Design, synthesis and immunological evaluation of 1,2,3-triazole-tethered carbohydrate–Pam3Cys conjugates as TLR2 agonists

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

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Design, synthesis and immunological evaluation of 1,2,3-triazoletethered carbohydrate–Pam3Cys conjugates as TLR2 agonists Naresh Nalla a, Preethi Pallavi a, Bonam Srinivasa Reddy a, Sreekanth Miryala a, V. Naveen Kumar a, Mohammed Mahboob b, Halmuthur M. Sampath Kumar a,⇑ a b

Vaccine Immunology Laboratory, Natural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India Toxicology Laboratory, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India

a r t i c l e

i n f o

Article history: Received 25 May 2015 Accepted 29 June 2015 Available online xxxx Keywords: Click chemistry Carbohydrate Pam3Cys Adjuvants Cytokines

a b s t r a c t Novel triazolyl Pam3Cys conjugates encompassing various carbohydrate entities have been synthesized by copper mediated azide-alkyne click chemistry protocol with a view to probe the SAR pertaining to their adjuvant activity in conjunction with OVA as antigen. The preliminary ex vivo cytokine profiling revealed optimal Th1 activation and the in vivo adjuvant studies of ribose derived hybrid (6e) revealed a marked improvement in the OVA specific antibody IgG response and Th1 cytokine expressions. The triazolyl Pam3Cys carbohydrate conjugates were found to be the hTLR2 agonists as revealed by their SEAP activity due to NFKB activation. The described protocol is the first successful attempt of the amalgamation of carbohydrate–Pam3Cys motifs tethered to a triazole linker as a peptide free adjuvant. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Adjuvants have become important components of new generation vaccines. Due to lack of sufficient varieties of safe and potent immunoadjuvants with varying degree of Th1 polarization required for human and veterinary vaccine use, the interest in the co-development of vaccine adjuvants is steadily increasing.1 Adjuvants initiate early innate immune responses, which lead to the induction of robust and long-lasting adaptive immune responses. During the last 95 years many adjuvants have been developed among which aluminum salts2 have been first licensed for human use. Recently MF 59 a squalene based o/w emulsion and ASO4-containing TLR4 agonist MPL (3-O-desacyl-40 monophosphoryl lipid A) together with aluminum salt, have been approved. Because of higher toxicity no other adjuvants have been licensed for human vaccination programme wherein the safety of adjuvant is the prime importance for routine vaccination. Thus, the ideal adjuvant would be non-toxic, biodegradable, inexpensive, non-immunogenic by itself and must not have any interaction with the antigen. Adjuvants augment the immune response of an antigen by mimicking specific sets of evolutionarily conserved pathogen associate molecular patterns (PAMPs), which include liposomes, lipopolysaccharide (LPS), molecular cages for antigen, components ⇑ Corresponding author. Tel.: +91 40 27191824; fax: +91 40 27160512. E-mail address: [email protected] (M.S.K. Halmuthur).

of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA(dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA. Because immune systems have evolved to recognize these specific antigenic moieties, the presence of an adjuvant in conjunction with the vaccine can greatly increase the innate immune response to the antigen through activation of dendritic cells (DCs), lymphocytes, and macrophages by mimicking a natural infection. Thus PAMPs provide clues for the design of Th1 dominant adjuvants capable of inducing both cellular immunity as well as humoral immunity and several such PAMP based natural ligands/synthetic PRR agonists3 are currently being explored as adjuvants either alone or as formulations with other ingredients for various subunit vaccines against cancers and infectious diseases. Hence, diacyl and triacyl lipopeptides such as macrophage-activating lipopeptide-2 (MALP-2) and N-palmitoylated cysteine analogs derived from the N-terminal moiety of mycoplasma and Escherichia coli (E. coli) lipoproteins, respectively, are such TLR agonists which evoke powerful adjuvant action when conjugated to the antigens and they have been used successfully to trigger Th1 activation.4 S-[2,3-Bis(palmitoyloxy)propyl] cysteine, (Pam2Cys), which corresponds to the lipid component of MALP-2 from Mycoplasma fermentans and its N-palmitoylated synthetic analog viz., Pam3Cys are known to be potent immuno-adjuvants (Fig. 1).5 Their mode of action appears to rely on the ability of palmitoylated cysteine lipid head group to activate the downstream signaling cascade by binding and activating TLR2 on DC’s which trigger

http://dx.doi.org/10.1016/j.bmc.2015.06.070 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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N. Nalla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

NH2 O C15 H31 O C15 H31

C15 H31 O

O S

NH

O

O

NH2 H N

O N H OH

O HN

O N H

NH2

H N

O OH

O

NH 2

Figure 1. ‘Pam3CSK4’, N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine.

maturation of DCs resulting in increased efficiency of antigen processing and presentation. The responses produced by activation through a TLR are determined by many factors specific to individual cell types, as well as quantitative and qualitative parameters of the TLR–ligand interaction itself. Thus, the response to TLR signaling can include cell differentiation, proliferation or apoptosis, as well as induction of many secreted mediators, prominently IFNs, TNF and interleukins viz., IL-2, IL-4, IL-6, IL-10, IL-12, and many different chemokines. MALP-2 analogs that are complex lipopeptides viz., Pam2Cys-GDPKHPKSF and Pam2Cys-GDPKHPKSFTGWVA representing the N-terminal part of the 44-kDa lipoprotein LP44 of Mycoplasma salivarium, or Braun lipoprotein have all been described as potent TLR2 agonists.6 Whereas, structurally simple synthetic counterparts like S-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl lipopeptides, like Pam3CSK4, have also shown TLR2 agonist activity which makes them commercially affordable adjuvants. Pioneering work by Bessler7 with the Braun lipoprotein of E. coli established that the N acylated lipid head group Pam3Cys enhances immunogenicity when coupled to a variety of polypeptides. In these constructs, the Pam2Cys or Pam3Cys template is necessary but not enough to stimulate TLR2 and a substitution of the cysteine is required to establish agonistic activity. While it has been established that derivatives with at least two lysine residues usually needed to evoke sufficient cytokine activity attributed to polycationic nitrogens that impart high amphiphilicity to the peptide attached to C-terminus of the cysteinyl lipid head group. Our continuing efforts to rationally design and synthesize new immunoadjuvants,8 we aimed to develop a focused library of novel peptide free Pam3Cys derivatives that can be structural and functional mimics of classical lipopeptide based adjuvants. 2. Results and discussion 2.1. Design and synthesis of 1,2,3-triazolyl carbohydrate– Pam3Cys adjuvant conjugates In the light of the SAR of various palmitoylated cysteine analogues available in the literature, we envisioned the design and synthesis of hybrid molecules of Pam3Cys lipid head group attached to a variety of carbohydrate entities through a spacer encompassing a 1,2,3-triazole moiety. After having embarked upon this goal, we came across a recent report9 that revealed the improved TLR2 agonistic activity of a-galactosyl Pam2Cys, without any mention of any detailed studies pertaining in vivo antibody response and surface marker expression due to these altered structures. However, this further reinforced our own view that the peptide part can be swapped with other suitable non peptide hydrophilic entities to examine the modulation of adjuvant activity. Furthermore, presence of triazolyl moiety along with the carbohydrate should greatly enhance the water solubility of the adjuvant. The carbohydrate entity present in the hybrid should impart the required hydrophilicity whereas the 1,2,3-triazolyl

