N-Tetradecylcarbamyl Lipopeptides as Novel Agonists for Toll-like

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Brief Article

N-tetradecylcarbamyl lipopeptides as agonists for Toll-like receptor 2 Marian M.J.H.P Willems, Gijs G. Zom, Selina Khan, Nico Meeuwenoord, Cornelis J. M. Melief, Mario van der Stelt, Herman S. Overkleeft, Jeroen D. C. Codée, Gijsbert A. van der Marel, Ferry Ossendorp, and Dmitri V. Filippov J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500722p • Publication Date (Web): 14 Jul 2014 Downloaded from http://pubs.acs.org on July 15, 2014

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Journal of Medicinal Chemistry

N-tetradecylcarbamyl lipopeptides as novel agonists for Toll-like receptor 2 Marian M. J. H. P. Willemsa, Gijs G. Zomb, Selina Khanb, Nico Meeuwenoorda, Cornelis J.M. Meliefb# Mario van der Stelta, Herman S. Overkleefta, Jeroen D. C. Codéea, Gijsbert A. van der Marela, Ferry Ossendorpb, Dmitri V. Filippova* a

Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands, bDepartment of Immunohematology and Bloodtransfusion, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands TLR2, synthetic ligand, lipopeptides, dendritic cell, solid phase synthesis

ABSTRACT: New analogues (UPam) of triacylated lipopeptide Pam3CysSK4, a popular agonist of Toll-like receptor 2 (TLR2) were designed making use of the co-crystal structure of a TLR2 heterodimer (with TLR1) with Pam3CysSK4. An array of 22 UPam derivatives that feature an N-tetradecylcarbamyl chain to mimic the native N-palmitoyl moiety and various small amino acids residues at the penultimate N-terminal position was prepared via solid-phase synthesis. In vitro evaluation of immunostimulatory properties revealed new potent TLR2 ligands.

INTRODUCTION Lipoproteins that are part of the outer membrane of Grampositive and gram-negative bacteria share an unusual Nterminal amino acid S-(2,3-dihydroxypropyl)-L-cysteine acylated by fatty acids. The pattern recognition receptor of the innate immune system for these lipoproteins is Toll-like Receptor 2 (TLR2) and it was discovered that activation of TLR2 can be attained with S-(2,3-bispalmitoyloxypropyl)-Npalmitoyl-Cys-Ser-Ser-Asn-Ala (Pam3CSSNA, 1A), that originally was isolated from natural sources.1,2 On the basis of this finding, a series of lipopeptide derivatives have been designed to assess structure-activity relations (SAR) for optimal ligand recognition (Figure 1, 1A-D). 1,3 These studies led to the synthesis of a number of derivatives bearing two to six amino acids that showed an activity comparable to the natural lipoprotein. 3-8 In contrast, truncated derivatives of 1A bearing only one or no amino acid in the peptide fragment were found to be inactive. These results suggest that elongation of the S-(2,3-bispalmitoxyloxypropyl)-N-palmitoylCys moiety by an oligopeptide of a certain length and composition is necessary for immunological activity.In this framework Reitermann reported the synthesis and immunological evaluation of a series of triacylated lipopeptide

derived ligands, including Pam3CysSSNA (1A), Pam3CysSK4 (1B), Pam3CysS (1C) and Pam3CysAG (1D).9 It appeared that Pam3CysSK4 (Figure 1, 1B) was the most potent immunostimulating agent out of this series with the additional advantage of improved solubility in water due to the presence of four hydrophilic lysine residues.9 These advantageous properties have made Pam3CysSK4 one of the most applied TLR2 ligands.10,11 A later attempt to improve the potency of O

H N

NH

13

O 14

O

peptide

S

O O 14

O

1A: 1B: 1C: 1D:

peptide peptide peptide peptide

= = = =

Ser-Ser-Asn-Ala Ser-Lys-Lys-Lys-Lys Ser Ala-Gly

1B did not yield a more potent Pam3Cys derivative.8 Figure 1. Pam3Cys-peptides as TLR2 ligands

