Synthesis and evaluation of novel TLR2 agonists as potential

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Synthesis and evaluation of novel TLR2 agonists as potential adjuvants for cancer vaccines Benjamin Lu, Geoffrey M Williams, Dan Verdon, P Rod Dunbar, and Margaret A. Brimble J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01044 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 17, 2019

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Journal of Medicinal Chemistry

Synthesis and evaluation of novel TLR2 agonists as potential adjuvants for cancer vaccines Benjamin L. Lua,c, Geoffrey M. Williamsb,c, Daniel J. Verdonb,c, P. Rod Dunbarb,c and Margaret A. Brimblea,b,c* a

School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1010, New Zealand School of Biological Sciences, The University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand c Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3A Symonds Street 1010, Auckland, New Zealand b

ABSTRACT: Cancer immunotherapy has gained increasing attention due to its potential specificity and lack of adverse side-effects when compared to more traditional modes of treatment. Toll-like receptor 2 (TLR2) agonists are lipopeptides possessing the S-[2,3-bis(palmitoyloxy)propyl]-L-cysteine (Pam2Cys) motif and exhibit potent immunostimulatory effects. These agonists offer a means of providing “danger signals” in order to activate the immune system towards tumor antigens. Thus, the development of TLR2 agonists is attractive in the search of potential immunostimulants for cancer. Existing SAR studies of Pam2Cys with TLR2 indicate that the structural requirements for activity are, for the most part, very intolerable. We have investigated the importance of stereochemistry, the effect of N-terminal acylation, and homologation between the two ester functionalities in Pam2Cys-conjugated lipopeptides on TLR2 activity. The R diastereomer is significantly more potent than the S diastereomer and N-terminal modification generally lowers TLR2 activity. Most notably, homologation gives rise to analogues which are comparatively active to the native Pam2Cys containing constructs.

INTRODUCTION

B-cells specific to that antigen (adaptive immune system).8,9 The harnessing of these processes offers great potential for developing new immunotherapeutic agents for the treatment of disease, notably cancer, and research efforts have accordingly gained considerable momentum in recent years.10 Toll-like receptor 2 (TLR2) recognizes lipopeptides derived from bacterial cell wall components including lipoproteins, peptidoglycans, lipoteichoic acid, and the potent Pam2Cys 1 and Pam3Cys 2 motifs (Figure 1), all of which have been shown to promote immunomodulatory effects.11,12 Pam2Cys and Pam3Cys have been seen as attractive components of therapeutic vaccine constructs owing to their well-defined structures and synthetic accessibility. Indeed, there has been considerable exploration of the use of Pam2Cys and Pam3Cys as adjuvants in peptide-based vaccines;13 however, formal clinical investigation as yet remains limited in scope.14

Historically, cancer has been treated using a combination of conventional chemotherapy, radiotherapy and surgery. The development of these approaches has seen much success, leading to tumor regression and prolongation of life. Such techniques are invasive, often causing debilitating side-effects, furthermore certain tumor types remain persistent to most forms of treatment. Recently, immunotherapy has attracted attention as an upcoming mode of treatment.1 Therapeutic vaccinations have been used to restore and augment the body’s compromised immune system to combat cancer.2 Vaccines include immune potentiators (adjuvants) along with the antigen; adjuvants activate an immune response, leading to the mounting of long-term adaptive immune responses.3,4 The Toll-like receptors (TLRs) are a family of well-known transmembrane pattern recognition and immunostimulatory receptors named from the Toll protein of Drosophila melanogaster that was originally shown to engage immune gene expression via the NF-κB pathway.5 These pattern recognition receptors are present on antigen-presenting cells (APCs) such as monocytes, dendritic cells, and macrophages.6 Ten members of the human TLR family have been identified; these recognize a diverse range of agonists, including viral genetic material, microbial nucleic acids and membrane components (pathogen-asFigure 1. General structures of Pam2Cys 1, Pam3Cys 2, and Pam2Cys sociated molecular patterns), as well as molecular motifs arising lipopeptides 3 from distressed or damaged tissue (danger-associated molecular 7 patterns) as may arise in cancerous cells. Agonism at the TLRs The general structure of Pam2Cys lipopeptides 3 (Figure 1) can on APCs ultimately results in the production of pro-inflammabe separated into three components: the two palmitoyl fatty acid tory cytokines, expression of various costimulatory signals, and chains (blue), the core N-terminal S-(2,3-dihydroxypropyl)-Lpresentation of antigenic material on major histocompatibility cysteine motif to which the two fatty acid chains are linked complex (MHC) molecules. These processes activate natural (red), and the peptide component (black). killer cells (innate immune system) while also priming T- and ACS Paragon Plus Environment

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The importance of these molecular features in the structure-activity relationship (SAR) of Pam2Cys with TLR2 has been investigated by several groups.15-21 In a study carried out by Ulmer et al.15 the minimum length of the fatty acids required for immunogenicity was determined to be C8. They also established that the strength of immunogenicity increases with carbon chain length, plateauing at C16 (C18 = C16 > C12 > C8). Another study by David et al.16 established that the introduction of unsaturation by replacement of the palmitoyl fatty acids with linoleoyl (cis,cis-9,12-octadecadienoic acid) and α-linoleoyl (all-cis-9,12,15-octadecatrienoic acid) unsaturated fatty acids was accompanied by mild loss in TLR2 activity, whereas substitution of the fatty acid chains with polyether and amine functionalities resulted in a dramatic loss in TLR2 activity.

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Pam1Cys was originally synthesized from L-cystine in 7 steps16 but in 2013, Brimble et al.23 showed that the efficiency of the process could be significantly improved using a thiol-ene strategy wherein Pam1Cys was directly installed into a peptide antigen via reaction between an N-terminal cysteine peptide 18 and vinyl palmitate 17 to give peptide 20 (Scheme 1, A). During the optimization of this chemistry, the formation of a significant byproduct was observed with a mass indicating incorporation of a second equivalent of vinyl palmitate (Scheme 1, B). It was hypothesized that telomerization was responsible and the product was determined to be 21 resulting from reaction of radical intermediate 19 with a second equivalent of vinyl palmitate 17.24 Novel chemical entity 21 (a homologue of Pam2Cys, hence named homoPam2Cys) was isolated as a mixture of diastereomers at the glyceryl moiety, tested for NF-κB induction (HEK BlueTM assay), and found to exhibit the same level of activity as native Pam2Cys.25 This was an unexpected finding and offered the opportunity to explore new SAR in an otherwise well-described motif. In this context, of key interest was assessing the extent to which the activity of the individual diastereomers of the new agonist mirrored that of native Pam2Cys as well as the effect of further homologating the carbon chain.

