Block Synthesis and Step-Growth Polymerization of C-6-Sulfonatomethyl-Containing Sulfated Malto-Oligosaccharides and Their Biological Profiling

Highly sulfated malto-oligomers, similar to heparin and heparan-sulfate, have good antiviral, antimetastatic, anti-inflammatory and cell growth inhibitory effects. Due to their broad biological activities and simple structure, sulfated malto-oligomer derivatives have a great therapeutic potential, therefore, the development of efficient synthesis methods for their production is of utmost importance. In this work, preparation of α-(1→4)-linked oligoglucosides containing a sulfonatomethyl moiety at position C-6 of each glucose unit was studied by different approaches. Malto-oligomeric sulfonic acid derivatives up to dodecasaccharides were prepared by polymerization using different protecting groups, and the composition of the product mixtures was analyzed by MALDI-MS methods and size-exclusion chromatography. Synthesis of lower oligomers was also accomplished by stepwise and block synthetic methods, and then the oligosaccharide products were persulfated. The antiviral, anti-inflammatory and cell growth inhibitory activity of the fully sulfated malto-oligosaccharide sulfonic acids were determined by in vitro tests. Four tested di- and trisaccharide sulfonic acids effectively inhibited the activation of the TNF-α-mediated inflammatory pathway without showing cytotoxicity.


Introduction
In recent decades, researchers have paid increasing attention to the highly negatively charged polysaccharides and higher oligosaccharides on cell surfaces or in the extracellular matrix, as these carbohydrates play important roles in many vital biological processes.An important class of these structures includes glycosaminoglycans (GAGs), which are linear, high molecular weight, polydisperse heteropolysaccharides consisting of repeating disaccharide units and often containing sulfate ester groups in various patterns [1][2][3][4][5].The most well-known representatives of GAGs are heparin (1, HP) and heparan sulfate (2, HS) (Figure 1), which are copolymers built up alternately from D-glucosamine and a hexuronic acid [6].Heparin has been used as an anticoagulant in the medical field since the late 1930s [7].
pentasaccharide 3 as the main component (~60%), has received significant attention due to its remarkable antiangiogenic, tumor growth inhibitory and anti-metastatic activity (Figure 1) [17,18].PI-88, obtained from the exopolysaccharide of a diploid yeast, Pichia holstii, by acid-catalysed hydrolysis followed by exhaustive sulfation and its analogues is considered a promising antitumor drug candidate [11].
Another structurally simplified heparan sulfate mimetic is PG545 (4, pixatimod), a fully sulfated synthetic maltotetraose derivative linked to a cholestanol aglycone (Figure 1), which has shown strong in vivo efficacy in angiogenesis, solid tumor and metastasis models [19,20].As part of our efforts to produce heparin analogues, we have synthesized a number of non-glycosaminoglycan heparinoid oligosaccharides [21,22] and malto-oligomers [23] containing sulfonatomethyl groups at different positions of the glucose units.Sulfonic acids are resistant to cleavage by esterases and are therefore more stable in vivo than their sulfated counterparts and may exhibit stronger binding affinity to target proteins.Continuing this line of research, we aimed to synthesize malto-oligomeric derivatives that contain a sulfonatomethyl group at the C-6 position of each glucose unit and their hydroxyl groups are sulfated, thus capable of forming strong ionic interactions (Figure 2).Here, we describe our oligomerization, block synthetic and step-by-step synthesis approaches for fast and facile preparation of the designed oligomers.While conventional oligosaccharide synthesis routes, such as stepwise and block synthesis, involve multiple reaction steps (glycosylation reactions; work-up procedure and deprotection after each glycosylation step), the oligomerization method offers a faster and more convenient way to produce Both heparin and heparan sulfate have many other biological effects including antiinflammatory, cardiovascular and tissue protective, kidney and nerve protective, angiogenic, metastasis and growth factor inhibitory as well as antimalarial, antibacterial and antiviral activity [8][9][10][11][12][13][14][15][16].
As part of our efforts to produce heparin analogues, we have synthesized a number of non-glycosaminoglycan heparinoid oligosaccharides [21,22] and malto-oligomers [23] containing sulfonatomethyl groups at different positions of the glucose units.Sulfonic acids are resistant to cleavage by esterases and are therefore more stable in vivo than their sulfated counterparts and may exhibit stronger binding affinity to target proteins.Continuing this line of research, we aimed to synthesize malto-oligomeric derivatives that contain a sulfonatomethyl group at the C-6 position of each glucose unit and their hydroxyl groups are sulfated, thus capable of forming strong ionic interactions (Figure 2).Here, we describe our oligomerization, block synthetic and step-by-step synthesis approaches for fast and facile preparation of the designed oligomers.While conventional oligosaccharide synthesis routes, such as stepwise and block synthesis, involve multiple reaction steps (glycosylation reactions; work-up procedure and deprotection after each glycosylation step), the oligomerization method offers a faster and more convenient way to produce higher oligosaccharides in a single step, one-pot manner.We also report on the anti-cell growth, anti-inflammatory and antiviral evaluation of the new malto-oligosaccharide sulfonic acids.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 3 of 30 higher oligosaccharides in a single step, one-pot manner.We also report on the anti-cell growth, anti-inflammatory and antiviral evaluation of the new malto-oligosaccharide sulfonic acids.

Synthesis Using Benzyl Protecting Groups
The preparation of the planned C6-sulfonatomethyl-containing malto-oligomers requires suitable monosaccharide building blocks, which can be used to create the desired α-(1-4)-glycosidic bond with full regio-and stereoselectivity.The key monosaccharide, the 6-deoxy-6-sulfonatomethyl-containing thioglucoside 5 (Scheme 1), was obtained by the reaction of the corresponding primary triflate derivative with lithiated ethyl methanesulfonate according to our previous work [24,25].The substitution pattern of this compound enables it to be converted into an acceptor through the selective release of 4-OH, and when used as a donor in glycosylation reactions, the presence of the non-participating benzyl group at the C-2 position along with the shielding effect of the bulky sulfonic acid ethyl ester group from the β-side ensures full α-selectivity [25,26].Furthermore, 2-O-benzylated glycosyl donors are very reactive, so-called armed donors in glycosylation reactions, which presumably results in efficient glycosylation steps during the synthesis [27,28].
Thioglycoside 5 was converted to trichloroacetimidate 6 and glycosyl bromide 7, respectively, to obtain glycosyl donors suitable for the block synthetic approach.Synthesis of imidate 6 was accomplished in two steps, including removal of the ethylthio aglycone by N-bromosuccinimide (NBS) in an in acetone/H2O mixture and reaction of the resulting hemiacetal with trichloroacetonitrile under alkaline conditions [29].
Due to its degradability, the bromosugar derivative (7) was prepared in situ before the glycosylation reaction using elemental bromine in dry CH2Cl2 [30].