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In more recent studies, the structures of TLRs in the absence and in complex with the corresponding ligands have been investigated with X-ray crystallography. TLR2 is a unique member of the TLR family as it can form heterodimers with TLR1 and TLR6.12-14 The heterodimer combination determines the ligand specificity. The TLR2/6 complex recognizes the di-acylated ligand Pam2CysSK4, whereas the TLR1/2 complex recognizes tri-acylated lipopeptides such as Pam3CysSK4 (1B). The crystal structure (PDB entry 2Z7X) of the TLR1/2 heterodimer co-crystallized with the Pam3CysSK4 ligand12 shows that the two fatty esters at the glycerol moiety in 1B interact with a pocket formed at the convex side of the TLR2 protein while the N-terminal fatty chain fits in a hydrophobic site of the TLR1 protein (Figure 2). Overall, binding of Pam3CysSK4 with the receptor results in an M-shaped TLR1/2 dimer that is further stabilized by internal hydrogen-bonding and ionic interactions.

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H 2N H N 13 14

O

H N O

R N H

H 2N O

H N

N H

O

S

O NH 2

O

O

H N

O NH 2

N H

O NH 2 K4

O O

14

OH

1, R:

16, R:

9, R:

SH

N 2, R = H CH3

3, R:

17, R:

10, R: NH 2

11, R:

NH 2 18, R:

4, R:

S

S

12, R: O

5, R:

13, R:

6, R:

NH 2 19, R:

SH

20, R:

14, R:

7, R:

H N 13

H N O

O

O N H

S

n * R

N H

K4

O 14

O

14

n=0, * = D-configuration

8, R:

O

SH O

15, R:

n=0, * = D-configuration

21, R = H, n = 1 NH 2

Figure 2. Parts of the crystal structure of TLR1/2 co-crystallized with Pam3CysSK4 (2Z7X). Top image: The carbonyl of the backbone amide of Phe312 is in close proximity with α-CH2 of Npalmitoyl chain of the ligand (steel gray) as visualized by the red line of 2.99 Å length. Bottom image: The Ser residue of the ligand resides in a fairly small pocket that possesses both a hydrophobic (orange) and a hydrophilic (blue) face. This pocket can be filled with small amino acid side chains of different nature. (The images were rendered with Chimera software package15)

As a part of our on-going effort16-19 to develop TLR-ligands for application as immuno-potentiating components (adjuvants) of synthetic antitumor vaccines20 we set out to analyse the co-crystal structure published by Jin et al. in order to design a more active TLR2-agonist.

n=1, * = L-configuration 22, R: Figure 3. Structures of the members of UPam library. The letter ”U” in “UPam” refers to the urea moiety inserted between the C14-alkyl chain and the α-CH of the N-terminal modified Cys residue of the peptide segment.

RESULTS AND DISCUSSION Inspection of the crystal structure of the TLR1/2 heterodimer cocrystallized with the Pam3CysSK4 ligand reveals that an extra hydrogen bridge with the oxygen of the phenylalanine-312 residue can be formed (Figure 2, Top image) by substitution of the α-CH2 of the fatty acid amide in the ligand with a NH, resulting in the N-alkylcarbamyl derivative UPam-Ser (Figure 3, 1, Supplementary Figure S3). The estimated distance of 2.99 Å (Figure 2, Top Image) matches perfectly the typical distance between a donor and an acceptor atom of hydrogen bond. We hypothesized that the potential formation of an additional hydrogen bridge by UPam-Ser (see Supplementary Figure S3) would lead to an improved TLR2 agonist. Next to this, replacement of serine by another amino acid may as well improve the binding of the ligand with the receptor. To verify these assumptions a focused library of UPam derivatives (Figure 3) was


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projected in which the serine residue of UPam-Ser (1) is replaced by a number of natural and non-proteinogenic amino acids. Alongside serine (in 1) the other amino acids were selected on the basis of differences in the nature, length or bulkiness of the side chains to probe the steric, polar and electronic requirements for binding to TLR2 (Figure 2, Bottom). Amino acids with aliphatic chains of varying length were probed as Ser replacement

starting with a nearly perfect isostere of serine (Abu in 4).21 The length of the aliphatic side chain was either increased further by the use of norvaline and norleucine residues to provide 5 (UPam-NVal) and 6 (UPam-Nle) or decreased by incorporating an alanine residue to give 3 (UPam-Ala). To investigate the effect of a missing chain a glycine residue was implemented (UPam-Gly, 2). Allyl glycine, both as L- and Denantiomer, (in 7 and in 8), propargyl glycine (in 9) and cyanoalanine (in 10) were chosen by virtue of their unsaturated carbon chains. The presence of sp and sp2 hybridized carbons in the latter substituents would allow to probe subtle requirements the binding pocket of the receptor imposes on the ligand.