moiety should mimic the role of poly-cationic peptide in the lipopeptides based Pam3Cys adjuvants since the protonation of 1,2,3-triazole would lead to hydrophilic triazolium cation.10 Such non peptide adjuvants should also offer better hydrolytic stability in comparison with the respective peptide counterparts yet, should be easily metabolizable. Further, replacement of such hybrid entity should have profound effect on the binding and modulation of TLR2. With this aim we envisaged the synthesis of such moiety employing copper catalyzed click chemistry approach. Synthesis of triazole tethered hybrid conjugates of Pam3Cys involved two intermediates viz., azido carbohydrate and propargylamido Pam3Cys moieties. Preparation of azido carbohydrate11 entity is conversion of Corresponding peracetylated sugar was subjected to anomeric azidation by using PCl5/NaN3 or TMSN3/SnCl4 to get the anomeric(a/b) azides substituted acetylated sugar. Deacetylation under NaOMe condition affords completely deprotected azido sugar. On the other hand, the propargylated Pam3Cys fragment was prepared starting from cyclohexylidene mannitol which was subjected to vicinal diol cleavage using NaIO4 oxidation to afford the corresponding aldehyde which was reduced to alcohol using NaBH4/CH3OH which was converted to iodo derivative using I2/Triphenylphosphine and Imidazole in quantitative yields. The corresponding iodo derivative was reacted with N-Boc protected cysteine to get the corresponding protected cysteinyl glycerol unit. Deprotection of cyclohexyl moiety under acid treatment affords the diol which was subjected to palmitoylation using Palmitic acid/DIC and DMAP to afford the required fragment. Completely deprotected sugars bearing azide functionality at the anomeric position of the sugar unit has been subjected to cycloaddition with the corresponding acetylenic Pam3Cys moiety using click chemistry protocol to ligate both the entities through a triazolyl spacer, to afford the desired Pam3Cys triazolyl sugar hybrids. A focused library of 12 compounds has been generated by employing this approach with various sugar substitutions as shown in the Table 1. All compounds were formed in near quantitative yields which were isolated in high purity after crystallization. The compounds were characterized by 1H, 13C NMR, Mass, IR spectroscopy which confirmed the structure. It is noteworthy that all the novel triazolyl hybrids of Pam3Cys exhibited excellent solubility in PBS saline similar to the Pam3CSK4 lipopeptide adjuvant thereby required no extra formulations such as liposomes and nanoparticles or use DMSO to facilitate their in vitro testing. 2.2. Immunological evaluation of 1,2,3-triazolyl carbohydrate– Pam3Cys adjuvant conjugates We initially evaluated these conjugates (6a–l) for its toxicity, as the level of toxicity plays a major role in the process of the development of a successful vaccine adjuvant. Rat kidney cell line (NRK49F) was exposed to varying doses of test compounds in the range of 10 lM to 100 lM, to determine the toxicity and most of the analogs were non toxic. Wherein all the analogs retained about 80% viability at higher concentration, they also showed <15% hemolysis even at the maximum concentration (1000 lg). The conjugates at 6.25 lg/ml concentration were further examined by ex vivo studies for splenocyte proliferation and cytokine estimation viz., IL-2, IL-12 and IFN-c. The results showed that all the analogs exhibited varying degree of cytokine expression, b-galactose derived hybrid (6h) was most active in inducing all three cytokines at higher level followed by the corresponding ribose (6e) and both indicate predominant B cell mediate immunity specific to mitogen stimulation (shown in Supplementary data). Thus, the two primary leads 6h and 6e (20 lg/dose concentrations) were immunizing on 0th day to male BALB/c mice along with OVA as antigen (100 lg/dose) and compared with the standard Pam3CSK4 (20 lg/dose) for in depth assessment by in vivo

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N. Nalla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx Table 1 Pam3Cys based sugar triazoles Entry

Alkyne

Azide

a

C15H 31 C15H 31 O

O

C15H 31 O

O S

O

b

O

O S

O

C15H 31 C15H 31 O

C15H 31 O

O S

O

NH H N

C15H 31 C15H 31 O

C15H 31 O

O S

O

OH OH

NH H N

C15H 31 C15H 31 O

C15H 31 O

O S

O

NH H N

OH HO HO

HO

HO

O NH

OHN 3

C15H 31 C15H 31 O

C15H 31 O

O S

O

OH

N O N N NH

OH

O S

OH N 3

HO

O

O

O

C 15 H31 O C15H 31

S

HO

OH

O

NH

O

C 15 H31 O C15H 31

S

O

N

O

NN

O

O NH

NH

O

C15H 31 O C15H 31

S O

HO

O

O

C 15 H31 N

OH

HO

HO

O NH

N

N

O

NH

C 15 H31 O C 15 H31

O S

O

HO HO

O

O

C 15 H31 OH OH N N O N

O NH

NH

O

C 15 H31 O C15H31

S O

O h

C15H 31 C15H 31 O

C15H 31 O

O S

O

C15H 31 C15H 31 O

C15H 31 O

O S

O

NH H N

HO

OH O

N

NN

O NH

HO OH O HO

HO N3

O

NH

O

O NH

O

NH

O S

C15H 31 O C15H 31

O

j

C15H 31 C15H 31 O

C15H 31 O

O S

O

NH H N

HO

N3 O OH HO HO

N

O HO HO O

k

C15H 31 C15H 31 O

C15H 31 O

O S

O

O O

l

C15H 31 C15H 31 O O

NH H N

C15H 31 O

O S

NH H N O

OH O HO OH HO HO O O N3 HOHO OH OH O O O N3 HO HO HO HO OH

N

N

O

OH

O NH

O

NH

O S

C 15 H31 O C 15 H31

O

OH C 15 H31 O HO OH N HO O NH HO O N N NH S O HOHO O C15H31 OH OH N N O NH O O O N NH S HO HOHO HO O OH

93

O

C15H 31 OH

96

O

C 15 H31

O O

C15H 31 O C15H 31

S O

OH OH O HO HO N N N

94

O

C 15 H31

HO HO

O O

i

NH H N

HO OH O N 3 HO HO

93

O

OH

OH OH O N3

HO HO

97

O

OH

N3

94

O

C 15 H31 HO

93

O

OH O N N N NH

N3 OH

HO

NH H N

NH H N

NH

C 15 H31

HO HO

HO

96

O

O HO HO

C15H 31 O

C15H 31 O C 15 H31 O

OH

HO O

O S

O

O

C15H 31 C15H 31 O

NH

C15H 31

O O

g

N

NN

94

O

C15H 31

O

O

HO

C 15 H31 O C 15 H31 O

OH

HO

O S

OH OH HO HO

O

O

NH O

HO

f

O NH

OH OH OH O N3

O O

e

C15H 31 NN N

O O

d

NH H N

Yield

N3

O O

c

NH H N O C15H 31

O C15H 31 C15H 31 O

Triazole

96

O O O

C15H 31 O C15H 31

94

O O O

C 15 H31 O C15H 31 O

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97

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toxicity after 48h of incubation. Most of the analogs were non toxic wherein all the analogs retained about 80% viability at higher concentration (Fig. 6). 2.3.2. Hemolytic assay Different concentration of test compounds were tested with fixed volume concentration of rat RBCs. The vaccine adjuvants showed <15% hemolysis even at the maximum concentration (1000 lg) and no appreciable hemolysis observed at the least concentrations in any of the analogs (Fig. 7). 2.4. Structure activity relationship

Figure 2. (a) OVA-specific IgG antibodies in the treated mice sera. (b) Total IgG1 and IgG2a Isotype measurement from the treated mice sera. BALB/c mice were immunized sc with OVA (100 lg) alone or in combination with analogues (20 lg). Sera were collected 2 weeks after the last immunization and measured for OVA specific IgG and total IgG1 and IgG2a isotypes of IgG.

studies. A booster dose was given on 14th day and sacrificed on 28th day. OVA-specific IgG antibodies in the sera were measured compared to the gold standard Pam3CSK4, which significantly enhanced the IgG titers by 16 fold specific to OVA. But 6e showed a significant 8 fold enhanced titer in comparison to that of OVA injected mice and 6h with 2 fold increase (Fig. 2a). Isotype analysis (IgG1 and IgG2a) of antibody titers were measured to evidence Th1 or Th2 biased activity of the leads. 6e increased IgG2a as similar to Pam3CSK4 indicating Th1 biased and 6h showed IgG1 with Th2 biased response (Fig. 2b). To evaluate the cytokine influence in cell mediated immunity, serum samples were measured for IL-2, IL-4, IL-6, IL-10, IL-12, TNF-a and IFN-c. The analogue 6h has given higher IL-6 and TNF-a suggesting a path to both innate and adaptive immunity. They have given significantly lower IL-10 production than 6e which is vice versa in IL-6 production (Fig. 3). In terms of T cell stimulation CD8 splenic T cell response was dominant than CD4 T cells for 6e and 6h (Fig. 4). Human TLR-2 reporter gene assays (NF-jB induction): Compound Pam3CSK4 for TLR agonist activity using HEK-Blue™ hTLR2 cell line (InvivoGen, USA) designed for studying the stimulation of human hTLR2 by inducing the activation of NF-kB. We performed similar experiment for our selective leads (Fig. 5). Both these compounds exhibited hTLR2 agonistic activity even though Pam3CSK4 was thus found to be better activator of NFkb as revealed by the SEAP (secreted embryonic alkaline phosphatase) activity compared that of 6e and 6h. 2.3. Safety studies 2.3.1. In vitro cytotoxicity evaluation by MTT assay Rat kidney cell line NRK-49F was exposed to varying doses of test compounds in the range of 10–100 lM, to determine the