Synthesis Using Benzyl Protecting Groups
The preparation of the planned C6-sulfonatomethyl-containing malto-oligomers requires suitable monosaccharide building blocks, which can be used to create the desired α-(1-4)-glycosidic bond with full regio-and stereoselectivity.The key monosaccharide, the 6-deoxy-6-sulfonatomethyl-containing thioglucoside 5 (Scheme 1), was obtained by the reaction of the corresponding primary triflate derivative with lithiated ethyl methanesulfonate according to our previous work [24,25].The substitution pattern of this compound enables it to be converted into an acceptor through the selective release of 4-OH, and when used as a donor in glycosylation reactions, the presence of the non-participating benzyl group at the C-2 position along with the shielding effect of the bulky sulfonic acid ethyl ester group from the β-side ensures full α-selectivity [25,26].Furthermore, 2-O-benzylated glycosyl donors are very reactive, so-called armed donors in glycosylation reactions, which presumably results in efficient glycosylation steps during the synthesis [27,28].

Synthesis Using Benzyl Protecting Groups
The preparation of the planned C6-sulfonatomethyl-containing malto-oligomers requires suitable monosaccharide building blocks, which can be used to create the desired α-(1-4)-glycosidic bond with full regio-and stereoselectivity.The key monosaccharide, the 6-deoxy-6-sulfonatomethyl-containing thioglucoside 5 (Scheme 1), was obtained by the reaction of the corresponding primary triflate derivative with lithiated ethyl methanesulfonate according to our previous work [24,25].The substitution pattern of this compound enables it to be converted into an acceptor through the selective release of 4-OH, and when used as a donor in glycosylation reactions, the presence of the non-participating benzyl group at the C-2 position along with the shielding effect of the bulky sulfonic acid ethyl ester group from the β-side ensures full α-selectivity [25,26].Furthermore, 2-O-benzylated glycosyl donors are very reactive, so-called armed donors in glycosylation reactions, which presumably results in efficient glycosylation steps during the synthesis [27,28].
Thioglycoside 5 was converted to trichloroacetimidate 6 and glycosyl bromide 7, respectively, to obtain glycosyl donors suitable for the block synthetic approach.Synthesis of imidate 6 was accomplished in two steps, including removal of the ethylthio aglycone by N-bromosuccinimide (NBS) in an in acetone/H2O mixture and reaction of the resulting hemiacetal with trichloroacetonitrile under alkaline conditions [29].Scheme 1. Preparation of the C6-sulfonatomethyl-containing monosaccharide donors (6, 7) and acceptor (8).
Due to its degradability, the bromosugar derivative (7) was prepared in situ before the glycosylation reaction using elemental bromine in dry CH2Cl2 [30].
Thioglycoside 5 was converted to trichloroacetimidate 6 and glycosyl bromide 7, respectively, to obtain glycosyl donors suitable for the block synthetic approach.Synthesis of imidate 6 was accomplished in two steps, including removal of the ethylthio aglycone by N-bromosuccinimide (NBS) in an in acetone/H 2 O mixture and reaction of the resulting hemiacetal with trichloroacetonitrile under alkaline conditions [29].
Due to its degradability, the bromosugar derivative (7) was prepared in situ before the glycosylation reaction using elemental bromine in dry CH 2 Cl 2 [30].
The (2-naphthyl)methyl ether (NAP) of 5 was selectively removed with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), taking advantage of its sensitivity to oxidation by the single-electron transfer mechanism [31].The obtained thioglycoside 8 having a free 4-OH group served as a monomeric unit in the polymerization experiments and as an acceptor in the block synthetic approach.
It is important to note that polymerization of compound 8 enables the production of not only linear but also cyclic oligomers in a one-pot reaction, similarly to literature cyclooligomerizations observed in the polyglycosylation reactions of thioglycosides acting as both glycosyl donors and acceptors [32][33][34][35].

Block Synthesis
We first studied the block synthesis for the production of malto-oligomeric C6-sulfonic acids (Scheme 2).When acceptor 8 was reacted with the glycosyl bromide donor 7 in the presence of AgOTf, the desired disaccharide building block 9 was obtained in a very low yield of 18%, and significant degradation was observed.In order to increase the yield, the disaccharide was also prepared by glycosylation of acceptor 8 with the imidate donor 6 using TMSOTf catalyst in dry CH 2 Cl 2 .Although the reaction did not go to completion, disaccharide 9 was isolated with a good yield of 56%.Acceptor 8 was recovered with 13% yield and thioglycoside 5 was also isolated with 5% yield in the form of an anomeric mixture.The unexpected side product 5 could be formed by the reaction of the thio-aglycone of acceptor 8 with the anomeric carbon atom of the oxocarbenium ion from donor 6 in an aglycone transfer reaction [25,36,37].
Scheme 2. Block synthetic route to tetrasaccharide 12. Synthesis of the disaccharide building block 9 (A); transformation of 9 into donor and acceptor and their condensation to tetrasaccharide 12 (B); the mechanism of the formation of side product 9αβ (C).

Polymerization-Synthesis and MS Study
Next, we focused on the polymerization reactions of the 4-OH-containing ethyl thioglucoside 8, which can function both as a glycosyl donor and as a glycosyl acceptor.Monomer 8 was subjected to NIS-TfOH-mediated polyglycosylation, and the scope of the reaction was examined to create linear and cyclic malto-oligosaccharides with different degrees of oligomerization (Scheme 3, Table 1).The reactions were performed using different amounts of promoter, as listed in Table 1, and after work-up, the composition of the crude reaction mixture was analysed by MALDI-TOF mass spectrometry.The standard glycosylation reaction using 1.2 equiv. of NIS resulted in linear oligosaccharides up to decamer (19)(20)(21)(22)(23)(24)(25)(26)(27), in the form of hemiacetals (Table 1, Reaction 1, Figures S2-S4).The corresponding thioglycoside oligomers were also present in the reaction mixture to a small extent, but surprisingly formation of cyclic oligomers was not observed.
In order to shift the composition of the product mixture from the difficult-to-isolate Scheme 2. Block synthetic route to tetrasaccharide 12. Synthesis of the disaccharide building block 9 (A); transformation of 9 into donor and acceptor and their condensation to tetrasaccharide 12 (B); the mechanism of the formation of side product 9αβ (C).
The fully protected 9 was converted into a trichloroacetimidate derivative by NBSmediated removal of the anomeric thiol group followed by reaction with trichloroacetonitrile, thus obtaining the donor building block 10 in 47% yield.In parallel, starting from disaccharide 9, the removal of the NAP protecting group with DDQ in a CH 2 Cl 2 /H 2 O mixture provided the disaccharide acceptor 11 with a free hydroxyl group at the C-4 position.
Glycosylation reaction of the disaccharide acceptor 11 with the disaccharide donor 10 upon TMSOTf activation proceeded with full stereoselectivity but low efficiency.The expected tetrasaccharide was isolated from the reaction mixture with a 7% yield after double purification by column chromatography.The mass spectrometric analysis of the reaction mixture (Figure S1, Supporting information) revealed that in addition to the desired tetrasaccharide, compounds 13 and 9 were also formed in the reaction.The disaccharide glycal 13 was obtained by elimination from the donor, and the fully protected thioglycoside 9 resulted from an aglycone transfer reaction, which took place competitively with the glycosylation reaction.Due to the poor yield of the desired tetrasaccharide, the block synthetic approach was not investigated further.