Figure 4. Dendritic cell maturation by UPam derivatives at indicated concentrations as measured by IL-12p40 cytokine production, in ng/ml. Top and middle panels: UPam derivatives 1, 2, 4 - 9, 12 - 18, 20 - 22 and Pam3CysSK4; Bottom panel: UPam derivatives 1, 3, 10, 11, 19. Error bars represent standard deviation of triplicates.

To evaluate the effect of an amine and amide in the side chain the residues of 2,4-diaminopropanoic acid (in 11) 2,4diaminobutanoic acid (in 12), and asparagine (in 13) were incorporated. The library contains several lipopeptides featuring a sulphur atom in the side chains of the amino acid residues substituting for Ser of UPam-Ser (1). Beside L- and D-cysteine, which are isosteric to serine (in 14 and 15, respectively), the more bulky amino acid 3-mercaptovaline (in 16), 2-thienylglycine (in 17) and 3-thienylalanine (in 18) were incorporated as well. To study the effect of an extended peptide backbone β-alanine (in 21) and β-lysine (in 22) were employed. Finally, phenylalanine and naphthyl alanine residues containing UPam derivatives (19 and 20) were added to the library because the bulky aromatic side chains were expected to fit poorly in the receptor. The library of twenty-two UPam derivatives (1-22) was constructed with the aid of a straightforward SPPS protocol (Supporting Information, Scheme S1). Subsequent RP-HPLC purification provided pure UPam derivatives in 3% - 27% overall yield. To examine the immunostimulatory activity of the obtained UPam derivatives the compounds were tested for their ability to stimulate the maturation of dendritic cells (DCs). Towards this end all members of the UPam library were incubated with murine DCs at 4 different concentrations and the production of the immunostimulatory cytokine IL-12p40 was measured (Figure 4). The DC maturing capacity was also independently analyzed by the cell surface upregulation of co-stimulatory molecule CD86 (see Supplementary Information, Figure S1). The testing of the UPam library has been performed in two rounds. In the first round of testing the founding member of the library UPam-Ser 1 was compared to the prototype Pam3CSK4 (1B) together with the UPam variants 2, 4 - 9, 12 18, 20 – 22 (Figure 4, top panel). The newly synthesized ligand 1 turned out to mature the DC better than the parent Pam3CSK4, even in the picomolar range (see Figure 4, Top Panel). Therefore 1 was subsequently used in the second round of testing as a reference compound to evaluate the maturing capacity of the other compounds of the UPam library. It became apparent that the lipopeptides modified with amino acid residues containing small, mostly hydrophobic side chains showed significant bioactivity (4, 5, 7, 9). From all derivatives 2-aminobutanoic acid (4), containing an ethyl group instead of a hydroxymethyl proved to be most active. Larger alkyl chains such as propyl (5) or butyl (6) were less beneficial, while bulky side-chains were, as expected, detrimental for the immunostimulatory activity (16, 20). The UPam derivatives with unsaturated side chains such as in 7 and 9 were more active than the saturated counterpart 5, but did not quite reach the activity of 4. The high activity and potency of ligands 12 and 13 bearing polar side chains capable of strong non-covalent interactions such as hydrogen bonding (carboxamide in 13) or salt-bridges (amino group in 12) points to a priori unexpected but favourable interactions of these UPam derivatives and the receptor. When allylglycine containing UPam ligand (7) was compared with the corresponding D-amino acid containing derivative 8 it was shown that D-allyl glycine derived ligand 8 showed barely any activity. This is in line with previously reported findings that amino acids residues of D-configuration render lipopeptide based TLR2 ligands inactive.22 A similar drop in activity was