Several carbohydrate and related hydrophilic polyhydroxyl structures were tethered to Pam3Cys lipid head group through a triazolyl linker. The ensuing conjugates bear hydrophilic carbohydrate entity and an ionizable 1,2,3-triazolyl entity as replacement of poly-cationic peptide group present in Pam3CSK4 and similar lipopeptides. Compounds derived from glycerol, various pentose and hexose sugars and disaccharides were studied for their Th1 activation and most of the compounds exhibited appreciable Th1 activation ex vivo, as compared to Pam3CSK4. Galactosyl analogues shown highest IL-2 and IL-12 and IFN-c response followed by ribose and maltose derived hybrids. The Th1 cytokine expression was several folds higher than the standard adjuvant Pam3CSK4. b-Galactosyl analogue (6h) exhibited higher IgG titre as compared to its ribose counterpart (6e). Both these conjugates expressed pronounced Th1 activation in vivo as revealed by their cytokine expressions. Both galactose and ribose analogues expressed higher populations of CD4 and CD8 surface markers indicating activation of T-helper cells and cytotoxic T cells. The overall activity emerged in this study clearly demonstrate that peptide can be replaced with a suitable carbohydrate based hydrophilic entity appended with a triazole moiety. Further, we found that b-galactosyl derivative was found to be more active than the a-galactosyl form indicating the influence of stereochemistry of the sugar linkage on the ensuing immunogenicity. In the hemolysis studies, compounds derived from mannitol and maltose (6d and 6k) showed the hemolytic activity up to 15%, where as rest of the compounds were found to be least hemolytic even at higher dose levels of 1000 lg/ml. Both compounds exhibited SEAP activity revealing their TLR2 agaonistic traits. Most of the compounds exhibited least toxicity on normal rat kidney cell lines even at 100lM concentration level. 3. Conclusion To sum up, we have presented here the synthesis of novel 1,2,3triazole tethered carbohydrate–Pam3Cys conjugates, by employing click chemistry protocol. A focused library of 12 compounds prepared by structural variation of sugar azides. The novel carbohydrate–Pam3Cys hybrid conjugates, have been subjected to immunological evaluation and compounds 6e and 6h exhibited good IgG antibody response against ovalbumin in Balb/c mice and compound 6e shown 8 fold increase in antibody titer as compared to conventional alum adjuvant. Even though peptide free adjuvant compound 6e shown almost half the antibody response as compared to standard Pam3aCysSK4 adjuvant, the quality of the derived immune response was far superior in terms of their Th1 cytokine expression both ex vivo and in vivo. Furthermore triazolyl hybrids exhibited an excellent quality of antigen recognition through antigen specific activation affirmed by CD8+ cell indicating their possible application as vaccine adjuvants. Triazolyl Pam3Cys analogs were found to be the hTLR2 agonists as evidenced by their SEAP activity.

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Figure 3. Serum Cytokine estimation. BALB/c mice were immunized sc with OVA (100 lg) alone or in combination with analogues (20 lg). Cytokines in the sera (50 lL) were measured. The values are presented as mean ± SD (n = 3). ⁄⁄⁄p 6 0.0005, ⁄⁄p 6 0.005, ⁄p 6 0.05 compared to (Pam3) Pam3CSK4.

4. Experimental 4.1. General synthetic materials and methods All commercially available reagents were used as received. Air and moisture-sensitive reaction were performed under nitrogen atmosphere. The progress of all reaction was monitored by TLC on a 2–5 cm precoated silica gel 60 F254 plates of thickness 0.25 mm (Merck). Optical rotations were recorded using a Perkin–Elmer 241polarimeter. IR spectra were recorded on Bruker Vector 22 instrument. NMR spectra were recorded on a Bruker DPX 300 instrument in CDCl3 and DMSO-d6 with (CH3)4Si is an internal standard for 1H NMR spectra and solvent signals as internal standard for 13C NMR spectra. 1HNMR chemical shifts and coupling constants J are given on ppm (relative to (CH3)4Si and Hz, respectively. Mass spectra were recorded on ESIMS (Shimadzu) instrument and mass-spectrometric (MS) data are reported in m/z.

4.1.1. Preparation of (S)-2-oxo-2-phenylethyl-3-((R)-1,4-dioxaspiro[4,5]decan-2-ylmethylthio)-2-(tert-butoxycarbonlyamino) propanoate (2) N-(tert-Butoxycarbonyl)-L-cysteine methylester (3 g, 12.76 mmol) and iodo compound (Scheme 1, 1) (4.31 g, 15.31 mmol) and was dissolved in Dry DMF (30 mL) with stirring. To The resulting solution DIPEA (4.43 mL, 25.52 mmol) was added slowly at room temperature. The resulting mixture was stirred for 2 h at room temperature, followed by addition of ice cold water and the aqueous layer was extracted with EtOAc (2  50 mL). The organic layer washed with 1 N HCl (2  50 mL), then organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo and the resulting residue used for next step without further purification. Thus obtained methyl ester compound (4.8 g, 12.33 mmol) dissolved in THF/H2O (40 mL, 4:1) then LiOHH2O (0.3 g, 12.33 mmol) was added stirred for 30 min.TLC checked solvent removed by rotavacc, with 10% HCl neutralize the reaction mixture then extracted with EtOAc (2  50 mL). The organic layer

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Figure 4. T cell stimulation by cell surface markers. BALB/c mice were immunized sc with OVA (100 lg) alone or in combination with analogues (20 lg). Staining of spleen cells (3  105cells/FACS tubes) with T cell surface marker CD4 and CD8. Data represented as Mean Fluorescent Intensity.

washed with NaHCO3 (2  50 mL), Then organic layers was dried over anhydrous sodium sulfate and concentrated in vacuo and the resulting residue used for next step without further purification. The obtained acid (3.7 g, 9.86 mmol) and 2-bromo acetophenone (2.95 g, 14.8 mmol) were dissolved in dry DMF with stirring. KF (1.2 g, 19.72 mmol) added to the solution and stirred at room temperature for 2.5 h. Ice cold water was added to the reaction mixture and stirred for 30 min, then extracted with EtOAc (2  100 mL). The organic layers dried over anhydrous sodium sulfate and evaporated under reduced pressure to afford the crude product, which was subjected to column chromatography in 20% EtOAc–hexane. Yellow solid (3.85 g, 80%). IR (KBr, cm1): 2918, 2850, 2310, 1729, 1663, 1551, 1466, 1219, 1161, 1069, 772, 722, 688; 1H NMR (300 MHz, CDCl3): d 1.46 (s, 9H), 1.53–1.67 (m, 10H), 2.68–2.73 (dd, J = 6.4, 6.5 Hz, 1H), 2.84–2.89 (dd, J = 5.9,13.5 Hz, 1H), 3.14–3.26 (ddd, J = 4.5, 6.4, 6.5 Hz, 2H), 3.7– 3.7 (dd, J = 6.4, 8.2 Hz, 1H) 4.08–4.12 (dd, J = 6.1, 8.2 Hz, 1H), 4.25.4.31 Hz (m, 1H), 4.66–4.71 (m, 1H), 5.32–5.37 (d, J = 16.3 Hz, 1H), 5.47–5.53 (t, J = 8.5 Hz, 2H) 7.48–7.52 (t, J = 7.6 Hz, 2H), 7.60–7.64 (t, J = 7.3 Hz, 1H), 7.89–7.92 (d, J = 9.0 Hz, 2H). 13C NMR (75 MHz, CDCl3): d 23.7, 23.9, 25.08, 28.2, 34.9, 35.8, 36.4, 53.6, 66.5, 68.3, 75.3, 80.1, 110.2, 127.7, 128.8, 133.8, 134.03, 170.6, 191.1. MS (ESI): m/z 516 [M+Na]+. 4.1.2. Preparation of (R)-3-((S)-2-(tert-butoxycarbonylamino)3-oxo-3-(2-oxo-2-phenylethoxy)propylthio)propane-1,2-diyldipalmitate (3) Compound 2 (3.6 g,7.3 mmol) was dissolved in 70% AcOH:H2O, then stirred at room temperature for 8 h until the complete consumption of starting material as revealed by TLC examination.

Figure 5. Human TLR-2 reporter gene assays (NF-jB induction). HEK-Blue™ hTLR2 cells were stimulated with agonists: Pam3CSK4, 6e and 6h (10 lg/ml). After 24 h incubation NF-kB-induced SEAP activity was assessed using QUANTI-Blue™ reading at OD at 655 nm.