Polymerization-Synthesis and MS Study
Next, we focused on the polymerization reactions of the 4-OH-containing ethyl thioglucoside 8, which can function both as a glycosyl donor and as a glycosyl acceptor.Monomer 8 was subjected to NIS-TfOH-mediated polyglycosylation, and the scope of the reaction was examined to create linear and cyclic malto-oligosaccharides with different degrees of oligomerization (Scheme 3, Table 1).The reactions were performed using different amounts of promoter, as listed in Table 1, and after work-up, the composition of the crude reaction mixture was analysed by MALDI-TOF mass spectrometry.The standard glycosylation reaction using 1.2 equiv. of NIS resulted in linear oligosaccharides up to decamer (19)(20)(21)(22)(23)(24)(25)(26)(27), in the form of hemiacetals (Table 1, Reaction 1, Figures S2-S4).The corresponding thioglycoside oligomers were also present in the reaction mixture to a small extent, but surprisingly formation of cyclic oligomers was not observed.formed with a substoichiometric amount of promoter.Using 0.6 equiv. of NIS, the polymerization proceeded up to hexamers in 2 h, with the predominance of di-and trisaccharides, and the formation of the oligosaccharide hemiacetals was significantly suppressed (Table 1, Reaction 2, Figure S5).
To increase the degree of oligomerization, compound 8 was subjected to polyglycosylation in the presence of 0.6 equiv. of NIS for 24 h (Table 1, Reaction 3), and after work-up, the resulting reaction mixture was studied in detail by MALDI-TOF MS.As Figure 3 inset show, two series of oligomers with decreasing MALDI-TOF MS intensities were formed up to heptamers (n = 5, see Scheme 3).The presence of the hemiacetal oligomer series with the -OH end-group can be ascribed to the reaction of the activated -SC2H5 chain-end with water during the work-up of the reaction mixture (see the Experimental).Interestingly, the appearance of a very low intensity series of peaks with m/z values lower by 62 compared to those with the -SC2H5 end-group ([M-C2H5SH + Na] + ) can also be recognized in the MALDI-TOF MS spectrum (see Figure S6 in the Supporting Information).This series of peaks may be due to the insource fragmentation of the chain-end by elimination of a C2H5SH unit and/or formation of cyclic oligomers.Owing to the relatively large size of the repeat unit, formation of cyclic oligomers can only be expected at higher degrees of polymerization, e.g., when n ≥ 3.However, the [M-C2H5SH + Na] + series of peaks is also present at low degrees of polymerization even at m/z 471, i.e., in the case of the monomer 8.This finding may suggest that the [M-C2H5SH + Na] + series is due to the in-source fragmentation occurring under MALDI-TOF MS conditions, although the formation of cyclic oligomer at higher degrees of polymerization cannot be ruled out.
In order to confirm the structure of the main oligomer series formed, ESI-TOF MS/MS experiments were performed.For these experiments, the target precursor ions were selected and subjected to collision-induced dissociation (CID).The ESI-TOF MS/MS spectrum of the sodiated oligomer with the -SC2H5 end-group and with n = 4 is shown in Figure 4.As seen in the figure, the main fragmentations are due to the cleavages at the glycosidic bonds (Domon and Costello, 1988)   The difference between the m/z values of the neighbouring peaks in each oligomer series is 448, which corresponds to the mass of the repeat unit with a composition of C 23 H 28 O 7 S. Furthermore, based on the m/z values, our previous assumption was confirmed that one of the two oligomer series was formed with -SC 2 H 5 (-SEt) and the other with the -OH group on the anomeric carbon atom of the last monomeric unit as indicated in Figure 3 (the mass difference between the series is 44).
The presence of the hemiacetal oligomer series with the -OH end-group can be ascribed to the reaction of the activated -SC 2 H 5 chain-end with water during the work-up of the reaction mixture (see the Experimental).Interestingly, the appearance of a very low intensity series of peaks with m/z values lower by 62 compared to those with the -SC 2 H 5 end-group ([M-C 2 H 5 SH + Na] + ) can also be recognized in the MALDI-TOF MS spectrum (see Figure S6 in the Supporting Information).This series of peaks may be due to the insource fragmentation of the chain-end by elimination of a C 2 H 5 SH unit and/or formation of cyclic oligomers.Owing to the relatively large size of the repeat unit, formation of cyclic oligomers can only be expected at higher degrees of polymerization, e.g., when n ≥ 3.However, the [M-C 2 H 5 SH + Na] + series of peaks is also present at low degrees of polymerization even at m/z 471, i.e., in the case of the monomer 8.This finding may suggest that the [M-C 2 H 5 SH + Na] + series is due to the in-source fragmentation occurring under MALDI-TOF MS conditions, although the formation of cyclic oligomer at higher degrees of polymerization cannot be ruled out.
In order to confirm the structure of the main oligomer series formed, ESI-TOF MS/MS experiments were performed.For these experiments, the target precursor ions were selected and subjected to collision-induced dissociation (CID).The ESI-TOF MS/MS spectrum of the sodiated oligomer with the -SC 2 H 5 end-group and with n = 4 is shown in Figure 4.As seen in the figure, the main fragmentations are due to the cleavages at the glycosidic bonds (Domon and Costello, 1988)   After purification of the reaction mixture of (Reaction 3, Table 1) by column chromatography, disaccharide 11 was obtained with a yield of 7% in a completely pure form.
Compound 8 was also subjected to polymerization using 1.5 equiv. of NIS and 0.3 equiv. of TfOH (Table 1, Reaction 4).The reaction yielded oligomers up to dodecamers (n = 10), predominantly in the form of hemiacetals (see Figures S7 and S8 in the Supporting Information).Unfortunately, the formation of cyclic oligomers was not observed in this case either.
In order to increase the amount of easily isolated oligomers and reduce the amount of difficult-to-isolate hemiacetal oligosaccharides formed as anomeric mixtures, we changed our polymerization strategy and henceforth thioglycoside 8 was subjected to a polyglycosylation reaction in the presence of a glycosyl acceptor molecule (30) with a fixed anomeric centre (Scheme 4).Scheme 4. One-pot polyglycosylation reactions of the α-OMe glycoside acceptor 30 with the C-6sulfonatomethyl monomer 8.The capping monomer is highlighted in red.
We hypothesized that methyl glycoside 30 [25] can function as a capping unit at the reducing end, ensuring the efficient formation of the oligomer series in the form of methyl After purification of the reaction mixture of (Reaction 3, Table 1) by column chromatography, disaccharide 11 was obtained with a yield of 7% in a completely pure form.
Compound 8 was also subjected to polymerization using 1.5 equiv. of NIS and 0.3 equiv. of TfOH (Table 1, Reaction 4).The reaction yielded oligomers up to dodecamers (n = 10), predominantly in the form of hemiacetals (see Figures S7 and S8 in the Supporting Information).Unfortunately, the formation of cyclic oligomers was not observed in this case either.
In order to increase the amount of easily isolated oligomers and reduce the amount of difficult-to-isolate hemiacetal oligosaccharides formed as anomeric mixtures, we changed our polymerization strategy and henceforth thioglycoside 8 was subjected to a polyglycosylation reaction in the presence of a glycosyl acceptor molecule (30) with a fixed anomeric centre (Scheme 4).
We hypothesized that methyl glycoside 30 [25] can function as a capping unit at the reducing end, ensuring the efficient formation of the oligomer series in the form of methyl glycoside.First, the preactivation method [39,40] was used, that is, thioglycoside 8 was activated with the NIS-TfOH promoter system, then, the mixture was stirred for 2 h and only then was acceptor 30 added to the reaction mixture (Table 2, Reaction 1).Unfortunately, the expected capping did not occur, the methyl glycoside unit 30 was not incorporated into the oligomers and only hemiacetal oligomers 40-44 were detected by MALDI-TOF MS (Figure S9).Therefore, we switched from the preactivation method to the standard glycosylation conditions.At a 2.2:1 donor:acceptor ratio, when thioglycoside 8 was added in two portions to the reaction mixture and a NIS-TfOH promoter system was used, the expected methyl-glycoside-capped oligosaccharide series was efficiently formed.(Table 2, Reaction 2, Figure S10).case either.
In order to increase the amount of easily isolated oligomers and reduce the amount of difficult-to-isolate hemiacetal oligosaccharides formed as anomeric mixtures, we changed our polymerization strategy and henceforth thioglycoside 8 was subjected to a polyglycosylation reaction in the presence of a glycosyl acceptor molecule (30) with a fixed anomeric centre (Scheme 4).Scheme 4. One-pot polyglycosylation reactions of the α-OMe glycoside acceptor 30 with the C-6sulfonatomethyl monomer 8.The capping monomer is highlighted in red.
We hypothesized that methyl glycoside 30 [25] can function as a capping unit at the reducing end, ensuring the efficient formation of the oligomer series in the form of methyl glycoside.First, the preactivation method [39,40] was used, that is, thioglycoside 8 was activated with the NIS-TfOH promoter system, then, the mixture was stirred for 2 h and   Disaccharide 31 and trisaccharide 32 were isolated from the reaction mixture with a 37% and 16% yield, respectively, and additional oligosaccharides up to octamers, both in the form of methyl glycosides and hemiacetals, were detected by MALDI-TOF MS.Using a NIS-AgOTf promoter system and a longer reaction time (Table 2, Reaction 3), but still adding the donor in two portions, the product ratio was slightly shifted to higher oligomers.Thus, disaccharide 31 was isolated with a yield of 16%, while trisaccharide 32 was isolated with a yield of 20%, and higher oligomers up to octasaccharides were identified in the MALDI mass spectra (Figure S11).The best result was achieved when polyglycosylation was started with three equivalents of thioglycoside excess (ratio of 8 and 30 was 3:1 from the beginning) using a NIS-TfOH promoter system (Table 2, Reaction 4).Both di-and trisaccharides were isolated with good yields (33% for 31 and 25% for 32) and further oligosaccharides up to decamers were detected by MALDI MS.
The polymerization was also performed with a 10:1 ratio of 8 and 30 using 1.5 equiv.NIS and 0.3 equiv. of TfOH (Table 2, Reaction 5).In this case, the degree of oligomerization increased slightly up to the dodecamer (n = 10), but the product ratio shifted significantly towards hemiacetals.Oligosaccharides larger than the trimer were predominantly present as hemiacetals in the reaction mixture (Figures S12 and S13).
The composition of the reaction mixture obtained by the oligomerization reaction of 8 in the presence of 30, with a 3:1 ratio of the monomeric units (Table 2, Reaction 4), was studied by MALDI MS (Figure 5).As seen in Figure 5 inset, a series of oligomers end-capped with acceptor 30 (-OMe end-group) ranging from n = 0 to n = 8 are formed in addition to an oligomer series with the -OH end-group that was most probably formed by the reaction of the activated -SC 2 H 5 chain-end with water during the work-up of the reaction mixture as discussed before.Interestingly, according to the MALDI-TOF MS, the presence of the starting acceptor (30) could be observed, however, the hydrolyzed product of the starting monomer (8) occurred with low intensity.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 10 of 30 formed in addition to an oligomer series with the -OH end-group that was most probably formed by the reaction of the activated -SC2H5 chain-end with water during the work-up of the reaction mixture as discussed before.Interestingly, according to the MALDI-TOF MS, the presence of the starting acceptor (30) could be observed, however, the hydrolyzed product of the starting monomer (8) occurred with low intensity.Size-exclusion chromatography (SEC) also supports the formation of oligomers with different degree of polymerization as shown in Figure 6.SEC trace presented in Figure 6a was deconvoluted into five different SEC traces corresponding to those of the starting acceptor (30), dimer, trimer, tetramer and pentamer as shown in Figure 6b.Note that due to the very small variation in size, oligomers with the same degree of polymerization but with different end-groups cannot be resolved by SEC.According to the deconvoluted SEC traces and assuming a similar refractive index increment (dn/dc) for each oligomers, the composition of the reaction mixture can be estimated, for which 15 wt% (donor + hydrolyzed monomer), 35 wt% (dimer), 28 wt% (trimer), 17 wt% (tetramer) and 4 wt% (pentamer) was obtained.Note that oligomers with a Size-exclusion chromatography (SEC) also supports the formation of oligomers with different degree of polymerization as shown in Figure 6.SEC trace presented in Figure 6a was deconvoluted into five different SEC traces corresponding to those of the starting acceptor (30), dimer, trimer, tetramer and pentamer as shown in Figure 6b.Note that due to the very small variation in size, oligomers with the same degree of polymerization but with different end-groups cannot be resolved by SEC.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 10 of 30 formed in addition to an oligomer series with the -OH end-group that was most probably formed by the reaction of the activated -SC2H5 chain-end with water during the work-up of the reaction mixture as discussed before.Interestingly, according to the MALDI-TOF MS, the presence of the starting acceptor (30) could be observed, however, the hydrolyzed product of the starting monomer (8) occurred with low intensity.Size-exclusion chromatography (SEC) also supports the formation of oligomers with different degree of polymerization as shown in Figure 6.SEC trace presented in Figure 6a was deconvoluted into five different SEC traces corresponding to those of the starting acceptor (30), dimer, trimer, tetramer and pentamer as shown in Figure 6b.Note that due to the very small variation in size, oligomers with the same degree of polymerization but with different end-groups cannot be resolved by SEC.According to the deconvoluted SEC traces and assuming a similar refractive index increment (dn/dc) for each oligomers, the composition of the reaction mixture can be estimated, for which 15 wt% (donor + hydrolyzed monomer), 35 wt% (dimer), 28 wt% (trimer), 17 wt% (tetramer) and 4 wt% (pentamer) was obtained.Note that oligomers with a According to the deconvoluted SEC traces and assuming a similar refractive index increment (dn/dc) for each oligomers, the composition of the reaction mixture can be estimated, for which 15 wt% (donor + hydrolyzed monomer), 35 wt% (dimer), 28 wt% (trimer), 17 wt% (tetramer) and 4 wt% (pentamer) was obtained.Note that oligomers with a higher degree of polymerization are also present in the reaction mixture as evidenced by MALDI-TOF MS, but their quantities are too low to be detected by SEC.
The work was continued by removing the protecting groups of 31 and 32 in order to transform the oligosaccharides into the final, sulfated form suitable for biological assays (Scheme 5).First, the sulfonic acid esters were deprotected by nucleophilic substitution reaction using NaI.The sulfonic acid sodium salt derivatives (51 and 52) were formed from both di-and trisaccharides 31 and 32 in excellent yields.Next, we attempted to remove the benzyl groups by catalytic hydrogenation.Although the debenzylation went to completion, hard-to-handle gel products were obtained, probably due to complex formation with Pd, from which the expected products 53 and 54 could be isolated with very low yields.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 11 of 30 higher degree of polymerization are also present in the reaction mixture as evidenced by MALDI-TOF MS, but their quantities are too low to be detected by SEC.
The work was continued by removing the protecting groups of 31 and 32 in order to transform the oligosaccharides into the final, sulfated form suitable for biological assays (Scheme 5).First, the sulfonic acid esters were deprotected by nucleophilic substitution reaction using NaI.The sulfonic acid sodium salt derivatives (51 and 52) were formed from both di-and trisaccharides 31 and 32 in excellent yields.Next, we attempted to remove the benzyl groups by catalytic hydrogenation.Although the debenzylation went to completion, hard-to-handle gel products were obtained, probably due to complex formation with Pd, from which the expected products 53 and 54 could be isolated with very low yields.Scheme 5. Deprotection of the di-and trisaccharide derivatives 31 and 32.