RESULTS AND DISCUSSION

Figure 2. SAR of the core structure of Pam2Cys and Pam1Cys17-21

Methodical SAR studies on the S-(2,3-dihydroxypropyl)-L-cysteine core revealed the strict structural requirements for TLR2 activity (Figure 2). Akira et al.17 established that the R stereoisomer 4 exhibits approximately 100-fold greater activity than the S 5. David et al.16,18 and Metzger et al.19 demonstrated that substitution of the thioether with either a methylene 6 or oxygen 7 resulted in a loss in TLR2 activity, whereas conversely, replacement with selenium 8 resulted in a slight increase. Modification of the space between the thioether and α center of cysteine (9 and 10) by David et al.16 resulted in dramatic losses in TLR2 activity. Jung et al.20 and Brandenburg et al.21 showed that substitution of the ester functionalities for amide bonds 11 and ethers 12 resulted in complete abolition of TLR2 activity. David et al.16 eventually discovered that the removal of a lipid chain gave the singly-palmitoylated (or Pam1Cys 13) compound that, remarkably, exhibited significant, albeit modest, activity. Further investigation of this new motif revealed that inversion of the ester bond (14 and 15) and extension of the carbon chain between the thioether and ester motif 16 resulted a significant loss in TLR2 activity.16 The retention in activity of Pam1Cys 13 is interesting especially considering the large reduction in molecular complexity when compared to Pam2Cys 1. This chemical motif continues to be a focus for structural investigations.22

The thiol-ene reaction that gives rise to the homologated Pam2Cys system (Scheme 1, B) does not enable stereocontrol at the newly generated chiral centre (giving instead an inseparable mixture of the R and S diastereomers) and is not amenable for the synthesis of analogues comprising homologation(s) of the carbon chain. It was therefore decided to make use of a lipidated amino acid building-block approach, in which the desired features of stereocontrol and chain length could be installed via organic synthesis. We decided to synthesize both the R and S diastereomers of Fmoc-Pam2Cys (22a and 22b) and Fmoc homoPam2Cys (23a and 23b) together with a series of analogues (24, 25, and 26) in which the chain length between the two ester functionalities was further homologated (Figure 3). Each of these well-defined building blocks could then be attached to a peptide via standard Fmoc solid phase peptide synthesis (SPPS) to give a series of constructs with the requisite properties.

Figure 3. Structures of Pam2Cys and related structural analogues investigated herein

Scheme 1. Previous work by Brimble et al. on thiol-ene reaction of a cysteine-containing peptide 18 with vinyl palmitate 1723,24

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Scheme 2. Synthesis of the Pam2Cys and homologous amino acid building blocksa

a Reagents and conditions: (i) Zn, CH2Cl2, MeOH/HCl/H2SO4 (100/7/1, v/v/v), 0 °C, 30 min, then epoxides 28-32 (4 equiv.), 70 °C, 19 h; (ii) palmitic acid, DIC, DMAP, THF, rt, 17-19 h; (iii) TFA, rt, 30 min.

Scheme 3. Synthesis of the peptide librarya

a Reagents and conditions: (i) (a) Fmoc L-Val-OCH2pC6H4OCH2CH2CO2H (2 equiv.), HATU, NMM, DMF, rt, 1 h, (b) piperidine/DMF (20% v/v), rt, 10 min; (ii) iterative Fmoc-SPPS. (iii) building blocks 22-26, PyBOP, collidine, DMF, rt, 1 h; (iv) piperidine/DMF (20% v/v), rt, 10 min; (v) DODT/TFA (5% v/v), rt, 3 h (for compounds 45a, 45b, 48a, 48b, 50-52); (a) Ac2O, rt, 15 min, (b) DODT/TFA (5% v/v), rt, 3 h (for compounds 46a, 46b, 49a, 49b); (a) palmitic acid, PyBOP, collidine, DMF, rt, 1 h, (b) DODT/TFA (5% v/v), rt, 3 h (for compounds 47a, 47b).

Our first objective was to establish the difference in relative agonist activity between Pam2Cys and homoPam2Cys containing lipopeptides. Accordingly, we prepared both the 6S and 6R diastereomers of Fmoc-Pam2Cys (22a and 22b) for subsequent incorporation into Fmoc-SPPS by utilizing a similar synthetic strategy to that reported by Metzger et al.26 (Scheme 2). Reaction of the thiol derived from the in situ reduction of disulfide 27 with either of the enantiopure epoxides 28a or 28b (available by hydrolytic kinetic resolution of the racemic epoxide27) and concomitant TBS deprotection gave diastereopure diols 33a or 33b, respectively. Elaboration to building blocks 22a and 22b proceeded smoothly without need for purification between the esterification and tert-butyl ester cleavage steps. Utilizing the same methodology, novel homoPam2Cys Fmoc building blocks 23a and 23b were then produced (Scheme 2). Enantiopure epoxy alcohols 29a and 29b were available from D- and L-as-

partic acid using literature methods.28 Using an approach analogous to the Pam2Cys building blocks, epoxy alcohols 29a or 29b were then reacted with disulfide 27 to give diols 34a and 34b, followed by conversion to Fmoc-homoPam2Cys building blocks 23a and 23b. Following the synthesis of the requisite building blocks 22a, 22b, 23a and 23b for use in Fmoc-SPPS, we then selected a peptide sequence that would serve as an example of how this motif might be incorporated into self-adjuvanting vaccines. The NY-ESO-1 protein is a well-studied tumor associated antigen that is known to illicit both humoral and cellular immune responses in a substantial number of patients.29,30 The potent immunogenicity and its broad expression in tumors make this protein an attractive component in anti-cancer vaccine strategies. Of the several of immunogenic regions of the ESO protein one epitope has been identified at residues 157-165



(SLLMWITQC), which has been shown to stimulate CD8 Tcells.31 It was shown in other studies that substitution of Cys165 with Val enhanced peptide binding to MHC class I molecules and antigenic character, increasing immunogenicity.32 Knowing this, we elected to synthesize a model peptide bearing the NY-ESO-1 epitope sequence SLLMWITQV which would be adjoined to the solubilizing tag SKKKK. This would improve the handling and ease the purification of the potentially lipophilic peptide. TentaGel® resin was first derivatized to give valinyl hydroxymethylphenoxypropionyl (HMPP) resin 38 (Scheme 3), then standard iterative Fmoc-SPPS ensued to furnish the desired peptidyl resin 39. Peptidyl resin 39 was accessible on a large scale which facilitated the investigation of optimal coupling conditions for incorporation the final palmitoylated building blocks 22a, 22b, 23a, and 23b. After testing several coupling agents and bases, it was apparent that racemization of the cysteinyl α center was particularly troublesome, however, it was found that use of (benzotriazol-1-yl-oxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) in conjunction with collidine as the base suppressed racemization affording the desired lipopeptides in good yield. Consequently, peptidyl resin 39 was treated with a mixture of the appropriate diastereomer of Pam2Cys (22a or 22b), PyBOP, and collidine, immediately followed by Fmoc removal to give the respective peptidyl resins 40a and 40b (Scheme 3). At this point it was decided to examine the effect that N-terminal acylation had on activity as previous studies had indicated this modification did exert some influence on TLR2 agonism.33 Peptidyl resins 40a and 40b were therefore conveniently derivatized and cleaved to give corresponding Nacetylated (46a and 46b) and N-palmitoylated (Pam3Cys, 47a and 47b) constructs. The free N-terminal peptides 45a and 45b were also prepared for comparison. In the same manner, homoPam2Cys constructs 48a, 48b, 49a, and 49b were prepared (Scheme 3). Peptidyl resin 39 was elaborated to peptidyl resins 41a or 41b using the appropriate homoPam2Cys building block 23a or 23b, PyBOP and collidine, followed by Fmoc cleavage. Peptidyl resins 41a and 41b were then readily transformed into the free N-terminal peptides 48a and 48b or the N-acetylated peptides 49a and 49b.