The excess solvent evaporated under reduced pressure and the resulting residue was co-evaporated with hexane. Residue obtained was used in next step without further purification. The residue (3.01g,7.28 mmol) dissolved in dry THF to that palmitic acid (5.59 g, 21.8 mmol), DIC (3.6 g, 27.9 mmol), DMAP (0.35 g, 2.9 mmol), were added respectively, then the mixture was stirred for 2h at room temperature after which glacial acetic acid (0.9 mL) added. The mixture was concentrated under reducing pressure, residue re-crystallized from CH2Cl2/CH3OH (1:20 v/v) at 20°C to give 8 (6 g, 91%). Light yellow solid, IR (KBr, cm1): 2918, 2850, 2310, 1729.7, 1663, 1551, 1466, 1219, 1161, 1069, 772, 722, 688. 1H NMR (300 MHz, CDCl3): d 0.88 (t, J = 6.2 Hz, 6H), 1.17–1.35 (m, 48H), 1.46 (s, 9H), 1.56–1.65 (m, 6H), 2.26– 2.36 (q, J = 6.9, 7.1 Hz, 4H), 2.8 (d, J = 6.2 Hz, 2H), 3.09 (dd, J = 6.4,13.9 Hz, 1H), 3.26 (dd, J = 4.1, 4.3 Hz, 1H), 4.19 (dd, J = 5.8, 6.0 Hz, 1H), 4.36 (dd, J = 3.3, 3.5 Hz, 1H), 4.63–4.73 (m, 1H), 5.15– 5.24 (m, 1H), 5.3–5.56 (m, 3H), 7.50 (t, J = 7.5 Hz, 2H) 7.59–7.66 (t, J = 7.3 Hz, 1H), 7.91 (d, J = 7.3 Hz, 2H). 13C NMR (75 MHz, CDCl3): d 13.9, 22.5, 24.7, 24.7, 31.7, 32.4, 32.7, 33.4, 33.9, 34.18, 45.7, 52.1, 52.3, 63.4, 69.9, 116.5, 127.7, 128.8, 133.8, 134.03, 171.9, 173.6, 173.7, 173.8. MS (ESI): m/z 912 [M+Na]+. 4.1.3. Preparation of (R)-3-((S)-3-oxo-3-(2-oxo-2-phenylethoxy)-2-palmitamidopropylthio)propane-1,2-diyldipalmitate Compound 3 (6 g, 6.74 mmol) was dissolved in 20% TFA: CH2Cl2, stirred at room temperature for 30 min, after completion of starting material excess solvent was evaporated by rota vacuo. Residue co-evaporated with hexane, then subjected to azeotropic evaporation with toluene and the residue obtained was used in the next step. Resulting amine compound (5.32 g, 6.7 mmol) was dissolved in dry CH2Cl2 and the solution was added to the palmitic acid (2.05 g, 8.04 mmol), DIC (1.03 g, 8.04 mmol), HOBt (1.23 g, 8.04 mmol) in dry CH2Cl2, which was stirred at room temperature for 8 h, after which diluted with CH2Cl2, filtered washed with water (2  50 mL), satd NaHCO3 (2  50 mL). The pooled up aqueous layers extracted CH2Cl2 (2  20 mL) the combined organic layers dried over anhydrous Na2SO4, evaporated by rota vacuo. The residue was recrystalised from CHCl3 at 0 °C to give light yellow solid (4.9 g, 70%). IR (KBr, cm1): 2919, 2850, 2310, 1738, 1677, 1550, 1465, 1377, 1218, 1174, 1112, 1050, 772, 722, 688. 1H NMR (300 MHz, CDCl3): d 0.88 (t, J = 6.2 Hz, 9H), 1.17–1.35 (m, 72H), 1.56–1.65 (m, 6H), 2.26–2.36 (q, J = 6.9,7.1 Hz, 6H), 2.8 (d, J = 6.2 Hz, 2H), 3.09 (dd, J = 6.4,13.9 Hz, 1H), 3.26 (dd, J = 4.1, 4.3 Hz, 1H), 4.19 (dd, J = 5.8,6.0 Hz, 1H), 4.36 (dd, J = 3.3,3.5 Hz, 1H), 4.63–4.73 (m, 1H), 5.15–5.24 (m, 1H), 5.3–5.56 (m, 3H), 7.50 (t, J = 7.5 Hz, 2H) 7.59–7.66 (t, J = 7.3 Hz, 1H), 7.91 (d, J = 7.3 Hz, 2H). 13C NMR: (75 MHz, CDCl3): 13.9, 22.5, 24.7, 24.7, 31.7, 32.4, 32.7, 33.4,

Figure 6. Cytotoxic effect of triazolyl Pam3Cys–carbohydrate conjugates on NRK49F cell line. NRK-49F cells (1  104 cells) were incubated for 24 h in 96 well plate and then treated with triazolyl Pam3Cys–carbohydrate conjugates (0–100 lM) for 48 h. The percentage of viable cell proliferation was measured by the MTT method as described in the text, and shown as a OD630 nm. Values are mean ± SEM of triplicate samples.

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(0.785 g, 5.1 mmol) were added. The reaction mixture stirred for 10 min at room temperature then propargyl amine (0.235 g, 4.2 mmol), DIPEA (0.828 g, 6.4 mmol) were added at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred for 8 h, diluted with CH2Cl2 (50 mL). The organic layer washed with water (2  50 mL) and satd NaHCO3 (2  50 mL), dried over Na2SO4, then purified by column chromatography (20% EtOAc/hexane) to afford (2.63 g, 65%) as white solid, mp 81 °C; IR (KBr, cm1): 2919, 2850, 2310, 1738, 1677, 1550, 1465, 1377, 1218, 1174, 1112, 1050, 772, 722, 688.1H NMR (300 MHz, CDCl3): d 0.88 (t, J = 6.2 Hz, 9H), 1.17–1.37 (m, 72H), 1.54–1.64 (m, 6H), 2.27 (t, J = 2.6 Hz, 1H), 2.3–2.4 (m, 6H), 2.84 (dd, J = 5.2, 7.9 Hz, 2H), 2.93 (dd, J = 2.8, 4.9 Hz, 2H), 4.09 (dd, J = 2.4, 5.2 Hz, 2H), 4.2 (dd, J = 5.8, 6.0 Hz, 1H), 4.28 (dd, J = 5.8, 12.1 Hz, 1H), 4.62 (dd, J = 7.6, 13.2 Hz, 1H), 5.28–5.37 (m, 1H), 7.0 (t, J = 3.7 Hz, 1H), 7.5 (t, J = 6.7 Hz, 1H). 13C NMR (75 MHz, CDCl3): 14.1, 22.6, 24.7, 24.8, 24.8, 22.0, 29.1, 29.1, 29.2, 29.2, 29.3, 29.4, 29.4, 29.5, 29.6, 29.6, 31.9, 33.8, 34.1, 34.3, 34.3, 35.4, 53.1, 63.6, 70.5, 72.1, 78.3, 168.2, 173.5, 174.1, 178.4. MS (ESI): m/z 970 [M+Na]+.

Figure 7. Hemolytic assay of Triazolyl Pam3Cys–carbohydrate conjugates. Hemolytic percent of distilled water is used as maximal hemolytic controls. n = 3 tests. Mean ± SD.

33.9, 34.1, 45.7, 52.1, 52.3, 63.4, 69.9, 116.5, 127.7, 128.8, 133.8, 134.2, 171.9, 173.6, 173.7, 173.8. MS (ESI): m/z 1050 [M+Na]+. 4.1.4. Preparation of (S)-3-((R)-2,3-bis(palmioyloxy)propylthio)2-palmitamidopropanoic acid (4) To a stirred solution of compound 3 (4.9 g, 4.7 mmol) in acetic acid (70 mL) at room temperature was added activated Zn (4.95 g,76.2 mmol) and stirred the mixture for 2 h at room temperature. After complete consumption of the starting material, the reaction mass was filtered through celite and the filtrate was evaporated under reduced pressure to afford crude product. Purification of the crude product by column chromatography, using pure EtOAc as eluent (Rf = 0.5) gave (3.89 g, 90%) as white solid, mp 62–64 °C (lit 66 °C). IR (KBr, cm1): 2919, 2850, 2310, 1738, 1677, 1550, 1465, 1377, 1218, 1174, 1112, 1050, 772, 722, 688.1H NMR (300 MHz, CDCl3): d 0.9 (t, J = 6.0 Hz, 9H), 1.17–1.35 (m, 72H), 1.56–1.65 (m, 6H), 2.26–2.36 (q, J = 6.7, 7.5 Hz, 6H), 2.8 (d, J = 6.0 Hz, 2H), 3.09 (dd, J = 6.4,13.8 Hz, 1H), 3.26 (dd, J = 4.1, 4.3 Hz, 1H), 4.19 (dd, J = 5.8, 6.0 Hz, 1H), 4.36 (dd, J = 3.3,3.5 Hz, 1H), 4.63–4.73 (m, 1H), 5.09–5.18 (m, 1H), 7.43 (d, J = 7.5 Hz, 1H). 13C NMR (75 MHz, CDCl3): 13.9, 22.5, 24.7, 24.7, 31.7, 32.4, 32.7, 33.4, 33.9, 34.1, 45.7, 52.1, 52.3, 63.4, 69.9, 116.5, 171.9, 173.6, 173.7, 173.8. MS (ESI): m/z 932 [M+Na]+.

