Design, Synthesis, and Biological Evaluation of Desmuramyl Dipeptides Modified by Adamantyl-1,2,3-triazole

Muramyl dipeptide (MDP) is the smallest peptidoglycan fragment able to trigger the immune response. Structural modification of MDP can lead to the preparation of analogs with improved immunostimulant properties, including desmuramyl peptides (DMPs). The aim of this work was to prepare the desmuramyl peptide (L-Ala-D-Glu)-containing adamantyl-triazole moiety and its mannosylated derivative in order to study their immunomodulatory activities in vivo. The adjuvant activity of the prepared compounds was evaluated in a murine model using ovalbumin as an antigen, and compared to the reference adjuvant ManAdDMP. The results showed that the introduction of the lipophilic adamantyl-triazole moiety at the C-terminus of L-Ala-D-Glu contributes to the immunostimulant activity of DMP, and that mannosylation of DMP modified with adamantyl-triazole causes the amplification of its immunostimulant activity.


Introduction
Peptidoglycans are conserved polymeric constituents of Gram-positive and Gramnegative bacteria that act as agonists of pathogen-recognition receptors (PRRs) and, therefore, stimulate the immune response [1][2][3]. PRRs in general are classified into Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs) [4]. Muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine) is the smallest peptidoglycan fragment (Figure 1) able to trigger the immune response via activation of the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Structural details regarding the activation of NOD2 are still not fully understood but, based on the crystal structure of the rabbit NOD2 in the ADP-bound form, an MDP binding site is proposed [5]. Up to now, our research has been directed towards MDP analogs lacking the N-acetylmuramyl group, called desmuramyl peptides (DMPs). We have prepared DMPs containing lipophilic adamantane moieties attached at the N-and C-termini of L-Ala or D-isoGln, as well as their mannosylated derivatives. The results indicate that mannosylation of DMPs contributes to the stimulation of the immune response [10,11], possibly by Structure-activity studies of muropeptides have been performed in order to prepare an adjuvant with improved properties of the MDP molecule, and lipophilic analogs and derivatives of MDP have shown improved immunostimulant activity [6][7][8][9].
Up to now, our research has been directed towards MDP analogs lacking the Nacetylmuramyl group, called desmuramyl peptides (DMPs). We have prepared DMPs containing lipophilic adamantane moieties attached at the Nand C-termini of L-Ala or D-isoGln, as well as their mannosylated derivatives. The results indicate that mannosylation of DMPs contributes to the stimulation of the immune response [10,11], possibly by affecting the immune response via the mannose receptor family [12,13]. The immunomodulatory activities of all prepared DMPs and their mannosylated derivatives were estimated in vivo, in murine models, based on the secondary humoral response to ovalbumin as a test antigen. In structure-activity relationship (SAR) studies, we have shown that glycoconjugates that contain a glycolyl linker between mannose and the DMP moiety exhibit a stronger adjuvant activity than analogs containing a hydroxyisobutyryl linker [11]. The most active adjuvant in this class of compounds is ManAdDMP (Figure 1), which has adamantane in the form of adamantylglycine between mannose and L-Ala. We have confirmed that MDP and ManAdDMP with a free carboxyl group at the side chain of the isoGln form strong interactions with the NOD2 receptor. Specifically, free γ-COOH makes the greatest contribution to the NOD2 binding. Furthermore, a contribution of hydrophilic mannose and lipophilic adamantane to the NOD2 binding was also observed [14].
Therefore, within this study we synthesized DMP derivatives with the adamantane substituent attached to the C-terminus of the dipeptide, at α-COOH of D-Glu. Furthermore, herein we describe a strategy for the preparation of an adamantyl-triazole structure, which was then coupled with the dipeptide part. The immunomodulatory properties of the prepared adamantyl-triazole DMP and its mannosylated derivative were estimated in vivo in a well-established murine model [8,10,11], and compared with the reference ManAdDMP adjuvant.