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observed for L- and D-cysteine, in 14 and in 15, respectively. The low potency of 14 with its relatively small and hydrophobic mercaptomethyl side chain is surprising and hard to explain. It is, however, noteworthy that other sulphur containing side chains in the UPam derivatives 16 - 18 proved to be rather detrimental for the activity. The substitution with glycine (in 2) gave consistently lower activity than 1 and compounds with β-alanine (21) and β-lysine (22) modifications were almost inactive. The side chain in the βsubstituted amino acids is shifted in comparison with the αamino acids and likely unable to fit properly in the binding pocket of the receptor. The results of the first round of the biological testing described above were largely corroborated by the DC maturation assay that was performed in the second round of testing on an additional set of UPam derivatives 3, 10, 11, 19 (Figure 4, bottom panel). The small alkyl side chain (methyl in 3) and small unsaturated moiety (cyanomethyl in 10) showed improved activity while relatively bulky benzyl (19) demonstrated a significant activity drop in comparison with UPamSer 1. Somewhat counter-intuitively, and in sharp contrast to UPamAbu (4) and UPamDab (12), UPamDab (11) proved to be poorly active despite of its small aminomethyl side chain that is also capable to form salt or hydrogen bridges. The latter finding is unexpected and at this moment remains unexplained. The maturation capacity of the UPam derivatives has been independently corroborated by measuring surface expression of CD86 (Supplementary figure S1). We also show that the effect of the N-carbamyllipopeptides is TLR2 dependent by measuring the activation of TLR2transfected HEK293 reporter cells. (Supplementary Figure S2). The UPam derivatives that have shown activity in DC maturation assays also demonstrated dose-dependent activation of HEK293 cells transfected with human TLR2 following the same trend as observed for DC maturation. UPam derivatives 3, 4, 5, 7, 9 and 10, that contain small, mostly hydrophobic side chains substituting for the hydroxymethyl of 1 show an increased potency. In line with findings from Pam3Cys-series8 the amino acids with straight alkyl chains of moderate size terminated with a polar functional group (see 12 and 13) are favorable for the activity. The derivatives 8, 14, 15, 20 and 22 were hardly active (Supplementary Figure S2). CONCLUSION A novel analogue of Pam3Cys, termed UPam, containing a N-tetradecylcarbamyl moiety as a substitute for the native Npalmitoyl chain was designed on the basis of the X-ray structure of the TLR1/TLR2 dimer co-crystallized with a Pam3Cys based ligand. The new TLR2-ligand (UPam-Ser) proved to be more potent than the original ligand supporting the idea that replacing the amide moiety at the N-terminus of the original ligand for a urea improves the immunostimulatory activity. This is possibly due to gain of an additional hydrogen bridge to the TLR1 subunit of the receptor heterodimer.23 Modification of the serine in the UPam based ligand led to several derivatives (UPamAbu 4, UPamAla 3, UPamCNA 10 and UPamDab 12) with further improved immunostimulatory activity. The results are in line with the postulation that the small side chains are able to fit into a small pocket of TLR2 subunit. The new ligands are a valuable asset to the pallet of structurally defined TLR ligands that are useful as immune