4.2. General procedure for preparation of Pam3Cys based sugar triazoles [6(a–l)] Pam3Cys alkyne (Scheme 1, 5) (0.1 g, 0.01 mmol or 1 equiv) was stirred in tertiary butanol and water (1:1 mixture, 5 mL) to which copper sulfate (0.042 mmol) and sodium ascorbate (0.042 mmol) were added and the reaction mixture was stirred at room temperature, after 15 min, sugar azide (Scheme 2, a–l) (0.021 g, 0.01 mmol or 1 equiv) was added and the mixture was allowed to stir for 8 h at room temperature. Tertiary butanol was evaporated under reduced pressure, diluted with CHCl3 and water. The organic layer was separated and aqueous layer extracted with CHCl3 (2  100 ml). The combined organic layers were dried over Na2SO4 and evaporated under reduced pressure to afford the crude product which was purified by column chromatography (9:1 CHCl3/CH3OH) to afford pure product. 4.2.1. (R)-3-((S)-3-((1-((R)-2,3-Dihydroxypropyl)-1H-1,2,3-triazol-4-yl)methylamino)-3-oxo-2-palmitamidopropylthio)propane-1,2-diyldipalmitate (6a) 1 White solid, mp 70 °C; [a]20 ): D +3 (c 1.0 CHCl3), IR (KBr, cm 3744, 3609, 2919, 2850, 2310, 1738, 1677, 1550, 1465, 1377, 1218, 1174, 1112, 1050, 772,722, 668. 1H NMR (300 MHz, DMSOd6): d 0.84 (t, J = 5.0 Hz, 9H), 1.13–1.36 (m, 72H), 1.39–1.56 (m, 6H), 2.24 (t, J = 6.9 Hz, 6H), 2.63–2.84 (m, 3H), 2.97 (dd,

4.1.5. Preparation of (R)-3-((S)-3-oxo-2-palmitamido-3-(prop-2ynylamino)propylthio)propane-1,2-diyldipalmitate (5) Compound 4 (3.89 g,4.2 mmol) was dissolved in dry CH2Cl2 under N2 atmosphere to which EDCI (0.98 g, 5.1 mmol) and HOBt

O

D-Mannitol

O

I

a

O

O

S

NHBoc O

O b

O

C 15 H 31 C 15 H31 O

2

1

O O

NHBoc O

S

O

C 15 H31

O

C 15H 31

c

O

O

O

O 3

4 C 15 H31 C 15H 31

O O

O

O S

HN

d

O OH O

C 15 H 31

HN

5

O S

C 15 H 31

O NH O

Scheme 1. Synthesis of propargyl amido Pam3Cys. Reagents and conditions: (a) (i) N-(tert-Butoxycarbonyl)-L-cysteine methyl ester, DIPEA, DRYDMF 0 °C–rt, 2 h, 80%; (ii) (i) LiOH.H2O 0 °C–rt, 30 m, 80%; (iii) phenasyl bromide, KF, DRY DMF, 2 h, rt, 80%. (b) (i) AcOH/H2O, (70:30), 8 h, (ii) palmitic acid, DIC, DMAP, DRY, THF, rt, 2h, 90%. (c) (i) 20% TFA/DCM, rt, 30 m, 98%, (ii) Palmticacid, DIC, HOBt, Dry DCM, rt, 6 h, 70%. (iii) Zn:AcOH, rt, 2.5 h, 90%. (d) Propargyl amine, EDCI, HOBt, DIPEA, 12 h, 0 °C–rt, 65%.

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O C15H31 C15H31

C15H31

O O C15H31 NH

O

HO

OH

O

HO h

d HO

HO

O OH

g

OH

O HO HO

NH

O

S

HO

OH

O

O O

6 (a-l)

HO

O

C15H31

O

HO

c

OH

O NH

N

O

HO HO

OH

b

OH

OH

HO

HO

a

OH

O

O

OH HO

OH

OH

N N

R

2) CuSO4 5H2O Sodium ascorbate, tBuOH:H2O(1:1)

O

5

R=

HO HO

1) RN3

H N

S

O

C15H31

OH

HO

i

OH

HO

OH O

OH

HO O HO HO

HO

O HO HO

j

k

OH e

HO

f

OH

OH OH OH OH O O O O HO HO HO HO l

Scheme 2. Cu catalyzed click chemistry approach to triazole tethered carbohydrate analogues of Pam3Cys.

J = 7.5,4.9 Hz, 1H), 3.7 (dd, J = 7.5, 4.9 Hz, 1H), 3.99–4.15 (m, 3H), 4.23–4.59 (m, 8H), 5.1 (m, 1H), 7.86 (s, 1H), 8.81 (dd, J = 5.6, 5.8 Hz, 1H), 9.69 (d, J = 8.1 Hz, 1H). 13C NMR: (75 MHz, DMSOd6): 13.6, 14.1, 20.6, 22.1, 23.3, 23.5, 24.3, 24.5, 28.44, 28.5, 28.7, 29.1, 31.3, 33.2, 33.3, 33.5, 34.3, 35.8, 51.8, 59.6, 65.1, 65.5, 73.5, 79.1, 109.5, 123.5, 144.2, 168.3, 168.4, 170.2, 171.9, 172.1. MS (ESI): m/z 1087 [M+Na]+. 4.2.2. (R)-3-((S)-3-Oxo-2-palmitamido-3-(1-(b-D-glucopyranosyl)1H-1,2,3-triazol-4-ylamino)propylthio)propane-1,2-diyldipalmitate (6b) White solid, mp 120 °C; [a]D 20 +4 (c, 1.0, CHCl3); IR (KBr, cm1): 3744, 2920, 2851, 2310, 1710, 1550, 1531, 1515, 1219, 722,671. 1H NMR (300 MHz, DMSO-d6): d 0.82 (t, J = 6.7 Hz, 9H), 1.09–1.3 (m, 72H), 1.4–1.53 (m,6H), 2.22 (t, J = 7.1 Hz, 6H), 2.61–2.85 (m, 3H), 2.97–3.06 (dd, J = 3.5,13.4 Hz, 1H), 3.6–3.76 (m, 2H), 4.06 (dd, J = 6.6, 6.7 Hz, 1H), 4.19–4.33 (m, 2H), 4.38 (dd, J = 5.4, 5.2 Hz, 1H), 4.44–4.54 (m, 1H), 4.62 (t, J = 5.4 Hz, 1H), 5.08 (m, 1H), 5.14 (d, J = 5.4 Hz, 1H), 5.27 (d, J = 4.9 Hz, 1H), 5.34 (d, J = 6.1 Hz, 1H), 5.48 (d, J = 9.2 Hz, 1H), 8.06 (s, 1H), 8.78 (t, J = 5.4 Hz, 1H), 9.66 (d, J = 8.1 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): 13.1, 22.1, 24.3, 24.3, 28.3, 28.3, 28.6, 29.1, 31.2, 33.1, 33.3, 33.4, 34.3, 52.5, 60.6, 69.6, 71.9, 76.9, 79.8, 87.3, 121.9, 144.1, 168.4, 172.1, 172.1, 172.3. MS (ESI): m/z 1175 [M+Na]+. 4.2.3. (2R)-3-((2S)-3-((6-(6-Deoxy-D-glucofuranoside)-1H-1,2,3triazol-4-yl)ylamino)-3-oxo-2-palmitamidopropylthio)propane1,2-diyldipalmitate (6c) 1 White solid, mp 128 °C; [a]20 ): D +17 (c, 1.0, CHCl3); IR (KBr, cm 3745, 2918, 2850, 2310, 1729, 1663, 1551, 1466, 1219, 1161, 1069, 772, 722, 689. 1H NMR (300 MHz, DMSO-d6): 0.83 (t, J = 5.6 Hz, 9H), 1.1–1.34 (m, 72H), 1.38–1.53 (m, 6H), 2.23 (t, J = 6.8 Hz, 6H), 2.62–2.84 (m, 3H), 2.9 (dd, J = 3.1, 4.1 Hz, 1H), 3.47–3.67 (m, 2H), 3.82 (m, 1H), 4.07 (q, J = 6.9 Hz, 1H), 4.22–4.11 (m, 5H), 4.62 (d, J = 6.5 Hz, 1H), 4.7–4.8 (dd, J = 15.4, 3.9 Hz, 1H), 4.83–4.85 (m, 1H), 5.08 (m, 2H), 6.2 (d, J = 4.7 Hz, 1H), 6.59 (d, J = 6.7 Hz, 1H), 7.86 (s, 1H), 8.7 (m, 2H). 13C NMR; (75 MHz, DMSO-d6): 13.7, 22.1, 24.3, 24.4, 28.4, 28.4, 28.7, 29.1, 31.3, 33.1, 33.3, 33.5, 34.4, 52.3, 52.9, 60.3,63.3, 63.8, 68.3, 69.2, 69.4, 69.8, 73.6, 78.3, 87.9, 121.7, 144.1, 168.4, 168.4, 172.3, 172.5. MS (ESI): m/z 1175 [M+Na]+.