Chemistry
In this work, we describe the preparation and characterization of adamantyl-triazole DMP 8 and its mannose derivative 14. The key step in the synthesis of compound 8 was the conjugation of L-alanyl-D-isoglutamine and adamantyl-triazole 3.
Compound 3 was prepared as shown in Scheme 1, starting from commercially available 1-adamantanol using a known procedure [15]. A modified one-pot method [16] was used for the preparation of Boc-protected derivative 2, which was subsequently deprotected using TFA, with good yields. Peptide building blocks were prepared starting from unprotected D-Glu (Scheme 2). In the first step, according to the procedure previously described [17], the carboxyl group of the side chain of D-Glu was selectively protected to produce compound 4 ( Figure S3) with a good yield. Compound 4 was then coupled with commercially available Boc-L-Ala-OSu in the presence of triethylamine (TEA) to form dipeptide 5 ( Figure S4).  Peptide building blocks were prepared starting from unprotected D-Glu (Scheme 2). In the first step, according to the procedure previously described [17], the carboxyl group of the side chain of D-Glu was selectively protected to produce compound 4 ( Figure S3) with a good yield. Compound 4 was then coupled with commercially available Boc-L-Ala-OSu in the presence of triethylamine (TEA) to form dipeptide 5 ( Figure S4). The n-butyl chloroformate/N-methylmorpholine coupling method was used for the conjugation of 5 with adamantyl-triazole derivative 3. Thus, compound 6 was prepared with an excellent yield. Boc and benzyl protection were removed using standard and efficient procedures, and compounds 7 and 8, respectively, were obtained. Scheme 1. Reagents and conditions: (a) NaN3, H2SO4, CHCl3, rt, 3 h, 87%; (b) (i) Boc2O, TEA, 1,4-dioxane, H2O, 0 °C to rt, 90 min, (ii) 1, Na ascorbate (aq), CuSO4 × 5 H2O (aq), Ar, 35 °C, 24 h, 97%; (c) TFA, dry CH2Cl2, rt, 2 h, 87%.
Peptide building blocks were prepared starting from unprotected D-Glu (Scheme 2). In the first step, according to the procedure previously described [17], the carboxyl group of the side chain of D-Glu was selectively protected to produce compound 4 ( Figure S3) with a good yield. Compound 4 was then coupled with commercially available Boc-L-Ala-OSu in the presence of triethylamine (TEA) to form dipeptide 5 ( Figure S4). The n-butyl chloroformate/N-methylmorpholine coupling method was used for the conjugation of 5 with adamantyl-triazole derivative 3. Thus, compound 6 was prepared with an excellent yield. Boc and benzyl protection were removed using standard and efficient procedures, and compounds 7 and 8, respectively, were obtained. The preparation of mannose precursor 12 is shown in Scheme 3. Benzylated mannopyranose 10 was prepared using a slightly modified version of the procedure described by Koto et al. [18]. SN2 substitution of bromine from tert-butyl bromoacetate with 2,3,4,6-tetra-O-benzyl-α-D-mannopyranose 10 in the presence of potassium carbonate produced compound 11. The tert-butyl group of the resulting compound 11 was removed using trifluoroacetic acid (TFA), and the benzylated mannoside 12 with a free carboxyl group was obtained for coupling with the peptide precursor (Scheme 3). The preparation of mannose precursor 12 is shown in Scheme 3. Benzylated mannopyranose 10 was prepared using a slightly modified version of the procedure described by Koto et al. [18]. S N 2 substitution of bromine from tert-butyl bromoacetate with 2,3,4,6tetra-O-benzyl-α-D-mannopyranose 10 in the presence of potassium carbonate produced compound 11. The tert-butyl group of the resulting compound 11 was removed using trifluoroacetic acid (TFA), and the benzylated mannoside 12 with a free carboxyl group was obtained for coupling with the peptide precursor (Scheme 3). The synthesis of mannose conjugate 13 was performed using the standard EDC/HOBt coupling method with triethylamine as a base, with an excellent yield (Scheme 4). Debenzylation of 13 via catalytic hydrogenolysis produced the final product 14, unequivocally identified by HPLC analysis, 1 H NMR and 13 C NMR spectroscopies, and mass spectrometry.  The synthesis of mannose conjugate 13 was performed using the standard EDC/HOBt coupling method with triethylamine as a base, with an excellent yield (Scheme 4). Debenzylation of 13 via catalytic hydrogenolysis produced the final product 14, unequivocally identified by HPLC analysis, 1 H NMR and 13 C NMR spectroscopies, and mass spectrometry.