Table 1. Peptide library EC50 values Compound

Structure

(X = SKKKKSLLMWITQV)

EC50 (nM)

45a

155.693

45b

0.468

46a

150.598

46b

0.281

47a

161.006

47b

22.959

48a

46.156

48b

0.609

49a

17.381

49b

Figure 4. Human TLR2 agonistic activities of lipopeptide analogues for Pam2 and homoPam2Cys constructs. HEK-Blue™-hTLR2 cells transfected with TLR2-NF-κB-SEAP reporter-gene system were used (Invivogen). Data points represent normalised mean +/- SEM ABS determined on triplicate samples after 16 h.

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1.162

With the series of lipidated constructs in hand, their ability to stimulate the human TLR2 signaling pathway was examined using the well-established HEK-Blue™ hTLR reporter system (Figure 4, Table 1). The R-Pam2Cys based constructs 45b and 46b were found to be the most active analogues of the suite tested (EC50 0.468 nM and 0.281 nM respectively) with the relatively small difference in EC50 suggesting that N-acetylation has negligible effect on TLR2 agonism. The corresponding S diastereomers 45a and 46a, however, show a marked loss of


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TLR2 agonism (EC50 155.693 nM and 150.598 nM respectively) – this is supportive of literature observations that the R diastereomer is favored over the S.17 N-palmitoyl (Pam3Cys) analogues 47a and 47b showed less activity (EC50 161.006 nM and 22.959 nM respectively) than native R-Pam2Cys analogue 45b (EC50 0.468 nM), however, R-Pam3Cys analogue 47b (EC50 22.959 nM) was more active than both S-Pam2Cys analogues 45a and 46a (EC50 155.693 nM and 150.598 nM respectively). Of particular interest are the novel homoPam2Cys constructs which seem to follow the same trend as the Pam2Cys constructs, with the R diastereomers 48b and 49b (EC50 0.609 nM and 1.162 nM respectively) outperforming the S diastereomers 48a and 49a (EC50 46.156 nM and 17.381 nM respectively) and Nacetylation having a negligible effect on activity. However, most notably, both S-homoPam2Cys analogues 48a and 49a (EC50 46.156 nM and 17.381 nM respectively) show improved activity over their S-Pam2Cys 45a and 46a (EC50 155.693 nM and 150.598 nM respectively) counterparts as well as comparable activity to R-Pam3Cys 47b (EC50 22.959 nM). The reason for the difference in activity is unclear, but may reflect an ‘improved’ packing arrangement in the receptor binding site, possibly owing to the extra methylene in S-homoPam2Cys which compensates for the disfavored chirality. Given these initial findings with the first suite of lipopeptides investigated, we were interested to probe the effect that further homologation of the 2,3-dihydroxypropyl motif would have on TLR2 agonism. Given that historically, and according to our own findings, the 6R diastereomer of Pam2Cys was shown to be more active than the 6S, it was decided to prepare only the 6R diastereomers of Pam2Cys homologues 24, 25, and 26 (Scheme 2). Synthesis of these analogues employed an extension of the methodology used for the synthesis of the Pam2Cys and homoPam2Cys building blocks. Thus, enantiopure epoxides 30, 31, and 32 were obtained by hydrolytic kinetic resolution of the corresponding terminal racemic epoxides.27 The union of epoxides 30 and 31 with disulfide 27 was expectedly challenging. The competing intramolecular cyclisation of epoxides 30 and 31 to give the furan and pyran rings respectively was favored under acidic conditions (Figure 5). Eventually, it was found that addition of the epoxides to the reaction mixture under reflux minimized the extent of intramolecular cyclization, hence diols 35 and 36 were finally obtained in satisfactory yields. No issues were encountered during the reaction of epoxide 32 and disulfide 27 to give diol 37. Final elaboration to give building blocks 24, 25, and 26 proceeded uneventfully. With the novel building blocks 24, 25, and 26 in hand, lipopeptides 50, 51, and 52 were prepared (Scheme 3) with synthesis proceeding analogously to that described above.

Figure 5. Intramolecular cyclization of epoxides 30 and 31

Figure 6. Human TLR2 agonistic activities of lipopeptide homologues of Pam2Cys. HEK-Blue™-hTLR2 cells transfected with TLR2-NF-κBSEAP reporter-gene system were used (Invivogen). Data points represent normalised mean +/- SEM ABS determined on triplicate samples after 16 h.

Table 2. Peptide library EC50 values Compound

Structure

(X = SKKKKSLLMWITQV)

EC50 (nM)

45b

0.468

48b

0.609

50

0.383

51

0.304

52

4.445

With homologated constructs 50, 51, and 52 in hand, they were next evaluated alongside the previously tested Pam2Cys and homoPam2Cys constructs 45b and 48b for TLR2 agonism (Figure 6, Table 2). Interestingly, the second generation of analogues retained considerable activity given the strict SAR requirements that had previously been reported. It was found that similar agonism was retained between constructs 45b, 48b, 50, and 51 (EC50 0.468 nM, 0.609 nM, 0.383 nM, and 0.304 nM respectively), however, the increased chain length in construct 52 resulted in a subtle loss in TLR2 agonism (EC50 4.445 nM), this could be attributed to the increase in steric congestion in the already narrow binding pocket of the TLR2 receptor.34



In the TLR2/6 crystal structure, Lee et al.34 indicate a hydrogen bond (Figure 7) between the C-6 ester group of the glycerol moiety and Phe349 of TLR2 (in blue). However, there do not appear to be any formal interactions between residues of the TLR2 receptor and the second ester at C-7, which suggests that this group may be less important to binding and free to be translocated, which our positional changes confirm. Nevertheless, it is somewhat intriguing that the somewhat polar ester group itself can, by the process of homologation (n = 2, 3, 4), be forced into the hydrophobic pocket of the receptor without compromising binding activity.

Figure 7. The hydrogen bonding interaction between the C-6 ester and Phe349 of TLR234

CONCLUSION In conclusion, a serendipitous discovery was made of a homologated version of the natural TLR2 ligand Pam2Cys as a byproduct of the thiol-ene reaction of 17 with 18. This material was analyzed and found, unexpectedly, to be a highly active TLR2 agonist. This led us to the synthesis and analysis of the R- and S- diastereomers of both Pam2 and homoPam2Cys to further clarify the SAR. Having established the activity of this novel motif, more homologues were then synthesized, extending the carbon chain still further. Surprisingly, these compounds also exhibit high activity, similar to that of the wild-type ligand.