4.2.4. (R)-3-((S)-3-Oxo-2-palmitamido-3-((1-(1-deoxy-D-mannitol)-1H-1,2,3-triazol-4-yl)-ylamino)propylthio)propane-1,2-diyldipalmitate (6d) 1 White solid, mp 124 °C; [a]20 ): D +9 (c, 1.0, CHCl3); IR (KBr, cm 3019, 2921, 2852, 1727, 1661, 1550, 1531, 1464, 1215, 928, 770, 667. 1H NMR (300 MHz, DMSO-d6): 0.84 (t, J = 6.7, Hz, 9H), 1.17– 1.28 (m,72H), 1.44–1.53 (dd, J = 5.7, 6.7 Hz, 6H), 2.25 (t, J = 7.3 Hz, 6H), 2.72 (dd, J = 5.3,7.7 Hz, 1H), 2.8 (ddd, J = 3.1,5.1,6.7 Hz, 2H), 3.02 (ddd, J = 3.3, 4.2, 4.4 Hz, 1H), 3.43– 3.57 (m, 3H), 4.04–4.11 (m,1H), 4.28 (dd, J = 5.1,9.4 Hz, 2H), 4.34 (dd, J = 5.4, 12.4 Hz, 1H), 4.4 (dd, J = 5.7, 13.4 Hz, 1H), 4.5 (m,1H), 4.65 (dd, J = 3.8, 8.7 Hz, 1H), 4.69 (t, J = 5.6 Hz, 1H), 5.02 (d, J = 5.3 Hz, 1H), 5.08 (m, 1H), 5.45 (d, J = 9.1 Hz, 1H), 8.04 (s, 1H), 8.78 (dd, J = 5.3,5.4 Hz, 1H), 9.68 (dd, J = 7.4,7.7 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): 13.7, 22.1, 24.3, 24.4, 28.4, 28.4, 28.7, 29.1, 31.3, 33.1, 33.3, 33.5, 34.4, 52.5, 52.9, 60.3, 63.3, 63.3, 68.3, 69.2, 69.4, 69.8, 73.6, 78.3, 87.9, 121.7, 144.1, 168.4, 172.1, 172.2.MS (ESI): m/z 1177 [M+Na]+. 4.2.5. (2R)-3-((2S)-3-((1-(b-D-Ribofuranosyl)-1H-1,2,3-triazol-4yl)ylamino)-3-oxo-2-palmitamidopropylthio)propane-1,2-diyldipalmitate (6e) 1 White solid, mp 132 °C; [a]20 ): D +12 (c, 1.0, CHCl3); IR (KBr, cm 2920, 2851, 1726, 1550, 1463, 1418, 1219, 1161, 1053, 772, 722, 614. 1H NMR (300 MHz, DMSO-d6): 0.88 (t, J = 6.2 Hz, 9H),1.15– 1.34 (m, 72H), 1.47–1.57 (m, 6H), 2.18–2.33 (t, J = 7.1 Hz, 6H), 2.7–2.88 (m, 2H), 2.91–3.23 (m, 2H), 3.99 (dd, J = 8.0, 8.2 Hz, 1H), 4.13 (dd, J = 6.1, 6.8 Hz, 1H), 4.25–4.44 (m, 4H), 4.53 (br, 1H), 4.59–4.72 (m, 2H), 4.83–4.92 (dd, J = 4.7, 3.6 Hz, 1H), 4.96–5.06 (dd, J = 4.7 Hz, 1H), 5.13 (br, 1H), 5.28–5.38 (dd, J = 5.3, 5.4 Hz, 1H), 6.39 (d, J = 4.7 Hz, 1H), 6.74 (d, J = 6.5 Hz, 1H), 7.84 (s, 1H), 7.89 (s, 1H), 8.8 (dd, J = 4.8,5.1 Hz, 1H), 9.7 (s,1H). 13C NMR (75 MHz, DMSO-d6): 13.1, 22.2, 24.3, 24.3, 28.3, 28.3, 28,6, 29.0, 31.2, 33.1,33.3, 33.4, 34.3, 52.5, 60.6, 69.5, 71.7, 75.9, 79.1, 87.8, 121.9, 144.0, 168.4, 172.0, 172.1, 172.3. MS (ESI): m/z 1145 [M+Na]+. 4.2.6. (2R)-3-((2S)-3-((1-(a-D-Xylofuranosyl)-1H-1,2,3-triazol-4yl)ylamino)-3-oxo-2-palmitamidopropylthio)propane-1,2diyldipalmitate (6f) 1 White solid, mp 142 °C; [a]20 ): D +6 (c, 1.0, CHCl3); IR (KBr, cm 3744, 3610, 2921, 2851, 2822, 2310, 1692, 1550, 1515, 1219, 772,

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671, 612. 1H NMR (300 MHz, DMSO-d6): 0.82 (t, J = 6.1 Hz, 9H), 1.09–1.29 (m, 72H), 1.39–1.53 (m, 6 H),2.18–2.27 (m, 6H), 2.62– 2.84 (m, 3H), 3.0 (dd, J = 3.5, 3.9 Hz, 1H), 3.74 (dd, J = 6.1,7.3 Hz, 1H), 4.06 (dd, J = 6.7,6.9 Hz, 1H), 4.16–4.4 (m, 7H), 4.47 (dd, J = 5.1,7.3 Hz, 3H), 4.65 (dd, J = 1.8, 2.1 Hz, 1H), 4.99 (d, J = 6.7 Hz, 1H), 5.08 (br, 1H), 7.81 (s, 1H), 8.76 (dd, J = 5.1, 5.4 Hz, 1H), 9.65 (d, J = 8.3 Hz, 1H). 13CNMR (75 MHz, DMSO-d6): 13.8, 22.0, 24.3, 24.3, 28.3, 28.3, 28.6, 28.9, 31.2, 33.1, 33.3, 33.4, 34.4, 52.6, 53.5, 63.3, 63.7, 69.2, 69.6, 70.4, 71.0, 123.6, 143.6, 168.3, 168.5, 172.1, 172.3. MS (ESI): m/z 1145 [M+Na]+. 4.2.7. (2R)-3-((2S)-3-((1-(b-D-Mannopyranosyl)-1H-1,2,3-triazol4-yl)ylamino)-3-oxo-2-palmitamidopropylthio)propane-1,2diyldipalmitate (6g) 1 White solid, mp 178 °C; [a]20 ): D 2 (c,1.0,CHCl3); IR (KBr, cm 2955, 2920, 2851, 1695, 1680, 1551, 1518, 1460, 1219, 772. 1H NMR (300 MHz, DMSO-d6): 0.84 (t, J = 6.4 Hz, 9H), 1.12–1.32 (m, 72H),1.44–1.54 (m, 6H), 2.25 (t, J = 6.4 Hz, 6H), 2.66–2.86 (m, 3H), 2.98–3.06 (dd, J = 4.2, 4.5 Hz, 1H), 3.69–3.84 (m, 2H), 4.09 (m, 1H), 4.23–4.4 (m, 4H), 4.5 (m, 1H), 5.09 (s, 1H), 5.45 (d, J = 9.1 Hz, 1H), 8.05 (s, 1H), 8.81 (dd, J = 4.4, 4.8 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): 13.8, 22.0, 24.3, 24.4, 28.4, 28.4, 28.7, 29.1, 31.3, 33.0, 33.3, 33.5, 34.3,63.2, 63.3, 68.2, 69.0, 69.6, 69.8, 71.9, 77.0, 87.9, 121.9, 141.1, 168.4, 168.4, 172.0, 172.3. MS (ESI): m/z 1175 [M+Na]+. 4.2.8. (2R)-3-((2S)-3-((1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol4-yl)ylamino)-3-oxo-2-palmitamidopropylthio)propane-1,2-diyldipalmitate (6h) White solid, mp 195 °C; [a]20 D +8 (c, 1.0, CHCl3); IR (KBr, cm1):2921, 2851, 1695, 1713, 1658, 1550, 1517, 1462, 1219, 772. 1H NMR (300 MHz, DMSO-d6): 0.84 (t, J = 6.2 Hz, 9H), 1.11– 1.35 (m, 72H), 1.4–1.55 (m, 6H), 2.25 (t, J = 6.7 Hz, 6H), 2.64–2.86 (m, 3H), 2.95–3.08 (m, 1H), 3.66–3.85 (m, 2H), 4.04–4.13 (m, 1H), 4.22–4.56 (m, 3H), 5.09 (br, 1H), 5.2 (d, J = 4.7 Hz, 1H), 5.33 (d, J = 4.5 Hz, 1H), 5.42 (dd, J = 5.8, 9.2 Hz, 1H), 8.06 (s, 1H), 9.69 (d, J = 7.7 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): 13.8, 22.0, 24.4, 24.4, 28.4, 28.4, 28.7, 29.1, 31.3, 33.0, 33.3, 33.5, 34.3, 63.2, 63.3, 68.2, 69.0, 69.6, 69.8, 71.9, 73.0, 93.9, 121.9, 141.1, 168.4, 168.4, 172.6, 172.3. MS (ESI): m/z 1175 [M+Na]+. 4.2.9. (2R)-3-((2S)-3-((1-(a-D-Galactopyranosyl)-1H-1,2,3-triazol4-yl)ylamino)-3-oxo-2-palmitamidopropylthio)propane-1,2-diyldipalmitate (6i) 1 White solid, mp 165 °C; [a]20 ): D +12 (c,1.0, CHCl3); IR (KBr, cm 2920, 2851, 1693, 1661, 1629, 1515, 1550, 1464, 1219, 772.1H NMR (300 MHz, DMSO-d6): 0.85 (t, J = 6.4 Hz, 9H), 1.16–1.31 (m, 72H),1.44–1.54 (m, 6H), 2.25 (t, J = 7.1 Hz, 6H), 2.66–2.76 (m, 1H), 2.81 (dd, J = 3.8, 5.43 Hz, 2H), 3.01 (dd, J = 3.9, 4.2 Hz, 1H), 3.52–3.61 (m, 2H), 4.27 (d, J = 11.7 Hz, 1H), 4.39 (dd, J = 5.3, 5.6 Hz, 3H), 4.5 (br, 1H), 4.59 (t, J = 5.6 Hz, 1H), 5.01 (dd, J = 5.1, 5.3 Hz, 1H), 5.88 (d, J = 3.9 Hz, 1H), 8.04 (s, 1H), 8.8 (q, J = 4.4 Hz, 1H), 9.68 (dd, J = 7.4, 7.7 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): 13.0, 22.1, 24.3, 24.3, 28.3, 28.3, 28.6, 29.0, 31.2, 33.1, 33.3, 33.4, 34.3, 52.5, 60.6, 69.5, 71.9, 76.9, 79.8, 87.3, 121.9,144.0, 168.4, 172.0,172.1,172.3. MS (ESI): m/z 1175 [M+Na]+. 4.2.10. (2R)-3-((2S)-3-((6-(6-Deoxy-D-galactopyranose)-1H-1,2,3triazol-4-yl)ylamino)-3-oxo-2-palmitamidopropylthio)propane1,2-diyldipalmitate (6j) 1 White solid, mp 170 °C; [a]20 ): D +16 (c,1.0, CHCl3); IR (KBr, cm 2920, 2851, 1550, 1515, 1483, 1219, 772, 671. 1H NMR (300 MHz, DMSO-d6): 0.84 (t, J = 6.2 Hz, 9H), 1.14–1.34 (m,72H),1.41–1.56 (m, 6H), 2.25 (t, J = 6.9 Hz, 6H), 2.63–2.87 (m, 3H), 2.98–3.07 (dd, J = 4.5,3.7 Hz, 1H), 3.16 (d, J = 5.2 Hz,1H), 3.85–3.95 (m, 2H), 4.03–4.63 (m, 6H), 4.71 (d, J = 4.3 Hz,1H), 4.95 (d,