Evaluation of Immunostimulant Activity
The adjuvant activity was estimated by the immunostimulant effect on the secondary humoral response of a well-established model antigen ovalbumin (OVA) in BALB/c mice, according to previously described in vivo studies [11,19]. Overall anti-OVA IgG, and subclasses of IgG (anti-OVA IgG1 and anti-OVA IgG2a) as indicators of Th1 and Th2 of the immune response, were measured in the murine sera after a second booster. The adjuvant activity of the newly synthesized compounds was compared to the reference ManAdDMP, which is able to switch the immune response towards a pronounced Th2-type immune reaction, as was described for MDP [11]. The synthesis of mannose conjugate 13 was performed using the standard EDC/HOBt coupling method with triethylamine as a base, with an excellent yield (Scheme 4). Debenzylation of 13 via catalytic hydrogenolysis produced the final product 14, unequivocally identified by HPLC analysis, 1 H NMR and 13 C NMR spectroscopies, and mass spectrometry.

Evaluation of Immunostimulant Activity
The adjuvant activity was estimated by the immunostimulant effect on the secondary humoral response of a well-established model antigen ovalbumin (OVA) in BALB/c mice, according to previously described in vivo studies [11,19]. Overall anti-OVA IgG, and subclasses of IgG (anti-OVA IgG1 and anti-OVA IgG2a) as indicators of Th1 and Th2 of the immune response, were measured in the murine sera after a second booster. The adjuvant activity of the newly synthesized compounds was compared to the reference ManAdDMP, which is able to switch the immune response towards a pronounced Th2-type immune reaction, as was described for MDP [11].
In general, when compared to the group treated with no adjuvant (OVA alone), an enhancement in total anti-OVA IgG antibody production was observed in all groups ( Figure 2). High levels of IgG antibody were present even in the OVA-treated group, leading to relatively weak stimulation of total antibody production in the Ma-nAdDMP-injected group. Compound 8 elicited a stronger immune response than OVA alone or the ManAdDMP-injected group. The enhancement in total anti-OVA IgG antibody production by adamantyl-triazole derivative 8 was statistically significant (p < 0.05). Immunization with compound 14, which has compound 8 attached to mannose through the glycolyl linker, led to the highest and most statistically significant increase in the specific IgG response (p < 0.01), in comparison to the control group. These results lead to the conclusion that α-COOH is a suitable position for the attachment of adamantane in this class of compounds. In general, when compared to the group treated with no adjuvant (OVA alone), an enhancement in total anti-OVA IgG antibody production was observed in all groups ( Figure 2). High levels of IgG antibody were present even in the OVA-treated group, leading to relatively weak stimulation of total antibody production in the ManAdDMPinjected group. Compound 8 elicited a stronger immune response than OVA alone or the ManAdDMP-injected group. The enhancement in total anti-OVA IgG antibody production by adamantyl-triazole derivative 8 was statistically significant (p < 0.05). Immunization with compound 14, which has compound 8 attached to mannose through the glycolyl linker, led to the highest and most statistically significant increase in the specific IgG response (p < 0.01), in comparison to the control group. These results lead to the conclusion that α-COOH is a suitable position for the attachment of adamantane in this class of compounds. Since it is known that immune adjuvants can enhanc Since it is known that immune adjuvants can enhance or modulate the Th1/Th2 bias of the induced immune response, the isotype profiles of the antigen-specific anti-OVA IgG antibodies IgG1 and IgG2a were quantitatively determined ( Figure 3). Specifically, the type of the generated immune response can be indirectly estimated via the quantification of OVA-specific IgG1 (for activation of Th2 type) and IgG2a (for activation of Th1 type), and calculation of the relevant IgG1/IgG2a ratio. When the amount of anti-OVA IgG1 antibodies was measured (Figure 3a), it was observed in all groups that the trend of IgG1 antibody production was similar to those of overall anti-OVA IgG. The highest response, which was also statistically significant, was elicited by compound 8 (p < 0.05) and its mannosylated derivative 14 (p < 0.01). A statistically significant enhancement of anti-OVA IgG2a production (Figure 3b) was observed only in the group immunized with mannosylated derivative 14 (p < 0.01). The type of immune response was indirectly determined by the calculation of the IgG1/IgG2a ratio (Figure 3c). From the IgG1/IgG2a ratio, it was evident that all groups treated with the tested adjuvants had higher values than the group treated with OVA alone, indicating a slight shift toward a more pronounced Th2-type immune response. Compound 8 significantly (p < 0.01) switched the immune response towards a pronounced Th2 type, due to the predominance of IgG1 antibodies. Figure 2. The effect of synthesized DMPs on the production of anti-OVA IgG in BALB/c mice immunized with OVA as an antigen. Experimental groups: 1-control group,; 2-OVA alone; 3-OVA + ManAdDMP; 4-OVA + 8; 5-OVA + 14. Bar graphs represent average values from individual mice from each group (n = 5). * p < 0.05 and ** p < 0.01 denote statistical significance in comparison to the control group.
Since it is known that immune adjuvants can enhance or modulate the Th1/Th2 bias of the induced immune response, the isotype profiles of the antigen-specific anti-OVA IgG antibodies IgG1 and IgG2a were quantitatively determined ( Figure 3). Specifically, the type of the generated immune response can be indirectly estimated via the quantification of OVA-specific IgG1 (for activation of Th2 type) and IgG2a (for activation of Th1 type), and calculation of the relevant IgG1/IgG2a ratio. When the amount of anti-OVA IgG1 antibodies was measured (Figure 3a), it was observed in all groups that the trend of IgG1 antibody production was similar to those of overall anti-OVA IgG. The highest response, which was also statistically significant, was elicited by compound 8 (p < 0.05) and its mannosylated derivative 14 (p < 0.01). A statistically significant enhancement of anti-OVA IgG2a production (Figure 3b) was observed only in the group immunized with mannosylated derivative 14 (p < 0.01). The type of immune response was indirectly determined by the calculation of the IgG1/IgG2a ratio (Figure 3c). From the IgG1/IgG2a ratio, it was evident that all groups treated with the tested adjuvants had higher values than the group treated with OVA alone, indicating a slight shift toward a more pronounced Th2-type immune response. Compound 8 significantly (p < 0.01) switched the immune response towards a pronounced Th2 type, due to the predominance of IgG1 antibodies.