Synthesis Using a Cyclic Acetal Protecting Group
Due to the difficulties experienced during the removal of benzyl protecting groups, we changed the protecting group strategy in order to efficiently produce the designed free malto-oligomeric sulfonic acids.To protect the monomers, we chose the butane diacetal group (BDA, 2,3-dimethoxybutane-2,3-diyl) [41,42] because (i) it can be easily removed under acidic conditions, (ii) it is a non-participating group in glycosylation reactions and therefore enables the formation of the desired 1,2-cis α-glycosidic bond [43] and (iii) the BDA-protected donors are semi-disarmed donors (more reactive than their acylated congeners), thus, they function as effective glycosylating agents [43,44].The synthesis of the fully protected 2,3-O-BDA-thioglycoside 55 was described earlier [24].The NAP-deprotection of 55 with DDQ liberated the C4 hydroxyl group to obtain the glycosyl donor 56 suitable for oligomerization (Scheme 6).The methyl glycoside building block 59 was prepared from compound 57 [45].First, the sulfonic acid ethyl ester moiety was introduced to the primary position in a two-step reaction (triflate formation followed by chain-elongation with lithiated ethyl methanesulfonate).The C-4 position of the resulting 58 was freed under oxidative removal of the NAP group using DDQ to produce the acceptor building block 59.Scheme 5. Deprotection of the di-and trisaccharide derivatives 31 and 32.