9

J = 5.4 Hz,1H),5.05–5.15 (br, 1H), 5.27 (d, J = 5.1 Hz, 1H), 6.12 (d, J = 5.8 Hz, 1H), 8.01 (s,1H), 8.8 (t, J = 5.2 Hz, 1H), 9.69 (d, J = 7.3 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): 13.7, 22.1, 24.3, 24.4, 28.4, 28.5, 28.7, 28.9, 29.1, 31.3, 33.1, 33.3, 33.5, 34.2, 52.6, 60.1, 63.4, 66.8, 68.1, 69.2, 75.4, 85.2, 114.6, 125.1, 142.8, 168.4, 172.1, 172.3. MS (ESI): m/z 1175 [M+Na]+. 4.2.11. (2R)-3-((2S)-3-((4-O-(b-D-Glucopyranosyl) (1-deoxy-a-Dglucopyranoside)-1H-1,2,3-triazol-4-yl)ylamino)-3-oxo-2palmitamidopropylthio)propane-1,2-diyldipalmitate (6k) 1 White solid, mp 193 °C; [a]20 ): D +4 (c,1.0,CHCl3); IR (KBr, cm 3744, 2921, 2851, 1727, 1693, 1644, 1550, 1465, 1219, 772. 1H NMR (300 MHz, DMSO-d6): 0.84 (t, J = 6.7 Hz, 9H), 1.16–1.29 (m, 72 H), 1.44–1.53 (m, 6 H), 2.25 (t, J = 6.7, 7.1 Hz, 6 H), 2.66–2.84 (m, 3H), 2.97–3.06 (ddd, J = 4.4, 4.5,7.8 Hz, 1H), 3.58 (dd, J = 3.9– 4.4 Hz, 1H), 3.66 (m, 2H), 3.95 (m, 1H), 4.0 (s, 1H), 4.08 (m, 1H), 4.27 (dd, J = 2.5 Hz, 1H), 4.31–4.41 (m, 5H), 4.49 (br,1H), 4.91 (d, J = 6.1 Hz, 1H), 5.08 (dd, J = 1.1, 1.3 Hz, 2H), 5.16 (dd, J = 3.8 Hz, 1H), 5.6 (d, J = 9.1 Hz, 1H), 8.05 (s, 1H), 8.78 (dd, J = 3.3, 3.5 Hz, 1H), 9.67 (dd, J = 4.7, 4.8 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): 13.6, 22.1, 24.4, 24.4, 28.5, 28.6, 28.8, 29.2, 31.4, 33.4, 33.1, 33.5, 34.3, 52.6, 53.0, 60.0, 60.4, 63.3, 68.1, 69.6, 69.8, 70.57, 71.76, 73.23, 75.23, 75.58, 77.75, 79.78, 86.8, 103.78, 122.13, 144.18, 168.5, 168.5, 171.9, 172.1. MS (ESI): m/z 1337 [M+Na]+. 4.2.12. (2R)-3-((2S)-3-((4-O-(a-D-Galactopyranosyl)(1-deoxy-aD-glucopyranoside)-1H-1,2,3-triazol-4-yl)ylamino)-3-oxo-2-

palmitamidopropylthio)propane-1,2-diyldipalmitate (6l) 1 White solid, mp 178 °C; [a]20 ): D +13 (c,1.0, CHCl3); IR (KBr, cm 2920, 2851, 1550, 1515, 1483, 1219, 772, 671. 1H NMR (300 MHz, DMSO-d6): 0.84 (t, J = 6.2 Hz, 9H), 1.15–1.33 (m, 72H), 1.42–1.56 (m, 6H), 2.25 (t, J = 6.9 Hz, 6H), 2.65–2.84 (m, 3H), 2.97–3.03 (m, 1H), 3.7–3.85 (m, 3H), 4.03–4.13 (m, 1H), 4.21–4.56 (m, 5H), 4.67 (s, 2H), 4.81 (d, J = 4.7 Hz, 1H), 4.9 (s, 1H), 5.04–5.18 (m, 3H), 5.55 (d, J = 5.2 Hz, 1H), 5.6 (d, J = 9.4 Hz, 1H), 8.09 (s, 1H), 8.8 (dd, J = 3.9, 7.2 Hz, 1H), 9.6 (s, 1H). 13C NMR (75 MHz, DMSO-d6): 13.8, 22.0, 24.3, 24.4, 28.4, 28.4, 28.7, 31.3, 33.1, 33.3, 33.5, 34.3, 52.6, 60.7, 63.4, 69.5, 69.6, 72.0, 76.9, 79.9, 87.3, 103.7 122.0, 144.1, 164.4, 168.5, 172.1, 172.3. MS (ESI): m/z 1337 [M+Na]+. 5. Biological material and methods 5.1. Immunization The lead compound b-galactose derived hybrids (6h) and ribose (6e) were studied in detail further in BALB/c mice. Male BALB/c mice (6–8 weeks) were purchased from National Institute of Nutrition, Hyderabad. All animal experiments were approved by the local ethical committee and animals were kept in accordance with CPCSEA guidelines with an IAEC No IICT/BIO/TOX/PG/1/02/2013. They were immunized with different groups according to various concentrations of (analogues). A booster dose was given on 14th day and sacrificed on 28th day. 5.2. Antibody titration To measure the antibody against a particular antigen, serum from the immunized mice were collected. The collected serum is then checked against IgG and its isotypes IgG1 and IgG2a. 5.2.1. Indirect ELISA: (IgG) The plates were coated with OVA (1 lg/well) in carbonate buffer (pH 9.4). Plates were incubated at 4 °C overnight. They were then washed 3 times with PBS/Tween, and non-specific binding sites were blocked by adding 200 ll of blocking solution. Plates

