell lines such as A549, HBE, Calu-3 and Vero E6 cell lines up to a polymer concentration of 5.0 mg/mL. In addition, the anticoagulant activity of P22a was tested in comparison to the commercial anticoagulant heparin. It was observed that P22a showed only negligible anticoagulant activity in comparison to heparin.

Polymer P22a was then used to form a hydrogel together with a 4-arm PEG-tetrathiol corresponding to general formula (XIX) (having a molecular weight of approximately 10 kDa). The time required for hydrogelation was around 24 h which is longer than the time required for hydrogelation of polymer P22.

The polymer concentration was varied between approximately 2.0 weight percent (wt %) and 4.0 wt %, wherein the ratio between PDS and thiol groups was 1 :1.

The synthesized hydrogels were washed with water to remove the byproduct 2-pyridone thione Afterwards, the rheological properties were characterized. Frequency sweep was performed in the linear viscoelastic region (LVR), and it was observed that the storage modulus G’ of the hydrogels could be tuned by varying the concentration of P22a (Figure 12). The modulus data of the hydrogels was compared to that of healthy sputum. It was found that a 2 wt % polymer hydrogel showed a storage modus that is very similar to the storage modulus of healthy sputum. Changing the polymer percentage from 2 to 3 wt% led to a synthetic hydrogel having a storage modulus similar to that of diseased mucus. It is also well known that in diseased mucus additional disulfide linkages lead to an enhanced storage modulus.

As the synthesized hydrogels comprise disulfide bonds, their degradability was tested upon treatment with GSH as reducing agent. The hydrogel manufactured with 3 wt% of P22a was treated with 10.0 mM GSH, and the rheological properties were monitored (Figure 13). A significant reduction of storage modulus observed after 24 hours confirmed the degradation of a relevant portion of the disulfide bonds. This is very relevant for mucus modulator research. In this context, the developed hydrogels could be used as an in-vitro model system.

Generally, the P22a-based hydrogel having a P22a concentration of 2 wt% showed a mucuslike shear-thinning behavior (Figure 14A).

Self-healing is an important property of hydrogels if they shall be used in biological applications. Therefore, the self-healing properties of the P22a-based hydrogel having a P22a concentration of 2 wt% was tested. The performed amplitude sweep estimated the rapturing point of the hydrogel to be at 700 % strain (Figure 14B). Repetitive rheological measurements were then performed for four cycles with 1 % strain and 1000 % strain (Figure 14C). The results of these repetitive rheological measurements showed that the hydrogel is self-healing after having been ruptured at the strain of 1000 % (lying above the rupturing point 700 %). These findings were confirmed by macroscopic observations done on a hydrogel made from differently colored portions (dyed blue with methylene blue and yellow with fluorescent sodium salt). The results are depicted in Figure 14D, wherein the yellow portion is depicted in light grey and the blue portion is depicted in dark grey. The first panel shows the differently dyed portions of the hydrogel forming individual hydrogel units. The second panel shows a selfassembled hydrogel made from the differently colored portions. If this hydrogel is ruptured essentially perpendicular to the border between the yellow portion and the blue portion, two separate hydrogen units are formed in which the yellow-colored portion and the blue-colored portion form together the hydrogel unit but are not structurally separated from each other within their unit.

To reveal the microstructure of the hydrogel, the P22a-based hydrogel and healthy sputum were investigated by scanning electron microscopy (SEM). Slow ethanol evaporation technique was used for sample preparation. SEM images of the P22a-based hydrogel showed a network structure with pore sizes from 200 nm to several micrometers (using image J analysis). These pore sizes were very similar to that of the observed network structure of healthy sputum.

HSV-1 binding of the hydrogels was evaluated by plaque reduction assays. For this purpose, P22a-based hydrogels crosslinked with different PEG crosslinkers were initially incubated with a virus solution under moderate shaking for 1 h. Afterwards, the number of viral particles in the supernatant was titrated by plaque assays on Vero E6 cells. The binding of virus was revealed by reduced virus titer in the supernatant as shown in Figure 15. The hydrogels showed a remarkably high binding ability of up to 1000 times higher than the virus control (without hydrogel), irrespective of the used crosslinker (Gel 1 : PEG-4SH (5 kDa); Gel 2: PEG-4SH (10 kDa); Gel 3: PEG-2SH (5 kDa).

The virus inhibition activity of P22a (as a polymer, not in form of a hydrogel) was also tested on the omicron variant of SARS-CoV-2 by a pre-infection assay. For this purpose, cells were incubated with P22a at different concentrations (between 0.5 pg/mL and 500 pg/mL) for 45 minutes. The cells were then infected with the omicron subvariant BA.5 of SARS-CoV-2 for 24 h. Infected cells were stained with SARS-CoV-2 N GFP proteins and quantified by fluorescence microscopy. The reduction of infected cells in presence of P22a (Figure 16) confirmed the inhibition activity of P22a against the SARS-CoV-2 omicron variant. Considering its long fiber structure, P22a probably interacts via polyvalent interactions with the receptor binding domain (RBD) of SARS-CoV-2 and provides enough steric shielding for the observed excellent inhibition against SARS-CoV-2.

Different polymers, namely P1 , P22a, P16, and P18 were tested in a comparable assay with respect to their inhibition capability against the respiratory syndrome virus (RSV). The corresponding results are depicted in Figure 17. IC50 values in a range of from approximately 0.2 and 0.35 mcg/mL were observed for the tested polymers, thus indicating a particularly appropriate anti-RSV activity of these polymers.

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