Synthesis Using a Cyclic Acetal Protecting Group
Due to the difficulties experienced during the removal of benzyl protecting groups, we changed the protecting group strategy in order to efficiently produce the designed free malto-oligomeric sulfonic acids.To protect the monomers, we chose the butane diacetal group (BDA, 2,3-dimethoxybutane-2,3-diyl) [41,42] because (i) it can be easily removed under acidic conditions, (ii) it is a non-participating group in glycosylation reactions and therefore enables the formation of the desired 1,2-cis α-glycosidic bond [43] and (iii) the BDA-protected donors are semi-disarmed donors (more reactive than their acylated congeners), thus, they function as effective glycosylating agents [43,44].The synthesis of the fully protected 2,3-O-BDA-thioglycoside 55 was described earlier [24].The NAP-deprotection of 55 with DDQ liberated the C4 hydroxyl group to obtain the glycosyl donor 56 suitable for oligomerization (Scheme 6).The methyl glycoside building block 59 was prepared from compound 57 [45].First, the sulfonic acid ethyl ester moiety was introduced to the primary position in a two-step reaction (triflate formation followed by chain-elongation with lithiated ethyl methanesulfonate).The C-4 position of the resulting 58 was freed under oxidative removal of the NAP group using DDQ to produce the acceptor building block 59.
Next, polymerization reaction was carried out with a 3:1 ratio of the repeating unit 56 and the capping glycosyl acceptor 59 using the NIS-TfOH promoter system (Scheme 6).After work-up, the series of oligosaccharides in both the methyl glycoside form and the hemiacetal form were detected by MALDI-TOF MS measurement up to the heptamers (Figure S14), and the di-and trisaccharides were isolated with acceptable yields (29% for 60 and 27% for 61).According to our expectations, the glycosylation took place with full stereoselectivity; diastereoisomers of 60 and 61 with a β-interglycosidic bond were not observed.Scheme 6. Synthesis of the BDA-protected C6-sulfonatomethyl-containing monosaccharide building blocks (56 and 59) and polymerization of 56 using 59 as a capping unit.The capping monomer is highlighted in red.
Next, polymerization reaction was carried out with a 3:1 ratio of the repeating unit 56 and the capping glycosyl acceptor 59 using the NIS-TfOH promoter system (Scheme 6).After work-up, the series of oligosaccharides in both the methyl glycoside form and the hemiacetal form were detected by MALDI-TOF MS measurement up to the heptamers (Figure S14), and the di-and trisaccharides were isolated with acceptable yields (29% for 60 and 27% for 61).According to our expectations, the glycosylation took place with full stereoselectivity; diastereoisomers of 60 and 61 with a β-interglycosidic bond were not observed.
In parallel to the oligomerization, the di-and trisaccharide derivatives were also produced by step-by-step synthesis (Scheme 7).First, acceptor 59 was glycosylated with the fully protected donor 55 using the NIS/TfOH promoter system in dry CH2Cl2.The expected protected disaccharide (72) was formed with excellent stereoselectivity, but the yield was moderate (48%) due to the partial loss of the acetal-protecting group during glycosylation.To increase the yield, the reaction was repeated in the presence of sym-collidine to prevent cleavage of the acid-sensitive BDA.In this case, disaccharide 72 was produced with an excellent yield of 70%.
Removal of the NAP group at position 4 with DDQ afforded derivative 60, part of which was used as an acceptor for the synthesis of the trisaccharide congener.Further deprotection of 60 included hydrolysis of the BDA groups with trifluoroacetic acid (TFA) and cleavage of the ethyl ester group of the sulfonic acid moieties with NaI.Finally, the free hydroxyl groups of 53 were sulfated with SO3•Et3N reagent in dry DMF to obtain the fully sulfated disaccharide derivative 73.Scheme 6. Synthesis of the BDA-protected C6-sulfonatomethyl-containing monosaccharide building blocks (56 and 59) and polymerization of 56 using 59 as a capping unit.The capping monomer is highlighted in red.
In parallel to the oligomerization, the di-and trisaccharide derivatives were also produced by step-by-step synthesis (Scheme 7).First, acceptor 59 was glycosylated with the fully protected donor 55 using the NIS/TfOH promoter system in dry CH 2 Cl 2 .The expected protected disaccharide (72) was formed with excellent stereoselectivity, but the yield was moderate (48%) due to the partial loss of the acetal-protecting group during glycosylation.To increase the yield, the reaction was repeated in the presence of sym-collidine to prevent cleavage of the acid-sensitive BDA.In this case, disaccharide 72 was produced with an excellent yield of 70%.
Removal of the NAP group at position 4 with DDQ afforded derivative 60, part of which was used as an acceptor for the synthesis of the trisaccharide congener.Further deprotection of 60 included hydrolysis of the BDA groups with trifluoroacetic acid (TFA) and cleavage of the ethyl ester group of the sulfonic acid moieties with NaI.Finally, the free hydroxyl groups of 53 were sulfated with SO 3 •Et 3 N reagent in dry DMF to obtain the fully sulfated disaccharide derivative 73.
Trisaccharide 74 was prepared analogously to the synthesis of the disaccharide congener by coupling of 55 and 60 upon NIS-AgOTf activation in the presence of sym-collidine.The protecting groups were then removed in three steps via NAP-deprotection (61) followed by acidic hydrolysis of the cyclic BDA group and conversion of the sulfonic acid esters into sulfonate salts (54).Treatment of 54 with SO 3 •Et 3 N resulted in the persulfated trisaccharide sulfonic acid final product 75 with good yield.
Trisaccharide 74 was prepared analogously to the synthesis of the disaccharide congener by coupling of 55 and 60 upon NIS-AgOTf activation in the presence of sym-collidine.The protecting groups were then removed in three steps via NAP-deprotection (61) followed by acidic hydrolysis of the cyclic BDA group and conversion of the sulfonic acid esters into sulfonate salts (54).Treatment of 54 with SO3•Et3N resulted in the persulfated trisaccharide sulfonic acid final product 75 with good yield.