EXPERIMENTAL SECTION Chemistry. All reactions were carried out in flame- or ovendried glassware under a dry nitrogen atmosphere. Solvents were obtained from a commercially sourced solvent purifier system from LC Technology Systems. Triethylamine was distilled from calcium hydride and stored over potassium hydroxide. All other reagents were used as received unless otherwise noted. Yields refer to chromatographically and spectroscopically (1H NMR) homogenous materials, unless otherwise stated. Reactions performed at low temperature were either cooled with an acetone/dry ice bath to reach -78 °C, or a water/ice bath to reach 0 °C. Flash chromatography was carried out using 0.0630.1 mm Riedel de Haen silica gel with the noted solvent system unless stated otherwise. Infrared (IR) spectra were recorded using a Perkin Elmer Spectrum One FTIR spectrometer on a film ATR sampling accessory. Absorption maxima are expressed in wavenumbers (cm-1) and recorded using a range of 450-4000 cm-1. NMR spectra were recorded as indicated on the Bruker DRX400 spectrometer operating at 400 MHz for 1H nuclei and 100 MHz for 13C nuclei. All NMR spectra were recorded at 300 K. All chemical shifts are reported in ppm relative to CDCl3 (δ = 7.26 for 1H NMR and δ = 77.0 for 13C NMR). 1H NMR is reported as chemical shift, relative integral, multiplicity (br s = broad singlet, s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, dq = doublet of quartets, m = multiplet), coupling constant (J in Hz), and assignment.

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The purity for all final tested compounds was confirmed to be 95% or greater by analytical HPLC using a 3 µ Phenomenex Gemini column (4.6 mm × 150 mm) reverse phase C18 column (see Supporting Information). N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((S)-2,3bis(palmitoyloxy)propyl)-L-cysteine (22a): To a stirred solution of diol 33a (0.42 g, 0.88 mmol) and palmitic acid (0.68 g, 2.64 mmol) in THF (8 mL) at r.t. was added N,N’-diisopropylcarbodiimide (0.41 mL, 2.64 mmol) and 4-dimethylaminopyridine (0.04 g, 0.35 mmol). The reaction mixture was allowed to stir at r.t. for 17 h. The mixture was then filtered through a pad of Celite®, diluted with EtOAc (50 mL), washed with 1M aq. citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (6 mL) and allowed to stir at r.t. for 45 min. The reaction mixture was again concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 9:1 → 0:1) to give the title compound 22a (0.61 g, 78%) as a colorless oil. [α]D23.4 +15.0 (c 0.80 in CHCl3) (lit26 +14.9 (c 0.55 in CHCl3)); 1H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 6.0 Hz), 7.40 (2H, t, J = 7.4 Hz), 7.30 (2H, td, J = 11.2, 0.9 Hz), 5.77 (1H, d, J = 7.8), 5.21-5.11 (1H, m), 4.71-4.63 (1H, m), 4.41-4.35 (3H, m), 4.23 (2H, t, J = 7.0 Hz), 4.15-4.10 (2H, m), 3.17 (1H, dd, J = 14.0, 4.2 Hz), 3.09 (1H, dd, J = 13.9, 5.0 Hz), 2.82-2.70 (2H, m), 2.32-2.27 (4H, m), 1.64-1.55 (4H, m), 1.25 (48H, m), 0.88 (6H, t, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 174.2, 173.7, 173.4, 143.7, 141.3, 127.7, 127.1, 125.2, 120.0, 70.3, 67.5, 63.6, 53.5, 47.1, 34.9, 34.3, 34.1, 32.9, 32.0, 29.9, 29.7, 29.5, 29.4, 29.3, 29.2, 24.9, 22.7, 14.1. Spectroscopic data was consistent with that reported in literature.26 N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((R)-2,3bis(palmitoyloxy)propyl)-L-cysteine (22b): To a stirred solution of diol 33b (0.50 g, 1.05 mmol) and palmitic acid (0.81 g, 3.16 mmol) in THF (9 mL) at r.t. was added N,N’-diisopropylcarbodiimide (0.49 mL, 3.16 mmol) and 4-dimethylaminopyridine (0.05 g, 0.42 mmol). The reaction mixture was allowed to stir at r.t. for 17 h. The mixture was then filtered through a pad of Celite®, diluted with EtOAc (50 mL), washed with 1M aq. citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (6 mL) and allowed to stir at r.t. for 45 min. The reaction mixture was again concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 9:1 → 0:1) to give the title compound 22b (0.65 g, 69%) as a colorless oil. [α]D23.4 +13.0 (c 0.51 in CHCl3) (lit26 +12.9 (c 0.58 in CHCl3)); 1H NMR (400 MHz, CDCl3) δ 7.75 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.1 Hz), 7.39 (2H, t, J = 7.4 Hz), 7.30 (2H, t, J = 7.4 Hz), 5.85 (1H, d, J = 7.4 Hz), 5.21-5.14 (1H, m), 4.59-4.52 (1H, m), 4.40 (2H, d, J = 5.5 Hz), 4.36 (1H, dd, J = 12.5, 3.2 Hz), 4.24 (2H, t, J = 7.0 Hz), 4.14 (1H, dd, J = 11.8, 5.7 Hz), 3.18 (1H, dd, J = 13.7, 3.8 Hz), 3.06 (1H, dd, J = 13.6, 5.9 Hz), 2.84-2.71 (2H, m), 2.33-2.28 (4H, m), 1.65-1.52 (4H, m), 1.34-1.19 (48H, m), 0.89 (6H, t, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 174.3, 173.7, 173.6, 156.2, 143.8, 141.4, 127.9, 127.2, 125.3, 120.1, 70.4, 67.6, 63.7, 53.8, 47.2, 34.4, 34.2, 32.0, 29.8, 29.6, 29.5, 29.4, 29.2, 25.0, 22.8, 14.2. Spectroscopic data was consistent with that reported in literature.26 Synthesis of N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S((S)-2,4-bis(palmitoyloxy)butyl)-L-cysteine (23a): To a stirred solution of diol 34a (1.68 g, 3.44 mmol) and palmitic acid (2.65 g, 10.31 mmol) in THF (50 mL) at r.t. was added N,N’-diisopropylcarbodiimide (2.13 mL, 13.75 mmol) and 4-