Discussion
Natural polymeric peptidoglycans-as well as smaller peptidoglycan fragments, such as MDP and its synthetic derivatives-exhibit immunomodulatory properties. To date, a number of structure-activity studies have been reported, and they have included synthetic modifications at the saccharide and peptide parts of the MDP [7,20,21]. Several synthetically modified MDPs have already been used in clinical settings. Furthermore, formulations of delivery systems-for example, liposomes-of these compounds have been found to be of interest. Mifamurtide is a clinically used therapeutic obtained by linking MDP and dipalmitoylphosphatidylethanolamine, because the phospholipid facilitates the incorporation of the peptide into liposomes [22]. Therefore, modifications with lipophilic substituents have been the mainstream of further research. Our research is focused on the adamantane substituent, because adamantane can act as a membrane anchor, and can enable the preparation of systems for targeted drug delivery [23]; it can also be incorporated into a β-cyclodextrin cavity-a powerful supramolecular nanoparticle carrier for targeted drug delivery [24].
As already mentioned in our previous research, we have demonstrated that the introduction of adamantane at γ-COOH significantly reduces the adjuvant activity by disabling key interactions with the NOD2 receptor [14]. Therefore, within this study, we have explored the modification of the D-Glu at the α-COOH group. In addition to better ligand binding to NOD2, the proposed design will also enable greater exposure of the adamantane structure for possible membrane anchoring, compared to those of ManAdDMP analogs [25,26].
Adamantane was connected to the L-Ala-D-Glu pharmacophore over a 1,2,3-triazole moiety, which was obtained via CuI-catalyzed azide/alkyne cycloaddition (CuAAC) of 1azidoadamanatane and simple propargylamine. The obtained adamantyl-triazole was then coupled to desmuramyl dipeptide using a standard procedure. The developed synthetic methodology, which includes potent CuAAC click chemistry, could be useful for further structural modification of DMPs (the azido component could be coupled with a wide range of structurally diverse alkynes).
The immunomodulatory properties of DMPs were explored in the BALB/c mouse model in two aspects: one aspect was the analysis of the overall production of specific anti-OVA IgG, and the other was the study of Th1-and Th2-type immune response bias. The results show that the adamantyl-triazole DMP derivative induces higher production of anti-OVA IgG than the reference compound, ManAdDMP, which was the most active adjuvant in this class of compounds. This indicates that the introduction of an adamantyl-triazole moiety at α-COOH is preferred for the amplification of immunostimulant activity, while MDP derivatives with a substituted triazole moiety at the C4 position of MurNAc showed lower affinity toward NOD2 [21]. Based on the IgG1/IgG2a ratio (Figure 3c), it is evident that compound 8 shifts the reaction toward a pronounced Th2-type immune response (p < 0.01). MDP dominantly induces IgG1 antibody production, and also stimulates Th2-polarized immune response [19,27]. Furthermore, it is well known that muropeptides act as NOD2 agonists, and can act synergistically. For example, NOD2 ligands can augment the adjuvant activity of TLR ligands, and can modulate innate and adaptive immune responses via NLR/TLR crosstalk [3,[28][29][30]. The application of multi-PRR activation, in general, represents a prospective approach in the design of vaccines [31]. Mannose receptors are soluble and transmembrane receptors in the CLR family [4]; they initiate an innate immune response, and also activate the acquired immunity. Therefore, mannose ligand binding could also affect the immune response. For this reason, adamantyl-triazole DMP was further modified by mannose, and glycopeptide 14 was prepared by coupling a corresponding DMP 7 and α-O-mannoside 12 functionalized by a carboxylic group, followed by debenzylation.
Mannosylation of peptide 8 amplified the production of anti-OVA IgG antibodies. Anti-OVA IgG levels reflect the ability of a potential adjuvant to increase the humoral immune response. Analysis of IgG1 and IgG2a showed that 14 reduced the switch toward the Th2 direction in comparison to parent compound 8. These results indicate that mannosylation can enhance the production of overall anti-OVA specific antibodies, but can also influence the polarization of the immune response. The new mannosylated DMP 14 with incorporated adamantyl-triazole will therefore be further explored in order to obtain a better insight into a possible PRR crosstalk. Additionally, it will be used for the preparation of liposome formulations in order to additionally affect the polarization of the immune response [32].