Biological Evaluation
The di-and trisaccharide sulfonic acid derivatives, both the ones with free hydroxyl groups (53 and 54) and the persulfated forms (73 and 75) were subjected to biological assays to study their cytotoxic, anti-inflammatory and antiviral effects.
It has been shown that heparin and heparinoid derivatives can prevent the binding of SARS-CoV-2 to human cells and inhibit the entry of different SARS-CoV-2 strains into cells [46][47][48].Therefore, we hypothesized that our heparin-analogue oligosaccharides may be effective against SARS-associated coronaviruses.The anti-SARS-CoV-2 effect of the compounds was tested in Vero E6 cells (Table S1/Figure S15).Unfortunately, neither oligosaccharides showed activity against the tested strain.
To examine the possible anti-inflammatory effects of compounds 53, 54, 73 and 75, the inhibitory potential of NF-κB pathway activation was investigated in HeLa cells (Fig-

Biological Evaluation
The di-and trisaccharide sulfonic acid derivatives, both the ones with free hydroxyl groups (53 and 54) and the persulfated forms (73 and 75) were subjected to biological assays to study their cytotoxic, anti-inflammatory and antiviral effects.
It has been shown that heparin and heparinoid derivatives can prevent the binding of SARS-CoV-2 to human cells and inhibit the entry of different SARS-CoV-2 strains into cells [46][47][48].Therefore, we hypothesized that our heparin-analogue oligosaccharides may be effective against SARS-associated coronaviruses.The anti-SARS-CoV-2 effect of the compounds was tested in Vero E6 cells (Table S1/Figure S15).Unfortunately, neither oligosaccharides showed activity against the tested strain.
To examine the possible anti-inflammatory effects of compounds 53, 54, 73 and 75, the inhibitory potential of NF-κB pathway activation was investigated in HeLa cells (Figure 7).Pretreatment of HeLa cells for 30 min with these compounds at a final concentration of 5 µM significantly reduced (p < 0.0001) the tumor necrosis factor alpha-induced (TNF-α) translocation of the NF-κB p65 subunit from the cytoplasm into the cell nucleus, inhibiting the activation of this main inflammatory pathway.According to these results, all four oligosaccharides can exert an anti-inflammatory effect on the tested cell line.
their sulfated counterparts (73 and 75).It can be assumed that the non-sulfated oligosaccharides are able to bind to the target protein with sufficient strength through their two or three sulfonic acid groups, thus creating the carbohydrate-protein interaction necessary for the biological effect.However, further studies are necessary for a deeper understanding of the anti-inflammatory effect and to reveal the structure-effect relationships.

Conclusions
During our block synthetic approach using thioglycosides as acceptors for the production of malto-oligosaccharides, aglycone transfer side reactions took place which impaired the efficiency of the syntheses.In the polymerization reactions, if the thioglycoside building block 8 was used as both donor and acceptor, the polymerization proceeded efficiently, but difficult-to-isolate hemiacetal oligosaccharides were predominantly formed.Unfortunately, contrary to our expectations, the formed oligomers showed no tendency to cyclize, and we could not detect cyclodextrin-type products in the reaction mixtures.
The most effective methods proved to be the polymerization reaction using a methyl glycoside acceptor (30 or 59) as a capping monomer.The polyglycosylation reactions of the methyl glycoside acceptors proceeded efficiently and with full stereoselectivity using both benzyl and butane diacetal-protecting groups, providing oligomeric series up to dodecasaccharides.In this work, only di-and trisaccharides were isolated from the polymerization mixtures, but according to size-exclusion chromatography (SEC) analysis, the method is suitable for the rapid production of higher oligomers, up to pentasaccharides, with good/acceptable yields.The most economical method for the preparation of small It is important to note that only sulfated oligosaccharides can be expected to have significant biological results based on the literature, so it was very surprising that the nonsulfated di-and trisaccharides (53 and 54, showed the same anti-inflammatory effect as their sulfated counterparts (73 and 75).It can be assumed that the non-sulfated oligosaccharides are able to bind to the target protein with sufficient strength through their two or three sulfonic acid groups, thus creating the carbohydrate-protein interaction necessary for the biological effect.However, further studies are necessary for a deeper understanding of the anti-inflammatory effect and to reveal the structure-effect relationships.

Conclusions
During our block synthetic approach using thioglycosides as acceptors for the production of malto-oligosaccharides, aglycone transfer side reactions took place which impaired the efficiency of the syntheses.In the polymerization reactions, if the thioglycoside building block 8 was used as both donor and acceptor, the polymerization proceeded efficiently, but difficult-to-isolate hemiacetal oligosaccharides were predominantly formed.Unfortunately, contrary to our expectations, the formed oligomers showed no tendency to cyclize, and we could not detect cyclodextrin-type products in the reaction mixtures.
The most effective methods proved to be the polymerization reaction using a methyl glycoside acceptor (30 or 59) as a capping monomer.The polyglycosylation reactions of the methyl glycoside acceptors proceeded efficiently and with full stereoselectivity using both benzyl and butane diacetal-protecting groups, providing oligomeric series up to dodecasaccharides.In this work, only di-and trisaccharides were isolated from the polymerization mixtures, but according to size-exclusion chromatography (SEC) analysis, the method is suitable for the rapid production of higher oligomers, up to pentasaccharides, with good/acceptable yields.The most economical method for the preparation of small oligomers, including di-and trisaccharides, was stepwise synthesis, which provided the desired malto-oligosaccharides in 60-70% yield.
Our results show that, as expected, the polymerization method resulted in oligosaccharides up to dodecamers in a single step, thus significantly shortening the synthesis.However, from a preparative point of view, it was not superior to traditional methods, since higher oligosaccharides could not be isolated in pure form due to the complexity of the polymerization mixtures.
The produced maltooligomeric di-and trisaccharide sulfonic acids proved to be inactive against SARS-CoV-2 viruses; it is assumed that the antiviral effect requires maltooligosaccharides with a higher degree of oligomerization.
Cytotoxicity tests performed on five cell lines revealed that the di-and trisaccharide sulfonic acids tested are biocompatible, showing no significant toxicity up to a concentration of 50 µM.According to preliminary studies, the malto-oligomeric sulfonic acids deserve further investigation as potential anti-inflammatory agents, as they effectively inhibit the activation of a main inflammatory pathway induced by tumor necrosis factor alpha.