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dimethylaminopyridine (0.04 g, 0.34 mmol). The reaction mixture was allowed to stir at r.t. for 19 h. The mixture was then filtered through a pad of Celite®, diluted with EtOAc (50 mL), washed with 1M aq. citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and allowed to stir at r.t. for 30 min. The reaction mixture was again concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 9:1 → 0:1) to give the title compound 23a (2.02 g, 65%) as a colorless oil. [α]D23.4 -4.0 (c 0.2 in CHCl3); νmax(neat)/cm-1 2922, 2852, 1733, 1525, 1450, 1168, 1110; 1H NMR (400 MHz, CDCl3) δ 7.75 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.4 Hz), 7.39 (2H, t, J = 7.4 Hz), 7.40 (2H, t, J = 7.4 Hz), 5.86 (1H, d, J = 7.5), 5.13-5.03 (1H, m), 4.72-4.62 (1H, m), 4.40 (2H, d, J = 6.9 Hz), 4.24 (1H, t, J = 7.0 Hz), 4.11 (2H, t, J = 6.5 Hz), 3.14 (1H, dd, J = 12.9, 3.5 Hz), 3.08 (1H, dd, J = 13.8, 4.4 Hz), 2.82-2.69 (2H, m), 2.31-2.26 (4H, m), 2.10-1.87 (2H, m), 1.65-1.54 (4H, m), 1.331.22 (48H, m), 0.89 (6H, t, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 174.1, 173.7, 155.0, 143.7, 141.3, 127.8, 127.1, 125.2, 120.0, 69.7, 67.5, 60.5, 53.7, 47.1, 36.5, 34.8, 34.4, 34.3, 32.2, 32.0, 29.8, 29.7, 29.6, 29.4, 29.2, 25.0, 22.8, 14.1; HRMS (ESI+) [M + H]+ 908.6069 calc for C54H86NO6S 908.6065 observed, [M + Na]+ 930.5888 calc for C54H85NNaO8S 930.5875 observed. N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((R)-2,4bis(palmitoyloxy)butyl)-L-cysteine (23b): To a stirred solution of diol 34b (1.52 g, 3.12 mmol) and palmitic acid (2.40 g, 9.35 mmol) in THF (45 mL) at r.t. was added N,N’-diisopropylcarbodiimide (1.93 mL, 12.46 mmol) and 4-dimethylaminopyridine (0.04 g, 0.31 mmol). The reaction mixture was allowed to stir at r.t. for 19 h. The mixture was then filtered through a pad of Celite®, diluted with EtOAc (50 mL), washed with 1M aq. citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and allowed to stir at r.t. for 30 min. The reaction mixture was again concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 9:1 → 0:1) to give the title compound 23b (1.98 g, 70%) as a colorless oil. [α]D23.9 +8.4 (c 0.44 in CHCl3); νmax(neat)/cm-1 2922, 2852, 1733, 1525, 1450, 1168, 1110; 1H NMR (400 MHz, CDCl3) δ 7.75 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.2 Hz), 7.38 (2H, t, J = 7.4 Hz), 7.30 (2H, t, J = 7.4 Hz), 5.89 (1H, d, J = 7.8 Hz), 5.13-5.06 (1H, m), 4.694.63 (1H, m), 4.39 (2H, d, J = 6.5 Hz), 4.23 (1H, t, J = 7.0 Hz), 4.16-4.06 (2H, m), 3.16 (1H, dd, J = 13.8, 4.0 Hz), 3.03 (1H, dd, J = 13.8, 6.1 Hz), 2.81-2.70 (2H, m), 2.33-2.26 (4H, m), 2.10-1.89 (2H, m), 1.60-1.58 (4H, m, ), 1.35-1.20 (48H, m), 0.89 (6H, t, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 174.0, 173.6, 156.0, 143.6, 141.2, 127.6, 127.0, 125.1, 120.0, 69.5, 67.3, 60.3, 53.6, 47.0, 36.3, 34.5, 34.3, 34.1, 32.0, 31.8, 29.6, 29.4, 29.3, 29.2, 29.1, 25.0, 22.6, 14.0; HRMS (ESI+) [M + H]+ 908.6069 calc for C54H86NO6S 908.6065, [M + Na]+ 930.5888 calc for C54H85NNaO8S 930.5875 observed. N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((R)-2,5bis(palmitoyloxy)pentyl)-L-cysteine (24): To a stirred solution of diol 35 (0.114 g, 0.243 mmol) and palmitic acid (0.180 g, 0.702 mmol) in THF (3 mL) at r.t. was added N,N’-diisopropylcarbodiimide (0.145 mL, 0.936 mmol) and 4-dimethylaminopyridine (0.011 g, 0.094 mmol). The resultant mixture was allowed to stir at r.t. for 17 h. The mixture was then filtered through a pad of Celite®, diluted with EtOAc (30 mL), washed with 1M aq. citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and allowed to stir at r.t for 45 min. The reaction mixture was again concentrated in vacuo. The crude was purified by

flash column chromatography (hexanes-EtOAc, 9:1 → 0:1) to give the title compound 24 (0.220 g, 98%) as a colorless oil. [α]D21.3 +10.0 (c 0.08 in CHCl3); νmax(neat)/cm-1 2919, 2851, 1723, 1521, 1521, 1221, 1108, 1054; 1H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 7.5 Hz), 7.62 (2H, d, J = 7.4 Hz), 7.39 (2H, t, J = 7.4 Hz), 7.30 (2H, td, J = 11.2, 0.9 Hz), 5.78 (1H, d, J =7.6 Hz), 5.04-4.95 (1H, m), 4.60 (1H, dd, J = 12.2, 5.2 Hz), 4.38 (2H, d, J = 7.2 Hz), 4.24 (2H, t, J = 7.1 Hz), 4.13-3.99 (2H, m), 3.16 (1H, dd, J = 13.9, 4.5 Hz), 3.04 (1H, dd, J = 14.0, 5.3 Hz), 2.78-2.70 (2H, m), 2.34-2.25 (4H, m), 1.74-1.56 (8H, m, ), 1.32-1.22 (48H, m), 0.88 (6H, t, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3) δ 174.3, 174.0, 173.5, 156.0, 143.7, 141.3, 127.8, 127.1, 121.2, 120.0, 72.1, 67.5, 63.8, 53.6, 47.1, 36.5, 34.6, 34.5, 34.3, 31.9, 29.7, 29.5, 29.4, 29.3, 29.2, 25.0, 24.6, 22.7, 14.1; HRMS (ESI+) [M + Na]+ 944.6045 calc for C55H87NNaO8S 944.6028 observed. N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((R)-2,6bis(palmitoyloxy)hexyl)-L-cysteine (25): To a stirred solution of diol 36 (0.190 g, 0.370 mmol) and palmitic acid (0.284 g, 1.10 mmol) in THF (3 mL) at r.t. was added N,N’-diisopropylcarbodiimide (0.226 mL, 1.47 mmol) and 4-dimethylaminopyridine (0.018 g, 0.147 mmol). The resultant mixture was allowed to stir at r.t. for 17 h. The mixture was then filtered through a pad of Celite®, diluted with EtOAc (50 mL), washed with 1M aq. citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and allowed to stir at r.t. for 45 min. The reaction mixture was again concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 9:1 → 0:1) to give the title compound 25 (0.301 g, quant.) as a colorless oil. [α]D21.2 +10.0 (c 0.07 in CHCl3); νmax(neat)/cm-1 3331, 2917, 2850, 1728, 1692, 1532, 1467, 1451, 1244, 1221, 1198, 1175; 1 H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 7.5 Hz), 7.62 (2H, d, J = 7.2 Hz), 7.40 (2H, t, J = 7.4 Hz), 7.30 (2H, td, J = 11.2, 1.0 Hz), 5.82 (1H, d, J = 7.9), 5.03-4.92 (1H, m), 4.714.60 (1H, m), 4.40 (2H, d, J = 7.0 Hz), 4.24 (1H, t, J = 7.1 Hz), 4.11-4.00 (2H, m), 3.15 (1H, dd, J = 13.9, 4.4 Hz), 3.04 (1H, dd, J = 13.8, 5.8 Hz), 2.78-2.65 (2H, m), 2.31 (2H, t, J = 7.6 Hz), 2.28 (2H, t, J = 7.6 Hz), 1.74-1.55 (8H, m), 1.45-1.17 (50H, m), 0.88 (6H, t, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 174.3, 174.0, 173.9, 156.1, 143.7, 141.3, 127.8, 127.1, 125.2, 120.0, 72.4, 67.4, 64.0, 53.6, 47.1, 36.6, 34.6, 34.5, 34.4, 32.7, 32.0, 29.7, 29.5, 29.4, 29.3, 29.2, 28.3, 25.0, 22.7, 21.7, 14.4; HRMS (ESI+) [M + Na]+ 958.6239 calc for C56H89NNaO8S 958.6238 observed. N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-((R)-2,7bis(palmitoyloxy)heptyl)-L-cysteine (26): To a stirred solution of diol 37 (0.168 g, 0.317 mmol) and palmitic acid (0.244 g, 0.951 mmol) in THF (4.6 mL) at r.t. was added N,N’-diisopropylcarbodiimide (0.191 mL, 1.269 mmol) and 4-dimethylaminopyridine (0.016 g, 0.127 mmol). The resultant mixture was allowed to stir at r.t. for 17 h. The mixture was then filtered through a pad of Celite®, diluted with EtOAc (30 mL), washed with 1M aq. citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and allowed to stir at r.t. for 45 min. The reaction mixture was again concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 9:1 → 0:1) to give the title compound 26 (0.301 g, quant.) as a colorless oil. [α]D20.8 +7.5 (c 0.24 in CHCl3); νmax(neat)/cm-1 3319, 2919, 2851, 1722, 1521, 1471, 1450, 1221, 1055; 1H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 7.6 Hz), 7.61 (2H, d, J = 7.3 Hz), 7.40 (2H, t, J = 7.7 Hz), 7.30 (2H, td, J = 11.2, 1.1 Hz), 5.82 (1H, d, J = 7.7 Hz), 5.00-4.94 (1H, m), 4.64 (1H, dd, J = 12.3, 5.6 Hz),