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were incubated at room temperature for 1 h. After 3 washes, diluted standards and samples to desired concentrations in blocking solution were added to the plates. Incubate at 37 °C for 1 h or at 4 °C overnight. Plates were washed 3 times with PBS/Tween. Avidin-Horseradish Peroxidase (Av-HRP) was diluted and added. Incubated at room temperature for 30 min. Plates were washed 3 times with PBS/Tween. TMB Substrate was added, plates were incubated at room temperature for 15 min. Color reaction was stopped by adding 50 ll of stop solution. Optical density (OD) was read at 450 nm.13

5.6. Human TLR-2 reporter gene assays (NF-jB induction) The assay was performed as per the manufacturer’s instructions. HEK-Blue™ hTLR2 cells (280,000 cells per mL) were harvested in HEK-Blue™ selection medium containing normocin treated with the compounds and the plate were incubated at 37 °C in 5% CO2 for 24 h. SEAP (secreted embryonic alkaline phosphatase) can be observed with naked eye and determined using a spectrophotometer at 620–655 nm. 5.7. Statistical analysis

5.2.2. Sandwich ELISA: (IgG1 and IgG2a) The plates were coated with diluted unlabeled capture antibody and incubated at 4 °C overnight. They were then washed 3 times with PBS/Tween, and non-specific binding sites were blocked by adding 200 ll of blocking solution. Plates were incubated at room temperature for 1 h. After 3 washes, diluted standards and samples to desire concentrations in blocking solution were added to the plates. Incubate at 4 °C overnight. Plates were washed 3 times with PBS/Tween. Detection antibodies were added followed by incubation at room temperature for 1 h. Plates were washed 3 times with PBS/Tween. Avidin-Horseradish Peroxidase (Av-HRP) was added and incubated at room temperature for 30 min. Plates were washed 5 times with PBS/Tween. TMB Substrate was added, plates were incubated at room temperature for 15 min. Color reaction was stopped by adding 50 ll of stop solution. Optical density (OD) was read at 450 nm.13

The statistical significance of the experiment was determined by two-tailed Student’s test in Excel. ⁄⁄⁄p 6 0.0005, ⁄⁄p 6 0.005, ⁄ p 6 0.05 were considered statistically significant. Error bars represent mean ± SD (n = 3). Acknowledgements Authors thank CSIR-New Delhi for the support under the 12th FYP supra institutional project DENOVA (CSC0205). N.N. thanks CSIR for the award of fellowship. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.06.070.

5.3. In vivo cytokine estimation

References and notes

ELISA was carried out by BioLegend capture and detection antibodies. Briefly, 96-Well plates were coated with capture antibody dissolved in coating buffer per well incubated overnight at 4 °C. Wells were blocked with BSA for 1 h at RT. After blocking, 50 lL/well of serum was added and incubated for 3 h. After washing, Biotinylated secondary antibody was added along with enzyme. Plates were incubated for 1 h at RT. Then plates were washed and TMB substrate solution was added. The reaction was stopped after 30 min with a stopping solution. Absorbance was measured at 450 nm with plate reader.12

1. (a) Francoise, A. International Immunopharmacology 2003, 3, 1187; (b) Moingeon, P.; Haensler; Lindberg, A. Vaccine 2001, 19, 4363; (c) Dante, J. M. Drug Discovery Today 2003, 8, 934. 2. (a) Glenney, A. T.; Pope, C. G.; Waddington, H.; Wallace, U. J. Pathol. Bacteriol. 1926, 29, 38. 3. Guy, B. Nat. Rev. Microbiol. 2007, 5, 505. 4. Kumar, H. M. S.; Irfan, H.; Singh, P. P., In Medicinal Chemistry and Drug Design, Deniz Ekinci, Ed.; 2012, ISBN: 978-953-51-0513-8. Medicinal-Chemistryand-Drug Design/Pattern-Recognition-Receptors-Based-Immune-Adjuvants: Their-Role-and-Importance-in-Vaccine-Design, Available from: http://www. intechopen.com/books/Medicinal Chemistry and Drug Design. 5. (a) Adam, A.; Ciorbaru, R.; Ellouz, F.; Petit, J. F.; Lederer, E. Biochem. Biophys. Res. Commun. 1974, 56, 561; (b) Akira, S.; Hemmi, H. Immunol. Lett. 2003, 85, 85. 6. (a) Akira, S.; Uematsu, S.; Takeuchi, O. Cell 2006, 124, 783; (b) Basith, S.; Manavalan, B.; Lee, G.; Kim, S. G.; Choi, S. Expert Opin. Ther. Patents 2011, 21, 927; (c) Berg, M.; Offermanns, S. Am. J. Physiol. 1994, 266, 1684. 7. Bessler, W. G.; Cox, M.; Lex, A.; Suhr, B.; Wiesmüller, K. H.; Jung, G. J. Immunol. 1985, 135, 1900. 8. (a) Kumar, H. M. S.; Singh, P. P.; Naveed, A. Qazi; Srinivas, J.; Fayaz, Malik; Tabasum, Sidiq; Amit, Gupta; Anamika, Khajuria; Suri, K. A.; Satti, N. K.; Qazi, G. N. Vaccine 2010, 28, 8327; (b) Singh, P. P.; Naveed, A.; Qazi; Shafi, S.; Reddy, D. M.; Abid, H.; Banday; Reddy, P. B.; Suri, K. A.; Gupta, B. D.; Satti, N. K.; Basant, P.; Wakhloo; Kumar, H. M. S.; Qazi, G. N. J. Mol. Catal. B Enzym. 2009, 56, 46; (c) Singh, P. P.; Bhunia, D.; Verma, Y. K.; Tabasum, S.; Khajuria, A.; Amit, G.; Preethi, Pallavi M.; Surya, Vamshi S.; Qazi, G. N.; Kumar, H. M. S. Int. Immunopharmacol. 2013, 17, 489. 9. Thomann, J. S.; Monneaux, F.; Heurtault, B.; Habermacher, C.; Schuber, F.; Bourel-Bonnet, L.; Frisch, B. Eur. J. Med. Chem. 2012, 51, 174. 10. (a) Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin, V. V. J. Am. Chem. Soc. 2005, 127, 210 (b) Zekarias, Yacob; Jürgen, Liebscher. 2011. Scott Handy (Ed.), ISBN: 978-953-307-6348, InTech, Available from: http://www.intechopen.com/books/ionic-liquids-classes-andproperties/1-2-3-triazolium-salts-as-aversatile- new-class-of-ionic-liquids. 11. All sugar azides were prepared as per previous literature. (a) Ibatullin, F. M.; Seli, Ano S. I. Tetrahedron Lett. 2002, 43, 9577; (b) Benoist, E.; Yvon, C.; Mehdi, A.; Kovensky, J.; Vincent, M.; Picard, C.; Galaup, C. G.; Sébastien; Gouin Carbohydr. Res. 2011, 346, 26; (c) Chantelle, J. C.; John, Trant F.; Mathieu, L.; Robert, N. Ben Bioconjugate Chem. 2011, 22, 605. 12. Derek, Hogan T. Methods in Molecular Medicines; Humana press, 2000. 13. Ejima, D.; Yumioka, R.; Tsumoto, K.; Arakawa, T. Anal. Biochem. 2005, 345, 250. 14. Gonipeta, B.; Duriancik, D.; Kim, E. J.; Gardner, E.; Gangur, V. ISRN Allergy 2013, 5, 1. Article ID 509427.

5.4. Immunophenotyping 5.4.1. Staining for extracellular markers Staining was as per the manufacturer’s protocol and run on a BD FACSVerse flow cytometer. Compensation was established using BD Biosciences compensation beads. Post acquisition flow cytometry analysis was performed using FACS Suite software.14 5.5. Safety studies 5.5.1. In vitro cytotoxicity evaluation by MTT assay Rat kidney cell line NRK-49F was exposed to varying doses of test compounds in the range of 10 lM–100 lM, to determine the toxicity after 48 h of incubation. The colorimetric MTT assay was used to evaluate cell viability which is determined based on reduction of MTT to blue formazan crystals by the mitochondrial succinate dehydrogenase enzymes of viable cells. 5.5.2. Hemolytic assay Different concentration of test compounds were tested with fixed volume concentration of rat RBCs. After incubation for 30 min the percentage of hemolysis was determined by comparing the absorbance (k = 540 nm) of the supernatant with that of distilled water as positive control.

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