General Information
Optical rotations were measured at room temperature on a Perkin-Elmer 241 automatic polarimeter (PerkinElmer GmbH, Rodgau, Germany).TLC analysis was performed on Kieselgel 60 F 254 silica-gel plates (Merck KGaA, Darmstadt, Germany) with visualization by immersing in a sulphuric-acid solution (5% in EtOH) followed by heating.Column chromatography was performed on silica gel 60 (Merck 0.063-0.200mm).Organic solutions were dried over MgSO 4 and concentrated under vacuum. 1 H and 13 C NMR spectroscopy ( 1 H: 360, 400 and 500 MHz;  13 C nuclei).In D 2 O, the H 2 O and CD 3 OD signals were used as a reference.MALDI-TOF MS measurements were carried out with a Bruker Autoflex Speed mass spectrometer (Bruker, Bremen, Germany) equipped with a time-of-flight (TOF) mass analyser.In all cases, 19 kV (ion source voltage 1) and 16.65 kV (ion source voltage 2) were used.For reflectron mode, 21 kV and 9.55 kV were applied as reflector voltage 1 and reflector voltage 2, respectively.A solid phase laser (355 nm, ≥100 µJ/pulse) operating at 500 Hz was applied to produce laser desorption and 3000 shots were summed.A 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix and F 3 CCOONa as a cationising agent in DMF.HRMS measurements were carried out on a maXis II UHR ESI-QTOF MS instrument (Bruker, Bremen, Germany) in positive ionization mode.The following parameters were applied for the electrospray ion source: capillary voltage: 3.6 kV; end plate offset: 500 V; nebulizer pressure: 0.5 bar; dry gas temperature: 200 • C and dry gas flow rate: 4.0 L/min.Constant background correction was applied for each spectrum; the background was recorded before each sample by injecting the blank sample matrix (solvent).Na-formate calibrant was injected after each sample, which enabled internal calibration during data evaluation.Mass spectra were recorded by otofControl version 4.1 (build: 3.5, Bruker, Bremen, Germany) and processed by Compass DataAnalysis version 4.4 (build: 200.55.2969,Bruker Daltonics GmbH, Bremen, Germany).
SEC chromatograms were recorded at a flow rate of 0.5 mL/min of tetrahydrofuran (THF) using a Waters Alliance e2695 HPLC separation module (Waters Corp., Milford, MA, USA) equipped with four gel columns (4.6 × 300 mm, 5 µm Styragel columns: HR 0.5, 1, 2 and 4) and with a Waters 2414 refractive index detector (Waters Corp., Milford, MA, USA).SEC was calibrated with polystyrene standards.The concentration of the samples was 1 mg/mL.
Anti-SARS-CoV-2 activity was measured by determining the extent the compounds inhibited virus-induced cytopathic effect (CPE) and viral replication assessed by immunofluorescence assay (IFA) in Vero E6 cells (ATCC no.CRL-1586, LGC standards, Lomianki, Poland).For CPE-based assay, two-fold serial dilutions of compounds were added in triplicate to Vero E6 cells that were seeded one day before in the amount of 20,000 cells in 96-well plate in DMEM medium with 10% FBS, 100 U of penicillin/mL and 100 µg of streptomycin/mL (all Merck).After 1 h incubation, SARS-CoV-2 (strain hCoV-19/Czech Republic/NRL_6632_2/2020) was added in the multiplicity of infection (MOI) 0.01 IU/cell and the cells were incubated for 72 h at 37 • C, 5% CO 2 .After incubation, the cell viability was analyzed by formazan-based (XTT) cell proliferation assay (Thermo Fisher Scientific, Waltham, MA, USA).Briefly, mixture of XTT labelling reagent and PMS electron-coupling reagent (both Sigma-Aldrich, Budapest, Hungary) was added to the cells and incubated for 4 h at 37 • C, 5% CO 2 .The absorbance of newly formed orange formazan dye was measured in EnVision (Perkin Elmer GmbH, Rodgau, Germany) at 450 nm.The compound concentrations resulting in 50% reduction in CPE (EC 50 ) were calculated from plots of percentage of absorbance versus log 10 drug concentration using nonlinear regression using GraphPad Prism v.9.5.1 (GraphPad Software, GraphPad Software Inc, Boston, MA, USA).For immunofluorescence-based assay, Vero E6 cells and compounds were prepared same as above but SARS-CoV-2 was added at MOI 0.005 IU/cells.After three days incubation at 37 • C, 5% CO 2 , IFA was performed.Briefly, medium was removed and cells were fixed using 4% paraformaldehyde, permeabilized with 0.2% Triton-X100 (both Sigma-Aldrich, Budapest, Hungary), incubated with the first mouse anti-SARS-CoV-2 antibody (mouse monoclonal nucleoprotein IgG, ProSci Inc, Poway, CA, USA) for 2 h at room temperature followed by incubation with the second anti-mouse antibody conjugated with Cy-3 fluorophore (Jackson ImmunoResearch Europe LTD, Ely, UK) for 1.5 h.Signal was detected using fluorescent microscope with camera (Olympus, Olympus Corporation, Tokyo, Japan).Images were processed in ImageJ program (NIH, Bethesda, Maryland, USA) and compound concentrations required to reduce fluorescence signal by 50% (EC 50 ) were calculated from plots of percentage of fluorescent cells versus log 10 drug concentration as above.For cytotoxicity determination, the same setup as for CPE-based assay without the addition of virus was used.Vero E6 cytotoxicity was determined after 72 h incubation at 37 • C, 5% CO 2 using XTT proliferation assay performed same as above.The compound concentrations resulting in a 50% reduction in cell viability (CC 50 ) were calculated using nonlinear regression as above for EC 50 determination.Remdesivir was used as a control in all experiments.The immortalized hCMEC/D3 human endothelial cell line (Merck KGaA, Darmstadt, Germany, Cat.# SCC066) was cultured in Endothelial Cell Growth Medium MV (supplemented with the components of the Supplement Pack) (Sigma-Aldrich Ltd., Budapest, Hungary).
The anti-inflammatory study was performed in HeLa cells (ECACC, Salisbury, UK).In this experiment, 40,000 cells/well were seeded on round glass cover-slips placed into 24 well plates.Four days later, when the cells reached the appropriate confluence, cells were washed twice with HBSS and pre-incubated for 30 min at 37 • C with 5 µM solutions of 53, 54, 73 and 75 compounds in HBSS.After this, cells were washed twice with HBSS and incubated with TNF-α (50 ng/mL) to activate the NF-κB inflammatory pathway.After the incubation time cells were washed twice with HBSS and fixed with methanol: acetone 1:1 for 5 min at −20 • C.After this incubation time cells were washed three times with HBSS and the nonspecific binding sites were blocked with fetal bovine serum (FBS) for 15 min at room temperature.Cells were then incubated with 2 µg/mL anti-p65 antibody (RELA/NF-κB p65 Antibody (F-6) Alexa Fluor ® 488) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) for 1 h at 37 • C.After this incubation, cells were washed four times with HBSS and cell nuclei were stained with DAPI (283 nM) for 5 min at 37 • C.After this, cells were washed once with HBSS and the round glass coverslips were glued to the slides.Fluorescence microscopy measurements and analyses were carried out by a Zeiss Axioscope A1 (Zeiss, Jena, Germany) fluorescent microscope.The following filters were used to examine the samples: DAPI: excitation G 365 nm, emission BP 445/50 nm; fluorescein: e xcitation BP 470/40 nm, emission BP525/50 nm.Fluorescence intensities of the cytoplasms and cell nuclei were evaluated by ZEN 2011 software (Zeiss, Jena, Germany) and the nucleus/cytoplasm fluorescence intensity ratio was calculated.ANOVA staistical analysis with Tukey's multiple comparison test was done by GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