4.40 (2H, d, J = 7.1 Hz), 4.24 (1H, t, J = 7.1 Hz), 4.10-4.00 (2H, m), 3.14 (1H, dd, J = 13.8, 4.3 Hz), 3.04 (1H, dd, J = 13.8, 5.6 Hz), 2.76-2.67 (2H, m), 2.31 (2H, t, J = 7.6 Hz), 2.28 (2H, t, J = 7.6 Hz), 1.65-1.56 (8H, m), 1.39-1.18 (52H, m), 0.88 (6H, t, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3) δ 174.4, 174.1, 173.9, 156.2, 143.8, 141.3, 127.8, 127.2, 125.2, 120.0, 72.5, 67.5, 64.3, 53.8, 47.2, 36.6, 34.6, 34.4, 33.1, 32.0. 29.8, 29.6, 29.4, 29.3, 28.4, 25.7, 25.1, 25.0, 22.8, 14.2; HRMS (ESI+) [M + Na]+ 972.6358 calc for C57H91NNaO8S 972.6392 observed. Tert-Butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S((S)-2,3-dihydroxypropyl)-L-cysteinate (33a): To a stirred solution of Nα,Nα-bis-Fmoc-L-cystine bis(tert-butyl ester) (2735, 2.15 g, 2.69 mmol) in CH2Cl2 (17 mL) at 0 °C was added zinc powder (1.28 g, 19.50 mmol) and a freshly prepared mixture of methanol, conc. hydrochloric acid and conc. sulfuric acid (100:7:1, 8.5 mL). The resultant mixture was allowed to stir at 0 °C for 30 min after which was added (R)-tert-butyldimethyl(oxiran-2-ylmethoxy)silane (28a27,36, 2.10 g, 11.14 mmol). The reaction mixture refluxed at 70 °C for 19 h. The mixture was then diluted with CH2Cl2 (30 mL), filtered through a pad of Celite® and washed with brine (50 mL). The aqueous layer was extracted with CH2Cl2 (3 × 50 mL) and the combined organic extracts were dried over anhydrous MgSO4 and concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 1:3) to give the title compound 33a (2.08 g, 82%) as a colorless oil. [α]D23.4 +7.0 (c 0.21 in CHCl3) (lit26 +6.7 (c 0.56 in CHCl3)); 1H NMR (400 MHz, CDCl3) δ 7.78 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.4 Hz), 7.40 (2H, t, J = 7.4 Hz), 7.32 (2H, t, J = 7.5 Hz), 5.73 (1H, d, J = 7.6 Hz), 4.55-4.47 (1H, m), 4.46-4.36 (2H, m), 4.23 (1H, t, J = 6.9 Hz), 3.86-3.76 (1H, m), 3.68 (1H, dd, J = 11.3, 3.0 Hz), 3.55 (1H, dd, J = 11.5, 5.8 Hz), 3.02 (1H, dd, J =13.7, 4.8 Hz), 2.94 (1H, dd, J = 13.9, 6.1 Hz), 2.79 (1H, dd, J =13.5, 4.4 Hz), 2.67 (1H, dd, J =13.3, 8.0 Hz), 1.50 (9H, s); 13C NMR (100 MHz, CDCl3) δ 169.7, 156.1, 143.8, 141.3, 127.8, 127.1, 125.1, 120.0, 83.3, 70.5, 67.2, 65.1, 54.5, 47.1, 36.7, 35.9, 28.0. Spectroscopic data was consistent with that reported in literature.26 Tert-Butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S((R)-2,3-dihydroxypropyl)-L-cysteinate (33b): To a stirred solution of Nα,Nα-bis-Fmoc-L-cystine bis(tert-butyl ester) (2735, 1.93 g, 2.42 mmol) in CH2Cl2 (16 mL) at 0 °C was added zinc powder (1.15 g, 17.51 mmol) and a freshly prepared mixture of methanol, conc. hydrochloric acid and conc. sulfuric acid (100:7:1, 8 mL). The resultant mixture was allowed to stir at 0 °C for 30 min after which was added (S)-tert-butyldimethyl(oxiran-2-ylmethoxy)silane (28b27,36, 1.88 g, 10.01 mmol). The reaction mixture refluxed at 70 °C for 19 h. The mixture was then diluted with CH2Cl2 (30 mL), filtered through a pad of Celite® and washed with brine (50 mL). The aqueous layer was extracted with CH2Cl2 (3 × 50 mL) and the combined organic extracts were dried over anhydrous MgSO4 and concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 1:3) to give the title compound 33b (2.01 g, 88%) as a colorless oil. [α]D23.4 -9.0 (c 0.71 in CHCl3) (lit26 -8.8 (c 0.65 in CHCl3)); 1H NMR (400 MHz, CDCl3) δ 7.74 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.2 Hz), 7.38 (2H, t, J = 7.4 Hz), 7.30 (2H, t, J = 7.4 Hz), 6.02 (1H, d, J = 8.0 Hz), 4.53-4.48 (1H, m), 4.39 (2H, d, J = 7.0 Hz), 4.22 (1H, t, J = 7.0 Hz), 3.83-3.74 (1H, m), 3.67 (1H, dd, J = 11.3, 3.3 Hz), 3.52 (1H, dd, J = 11.3, 6.2 Hz), 3.02 (1H, dd, J = 13.9, 4.3 Hz), 2.95 (1H, dd, J = 13.9, 6.1 Hz), 2.75 (1H, dd, J = 13.6, 4.2 Hz), 2.62 (1H, dd, J = 13.6, 7.8 Hz), 1.48 (9H, s); 13C NMR (100 MHz, CDCl3) δ 169.8, 156.1, 143.8, 141.3, 127.7, 127.1, 125.1, 120.0 83.0, 71.0, 67.2,