Experiments In Vivo
Healthy, nulliparous, and non-pregnant BALB/c female mice 8-12 weeks of age at the initiation of the experiment were used. Mice were obtained from the Ruder Bošković Institute's breeding colony. During the experimental period, groups of five animals were kept per cage. The bottoms of the cages were covered with sawdust (Scobis Uno ® , Mucedola srl, Italy). Standard food for laboratory mice (4RF 21 GLP ® Mucedola srl, Italy) was used. Access to food and water was ad libitum. Animals were kept in conventional circumstances: light/dark rhythms of 12/12 h, temperature of 22 • C, and humidity of 55%. All experiments were performed according to the ILAR Guide for the Care and Use of Laboratory Animals, EU Directive 2010/63/EU, and Croatian animal protection law (NN 102/17). Experimental groups of five mice were immunized and boosted twice subcutaneously (s.c.) into the tail base at 21-day intervals. Mice were anesthetized with isoflurane prior to blood collection on the 7th day after the 2nd booster. Sera were collected, decomplemented at 56 • C for 30 min, and stored at -20 • C until tested. The dose of OVA (antigen) was 10 µg per mouse. The doses of PGM and the tested compounds were 200 µg per mouse. OVA and the tested substances were dissolved in water, and the injection volume in all experimental groups was 0.1 mL per mouse.

Enzyme Immunoassays
Enzyme immunoassays (ELISA) were performed on flat-bottomed high-binding microtiter plates (Costar, USA), using murine anti-OVA IgG, IgG1, and IgG2a antibody assay kits (Chondrex, USA), according to the manufacturer's instructions. The ratio of anti-OVA IgG1 and anti-OVA IgG2a (IgG1/IgG2a) was calculated as an indication of the Th1/Th2 bias of the induced immune response.

Statistics
Statistical analyses were performed using GraphPad Prism Software. Significant differences between experimental groups were evaluated via Kruskal-Wallis ANOVA, followed by Dunn's multiple comparisons test. Probability values less than 0.05 (p < 0.05) were considered significant.

Conclusions
The immunostimulant activities of adamantyl-triazole DMPs were evaluated and compared to the reference ManAdDMP. In experiments in vivo, adamantyl-triazole DMP 8 exhibited higher adjuvant activity than the reference ManAdDMP. Mannosylation of adamantyl-triazole DMP 8, which produced the glycoconjugate 14, resulted in the further amplification of immunostimulant activity. The obtained results indicate the α-COOH of D-Glu as being a suitable position for the attachment of a lipophilic adamantane group, which can also enable a preparation of liposome formulations. Additionally, it was confirmed that the attachment of mannose to the DMPs plays an important role in the stimulation of the immune response. Thus far, glycopeptide 14 has been identified as the most potent adjuvant in the class of mannosylated DMPs. It should also be noted that compound 14 is stable, non-pyrogenic, and water soluble, making it potentially applicable as an adjuvant for vaccines. There is no clear evidence of which PRRs are activated by the mannosylated DMPs, and the possibility of affecting the immune response by binding mannose to mannose receptors cannot be excluded. Therefore, further research on this topic is required.