General Methods General Method A for Introduction of the Trichloroacetimidate Group (6, 10)
To the solution of the thioglycoside (5, 9) (0.482 mmol) in acetone/H 2 O (9:1, 21 mL), NBS (0.819 mmol, 1.7 equiv.) was added.The reaction mixture was stirred at room temperature for 30 min.The mixture was neutralized with a saturated aqueous solution of NaHCO 3 (5 mL) and concentrated in vacuo.The residue was dissolved in EtOAc (100 mL), washed successively with saturated aqueous solution of NaHCO 3 (2 × 15 mL) and H 2 O (2 × 15 mL).The organic phase was dried over MgSO 4 and concentrated under reduced pressure.The crude product was purified by column chromatography (6:4 n-hexane/EtOAc) to give an αβ-mixture of the appropriate hemiacetal as a colourless syrup.The solution of the hemiacetal (0.469 mmol) in dry CH 2 Cl 2 (7.0 mL) was cooled to 0 • C and trichloroacetonitrile (8.77 mmol, 18.7 equiv.)and DBU (0.120 mmol, 0.25 equiv.)were added.The reaction mixture was stirred for 30 min, then concentrated in vacuo at 30 • C. The residue was purified by silica gel column chromatography.

General Method C for Removal of the Ethyl ester Groups (51, 52, 53, 54)
To a solution of the ethyl esters (31, 32, 60, 61) (0.426 mmol) in acetone (25 mL) NaI (1.278 mmol, 1.5 equiv./ester)was added at room temperature.After 24 h, the mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography.

30 Figure 3 .
Figure 3. MALDI-TOF MS spectrum of the reaction mixture obtained by oligomerization reaction of 8.The Figure inset shows the partial MALDI-TOF MS spectrum of the reaction mixture in the m/z range of 2700-3300.The oligomer series appeared in the MALDI-TOF MS spectrum are cationized by sodium ion ([M + Na] + ).

Figure 3 .
Figure 3. MALDI-TOF MS spectrum of the reaction mixture obtained by oligomerization reaction of 8.The Figure inset shows the partial MALDI-TOF MS spectrum of the reaction mixture in the m/z range of 2700-3300.The oligomer series appeared in the MALDI-TOF MS spectrum are cationized by sodium ion ([M + Na] + ).

Figure 4 .
Figure 4. ESI-TOF MS/MS spectrum of the sodiated oligomer of 8 with n = 4 ([M + Na] + ) recorded at a collision energy of 80 eV.The Figure inset shows the characteristics glycosidic bond cleavages designated according to Domon and Costello [38].

Figure 4 .
Figure 4. ESI-TOF MS/MS spectrum of the sodiated oligomer of 8 with n = 4 ([M + Na] + ) recorded at a collision energy of 80 eV.The Figure inset shows the characteristics glycosidic bond cleavages designated according to Domon and Costello [38].

Scheme 4 .
Scheme 4. One-pot polyglycosylation reactions of the α-OMe glycoside acceptor 30 with the C-6sulfonatomethyl monomer 8.The capping monomer is highlighted in red.

Figure 5 .
Figure 5. MALDI-TOF MS spectrum of the reaction mixture obtained by oligomerization reaction of 8 in the presence of 30.The Figure inset shows the partial MALDI-TOF MS spectrum of the reaction mixture in the m/z range of 3550-4150.The oligomer series appeared in the MALDI-TOF MS spectrum are cationized by sodium ions ([M + Na] + ).

Figure 6 .
Figure 6.SEC chromatogram of the reaction mixture obtained after oligomerization reaction of 8 and 30 (a) and the deconvoluted SEC chromatogram obtained after baseline correction (b) Purple indicates the unresolved SEC trace in panel (a).

Figure 5 .
Figure 5. MALDI-TOF MS spectrum of the reaction mixture obtained by oligomerization reaction of 8 in the presence of 30.The Figure inset shows the partial MALDI-TOF MS spectrum of the reaction mixture in the m/z range of 3550-4150.The oligomer series appeared in the MALDI-TOF MS spectrum are cationized by sodium ions ([M + Na] + ).

Figure 5 .
Figure 5. MALDI-TOF MS spectrum of the reaction mixture obtained by oligomerization reaction of 8 in the presence of 30.The Figure inset shows the partial MALDI-TOF MS spectrum of the reaction mixture in the m/z range of 3550-4150.The oligomer series appeared in the MALDI-TOF MS spectrum are cationized by sodium ions ([M + Na] + ).

Figure 6 .
Figure 6.SEC chromatogram of the reaction mixture obtained after oligomerization reaction of 8 and 30 (a) and the deconvoluted SEC chromatogram obtained after baseline correction (b) Purple indicates the unresolved SEC trace in panel (a).

Figure 6 .
Figure 6.SEC chromatogram of the reaction mixture obtained after oligomerization reaction of 8 and 30 (a) and the deconvoluted SEC chromatogram obtained after baseline correction (b) Purple indicates the unresolved SEC trace in panel (a).

Figure 7 .
Figure 7. Inhibition of the NF-κB inflammatory pathway in HeLa cells by the 30 min pre-treatment of 53, 54, 73 and 75 compounds at a final concentration of 5 µM.(A) p65 subunit of NF-κB was labeled with a monoclonal antibody (green).The p65 subunit localizes in the cytoplasm in control cells, however after the inflammatory stimuly of TNF-α it translocates into the cell nucleus (blue).(B) Compounds 53, 54, 73 and 75 significantly reduced (p < 0.0001) the translocation of p65 subunit into the nucleus indicating a potential anti-inflammatory effect compared to the positive control TNF-α-treated cells, but not reached the control, unstimulated level.Mean ± S.D. values are shown, n = 10 cells were analyzed/group.**** p < 0.0001.

Figure 7 .
Figure 7. Inhibition of the NF-κB inflammatory pathway in HeLa cells by the 30 min pre-treatment of 53, 54, 73 and 75 compounds at a final concentration of 5 µM.(A) p65 subunit of NF-κB was labeled with a monoclonal antibody (green).The p65 subunit localizes in the cytoplasm in control cells, however after the inflammatory stimuly of TNF-α it translocates into the cell nucleus (blue).(B) Compounds 53, 54, 73 and 75 significantly reduced (p < 0.0001) the translocation of p65 subunit into the nucleus indicating a potential anti-inflammatory effect compared to the positive control TNF-α-treated cells, but not reached the control, unstimulated level.Mean ± S.D. values are shown, n = 10 cells were analyzed/group.**** p < 0.0001.

Table 1 .
Polymerization reaction of compound 8. values of the neighbouring peaks in each oligomer series is 448, which corresponds to the mass of the repeat unit with a composition of C23H28O7S.Furthermore, based on the m/z values, our previous assumption was [a] Detected by MALDI-TOF measurements.[b]Isolated yield.The difference between the m/z

Table 2 .
Polymerization of compound 8 in the presence of 30.