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65.2, 54.5, 47.1, 36.4, 35.5, 28.0. Spectroscopic data was consistent with that reported in literature.26 Tert-Butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S((S)-2,4-dihydroxybutyl)-L-cysteinate (34a): To a stirred solution of Nα,Nα-bis-Fmoc-L-cystine bis(tert-butyl ester) (2735, 2.01 g, 2.53 mmol) in CH2Cl2 (14 mL) at 0 °C was added zinc powder (1.15 g, 17.51 mmol) and a freshly prepared mixture of methanol, conc. hydrochloric acid and conc. sulfuric acid (100:7:1, 7 mL). The resultant mixture was allowed to stir at 0 °C for 30 min after which was added (S)-2-(oxiran-2-yl)ethan1-ol (29a28, 0.89 g, 10.11 mmol). The reaction mixture refluxed at 70 °C for 17 h. The mixture was then diluted with CH2Cl2 (30 mL), filtered through a pad of Celite® and washed with brine (50 mL). The aqueous layer was extracted with CH2Cl2 (3 × 50 mL) and the combined organic extracts were dried over anhydrous MgSO4 and concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 1:3) to give the title compound 34a (2.17 g, 88%) as a colorless oil. [α]D22 +8.5 (c 0.3 in CHCl3); νmax(neat)/cm-1 3347, 2976, 1703, 1518, 1449, 1413, 1369, 1335, 1249, 1151; 1H NMR (400 MHz, CDCl3) δ 7.74 (2H, d, J = 7.5 Hz), 7.60 (2H, d, J = 7.2 Hz), 7.38 (2H, t, J = 7.4 Hz), 7.30 (2H, t, J = 7.5 Hz), 6.04 (1H, d, J = 8.0 Hz), 4.51-4.47 (1H, m), 4.37 (2H, d, J = 8.2 Hz), 4.21 (1H, t, J = 7.1 Hz), 3.91-3.88 (1H, m), 3.83-3.73 (2H, m), 3.01 (1H, dd, J = 13.9, 4.6 Hz), 2.94 (1H, dd, J = 13.9, 5.9 Hz), 2.76 (1H, dd, J = 13.6, 3.8 Hz), 2.57 (1H, dd, J = 13.5, 8.4, Hz), 1.72-1.67 (2H, m), 1.48 (9H, s); 13C NMR (100 MHz, CDCl3) δ 169.8, 156.1, 143.8, 141.3, 127.7, 127.1, 125.1, 120.0, 83.0, 69.9, 67.2, 60.7, 54.7, 47.1, 40.9, 37.6, 35.5, 28.0; HRMS (ESI+) [M + Na]+ 510.1921 calc for C26H33NNaO6S 510.1921 observed. Tert-Butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S((R)-2,4-dihydroxybutyl)-L-cysteinate (34b): To a stirred solution of Nα,Nα-bis-Fmoc-L-cystine bis(tert-butyl ester) (2735, 1.59 g, 2.06 mmol) in CH2Cl2 (10 mL) at 0 °C was added zinc powder (0.94 g, 14.42 mmol) and a freshly prepared mixture of methanol, conc. hydrochloric acid and conc. sulfuric acid (100:7:1, 5 mL). The resultant mixture was allowed to stir at 0 °C for 30 min after which was added (R)-2-(oxiran-2-yl)ethan1-ol (29b28, 0.73 g, 8.24 mmol). The reaction mixture refluxed at 70 °C for 17 h. The mixture was then diluted with CH2Cl2 (30 mL), filtered through a pad of Celite® and washed with brine (50 mL). The aqueous layer was extracted with CH2Cl2 (3 × 50 mL) and the combined organic extracts were dried over anhydrous MgSO4 and concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 1:3) to give the title compound 34b (1.72 g, 88%) as a colorless oil. [α]D20.2 -3.5 (c 0.32 in CHCl3); νmax(neat)/cm-1 3347, 2976, 1703, 1518, 1449, 1413, 1369, 1335, 1249, 1151; 1H NMR (400 MHz, CDCl3) δ 7.77 (2H, d, J = 7.5), 7.61 (2H, d, J = 7.2 Hz), 7.40 (2H, t, J = 7.4 Hz), 7.32 (2H, t, J = 7.5 Hz), 5.81 (1H, d, J = 8.0 Hz), 4.53-4.50 (1H, m), 4.40 (2H, d, J = 6.8 Hz), 4.23 (1H, t, J = 7.0 Hz), 3.94-3.88 (1H, m), 3.85-3.81 (2H, m), 3.03 (1H, dd, J = 14.0, 4.2 Hz), 2.94 (1H, dd, J = 14.3, 6.1 Hz), 2.82 (1H, dd, J = 14.0, 2.9 Hz), 2.56 (1H, dd, J = 14.0, 9.0 Hz), 1.74-1.71 (1H, m), 1.50 (9H, s); 13C NMR (100 MHz, CDCl3) δ 169.7, 156.0, 143.6, 141.0, 127.5, 126.9, 125.0, 120.0, 82.6, 69.5, 67.0, 60.2, 54.5, 46.9, 40.5, 37.5, 35.2, 27.8; HRMS (ESI+) [M + Na]+ 510.1921 calc for C26H33NNaO6S 510.1921 observed. Tert-Butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S((R)-2,5-dihydroxypentyl)-L-cysteinate (35): To a stirred solution of Nα,Nα-bis-Fmoc-L-cystine bis(tert-butyl ester) (2735, 0.751 g, 0.94 mmol) in CH2Cl2 (5 mL) at 0 °C was added zinc powder (0.508 g, 7.78 mmol) and a freshly prepared mixture of


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methanol, conc. hydrochloric acid and conc. sulfuric acid (100:7:1, 2 mL). The resultant mixture was allowed to stir at 0 °C for 30 min. The mixture was then allowed to stir at 70 °C for 5 min after which was added (R)-tert-butyldimethyl(3-(oxiran2-yl)propoxy)silane (3037, 0.839 g, 3.88 mmol). The reaction mixture was allowed to stir at 65 °C for 19 h. The mixture was then diluted with EtOAc (50 mL), filtered through a pad of Celite® and washed with brine (50 mL). The aqueous layer was extracted with EtOAc (3 × 50 mL) and the combined organic extracts were dried over anhydrous Na2SO4 and concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 1:3) to give the title compound 35 (0.568 g, 60%) as a colorless oil. [α]D21.0 -26.7 (c 0.03 in CHCl3); νmax(neat)/cm-1 3321, 2931, 1706, 1532, 1450, 1369, 1248, 1152, 1050; 1H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.2 Hz), 7.40 (2H, t, J = 7.4 Hz), 7.31 (2H, t, J = 7.4 Hz), 5.90 (1H, d, J = 7.8 Hz), 4.51 (1H, dd, J = 12.3, 5.2 Hz), 4.39 (2H, d, J = 7.1 Hz), 4.23 (1H, t, J = 7.1 Hz), 3.733.58 (3H, m), 3.03 (1H, dd, J = 13.9, 4.4 Hz), 2.95 (1H, dd, J = 13.9, 5.7 Hz), 2.80 (1H, dd, J = 13.6, 2.9 Hz), 2.53 (1H, dd, J = 13.6, 8.9 Hz), 1.72-1.61 (4H, m), 1.49 (9H, s)); 13C NMR (100 MHz, CDCl3) δ 169.8, 156.1, 143.9, 141.1, 127.9, 127.2, 125.3, 120.1, 83.2, 70.1, 67.3, 62.8, 54.7, 47.2, 41.2, 35.5, 33.4, 29.2, 28.1; HRMS (ESI+) [M + Na]+ 524.2077 calc for C27H35NNaO6S 524.2075 observed. Tert-Butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S((R)-2,6-dihydroxyhexyl)-L-cysteinate (36): To a stirred solution of Nα,Nα-bis-Fmoc-L-cystine bis(tert-butyl ester) (2735, 0.500 g, 0.649 mmol) in CH2Cl2 (5 mL) at 0 °C was added zinc powder (0.300 g, 4.54 mmol) and a freshly prepared mixture of methanol, conc. hydrochloric acid and conc. sulfuric acid (100:7:1, 2 mL). The resultant mixture was allowed to stir at 0 °C for 30 min. The mixture was then allowed to stir at 70 °C for 5 min after which was added (R)-tert-butyldimethyl(4-(oxiran2-yl)butoxy)silane (3138, 0.600 g, 2.60 mmol). The reaction mixture was allowed to stir at 65 °C for 19 h. The mixture was then diluted with EtOAc (50 mL), filtered through a pad of Celite® and washed with brine (50 mL). The aqueous layer was extracted with EtOAc (3 × 50 mL) and the combined organic extracts were dried over anhydrous Na2SO4 and concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 4:1 → 1:3) to give the title compound 36 (0.553 g, 83%) as a colorless oil. [α]D21.2 -25.0 (c 0.07 in CHCl3); νmax(neat)/cm-1 3343, 2934, 2862, 1705, 1513, 1450, 1369, 1344, 1248, 1152; 1H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.0 Hz), 7.40 (2H, t, J = 7.4 Hz), 7.30 (2H, td, J = 11.2, 1.1 Hz), 5.88 (1H, d, J = 7.8 Hz), 4.52 (1H, dd, J = 12.5, 5.2 Hz), 4.39 (2H, d, J = 8.1 Hz), 4.23 (1H, t, J = 7.1 Hz), 3.70-3.59 (3H, m), 3.03 (1H, dd, J = 13.7, 4.7 Hz), 2.94 (1H, dd, J = 13.7, 5.4 Hz), 2.80 (1H, dd, J = 13.6, 3.4 Hz), 2.51 (1H, dd, J = 13.4, 8.7 Hz), 1.60-1.38 (15H, m); 13 C NMR (100 MHz, CDCl3) δ 169.7, 156.0, 143.8 , 141.3 , 127.8 , 127.1 , 125.2 , 120.0 , 83.1 , 69.8 , 67.2 , 62.5 , 54.6 , 47.1 , 41.1 , 35.8 , 35.4 , 32.4 , 28.0 , 21.9 ; HRMS (ESI+) [M + Na]+ 538.2226 calc for C28H37NNaO6S 538.2234 observed. Tert-Butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S((R)-2,7-dihydroxyheptyl)-L-cysteinate (37): To a stirred solution of Nα,Nα-bis-Fmoc-L-cystine bis(tert-butyl ester) (2735, 0.30 g, 0.375 mmol) in CH2Cl2 (1 mL) at 0 °C was added zinc powder (0.20 g, 3.01 mmol) and a freshly prepared mixture of methanol, conc. hydrochloric acid and conc. sulfuric acid (100:7:1, 1 mL). The resultant mixture was allowed to stir at 0 °C for 30 min after which was added (R)-tert-butyldimethyl((5(oxiran-2-yl)pentyl)oxy)silane (3239, 0.344 g, 1.13 mmol). The

reaction mixture refluxed at 70 °C for 17 h. The mixture was then diluted with EtOAc (30 mL), filtered through a pad of Celite® and washed with brine (30 mL). The aqueous layer was extracted with EtOAc (3 × 30 mL) and the combined organic extracts were dried over anhydrous MgSO4 and concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 1:3) to give the title compound 37 (0.350 g, 88%) as a colorless oil. [α]D20.8 -20.0 (c 0.03 in EtOAc); νmax(neat)/cm-1 3365, 3933, 1703, 1514, 1450, 1369, 1343, 1248, 1151, 1046; 1H NMR (400 MHz, MeOD) δ 7.79 (2H, d, J = 7.5 Hz), 7.68 (2H, d, J = 7.4 Hz), 7.39 (2H, t, J = 7.4 Hz), 7.31 (2H, t, J = 4.7 Hz), 4.34 (2H, d, J = 7.1 Hz), 4.28 (1H, dd, J = 8.2, 5.1 Hz), 4.23 (1H, t, J = 7.0 Hz), 3.72-3.61 (1H, m), 3.57-3.79 (2H, m), 3.01 (1H, dd, J = 13.8, 5.0 Hz), 2.86 (1H, dd, J = 13.7, 8.3 Hz), 2.69 (1H, dd, J = 13.4, 4.9 Hz), 2.60 (1H, dd, J = 13.4 , 7.0 Hz), 1.57-1.34 (17H, m); 13C NMR (100 MHz, MeOD) δ 171.8, 158.1, 145.3, 142.6, 128.8, 128.2, 126.4, 121.0, 83.3, 71.9, 68.2, 62.9, 56.5, 50.2, 40.8, 37.3, 35.5, 33.6, 28.3, 26.9, 26.6; HRMS (ESI+) [M + Na]+ 552.2390 calc for C29H39NNaO6S 552.2393 observed. Biology. Human TLR2 agonism: Human TLR2 agonism by was investigated using HEK-Blue™-hTLR2 cells and HekBlue™ Detection medium (Invivogen) across an 8-log10 dilution series (109 fM to 102 fM). Agonists were diluted and incubated with HEK-Blue™-hTLR2 cells for 16h, and well absorbance (ABS) then determined at 655nm using an Ultramark™ microplate reader (BioRad). Data presented as normalized mean +/- SEM ABS, for >triplicate wells, following background subtraction. EC50 (nM) values were determined by nonlinear regression curve fit of normalised ABS (655nm) values using Prism 7 software (GraphPad).

ASSOCIATED CONTENT Supporting Information The Supporting information is available free of charge on the ACS Publications website at DOI: Molecular formula strings (CSV) Experimental procedures and characterization of intermediates and final compounds (1H, 13C spectra, and HPLC traces) and raw biological data (PDF)

AUTHOR INFORMATION Corresponding Author *Tel: +64 9 9238259. Fax: +64 9 3737422. E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT We thank the Maurice Wilkins Centre for Molecular Biodiscovery for financial support.

ABBREVIATIONS ABS, absorbance; DIC, N,N’-diisopropylcarbodiimide; DMAP, N,N-dimethylaminopyridine; DMF, dimethylformamide; Fmoc, 9fluorenylmethyloxycarbonyl; DODT, 2,2’-ethylenedioxy)diethanethiol; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; HEK, human



embryonic kidney; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NMM, N-methylmorpholine; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; SEAP, secreted embryonic alkaline phosphatase; SEM, standard error of the mean; TBS, tert-butyldiemthylsilyl; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLR, Toll-like receptor; Val, valine.

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Synthesis and evaluation of novel TLR2 agonists as potential

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