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stimulatory compounds. The clinical evaluation of a UPam derivative is at present ongoing. EXPERIMENTAL SECTION All reagents and solvents used in the solid phase peptide synthesis were purchased from Bachem and Biosolve and used as received. Palmitoyl-Cys((RS)-2,3-di(palimitoyloxy)propyl)-OH was purchased from Bachem, Fmoc-amino acids from Novabiochem and HATU and tetradecyl isocyanate from Tebu Bio. Tentagel based resins were ordered from Rapp Polymere. All tested compounds had a purity of ≥ 95%, as shown by RP HPLC. H-Lys-Lys-Lys-Lys-NH2 Peptide synthesis was performed on a 1 mmol scale using an ABI 433A automated instrument applying Fmoc based protocol starting form Rink Amide S Tentagel (loading 0.26 mmol/g). The resin, after final Fmoc deprotection, was washed with NMP and DCM and dried. On 23 µmol of the resin was performed a HCTU coupling with Fmoc-Phe-OH to increase retention for LCMS analysis. This aliquot was transferred to a tube and treated for 104 minutes with a cleavage cocktail TFA/TIS/H2O (95/2.5/2.5). The solution was filtered and the peptide was precipitated out of solution with Et2O (50 mL) and the resin was washed with neat TFA. The Et2O was centrifuged, removed and the precipitated was washed with Et2O. The washing was repeated (3x). After centrifugation the Et2O was removed and the precipitate of Fmoc-FKKKK-NH2 was air dried and dissolved in MeCN:H2O:tBuOH (1:1:1). LC/MS: Rt = 8.40 min (C18 Alltima, 0 - 50% MeCN, 15 min run); ESI-MS: m/z 899.55 [M+H]+. General procedure coupling amino acids Fmoc-Xxx-OH One mmol H-Lys-Lys-Lys-Lys preloaded resin was suspended in DCM : NMP (30 : 15 mL) in total volume of 50 mL to pipette 1 mL portions of 20 µmol resin, followed by draining the syringes. Appropriate Fmoc-amino acids (see Table S1, Supplementary Information) were pre-activated with a stock solution of 0.5 mL 0.22 M PyBOP in DCM:NMP (2:1) and 36.5 µL DiPEA and added to the 20 µmol resin in separate syringes. The resulting mixture was reacted for 1h by shaking followed by a NMP wash. The resin was capped 3 x 15 min with 1mL 0.5M Ac2O : DiPEA (2:1) in NMP followed by a NMP and DCM wash. The N-terminal Fmoc group was removed in a 3 x 3 min cycle of 20% piperidine in NMP continued with an NMP and DCM wash. General procedure coupling Fmoc-Cys((RS)-2,3di(palimitoyloxy)-propyl)-OH 20 µmol tentagel S Ram resin loaded with H-Xxx-Lys-LysLys-Lys was treated with a 0.5 mL stock solution of 0.18 M Fmoc-Cys((RS)-2,3-di(palimitoyloxy)-propyl)-OH in 0.22 M PyBOP in DCM : NMP (2 : 1). The resulting mixture was activated with 2 x 44 µmol DiPEA over 15 min. and reacted by shaking for 18h followed by NMP and DCM wash. The resin was swelled in DCM : NMP again and divided in portions of 10 µmol. General procedure coupling tetradecyl isocyanate The 10 µmol resin loaded with Fmoc-Cys((RS)-2,3-di(palimitoyloxy)propyl)-Xxx-Lys-Lys-Lys-Lys was swollen in DCM : NMP (1 : 1) and treated with 3 x 3 min 20% piperidine in NMP for


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Fmoc-deprotection. After thoroughly NMP wash the portions resin were suspended in 1 mL DCM : NMP (1 : 1) and treated with 25 µL tetradecyl isocyanate. The mixture was shaken for 18, washed with NMP and DCM and air dried. The resin was treated for 104 minutes with a cleavage cocktail of 95% TFA, 2.5% TIS and 2.5% H2O. The solution was filtered and precipitated with Et2O (50 mL) and stored at -200C for 18h. The Et2O was centrifuged, removed and the precipitated was dissolved by sonification in 1 mL MeCN : H2O : tBuOH (1 : 1 :1 ). A small amount (50 µL) was used for LCMS analysis (Vidac C4 column). Obtained sequences were diluted with another 0.5 mL MeCN : H2O : tBuOH (1 : 1 : 1) and were purified on a semiprep Vidac C4 column (10 x 250 mm, 5 µm particle size, flow 5.0 mL/min, 60 - 100% MeCN.). UPam-Ser, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy) -propyl)-Ser-Lys-Lys-Lys-Lys-NH2 (1) 0.89 mg (0.59 µmol, 6%); LC/MS: Rt = 8.23 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1510.2 [M+H]+; HRMS Calcd for [C80H156N12O12S + H]+ 1510.17592, found 1510.17670. UPam-Gly, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Gly-Lys-Lys-Lys-Lys-NH2 (2) 1.07 mg (0.72 µmol, 7%); LC/MS: Rt = 11.06 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1480.2 [M+H]+; HRMS Calcd for [C79H154N12O11S + H]+ 1480.16535, found 1480.16613. UPam-Ala, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Ala-Lys-Lys-Lys-Lys-NH2 (3) 3.72 mg (2.50 µmol, 25%); LC/MS: Rt = 8.52 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1494.18 [M+H]+; HRMS Calcd for [C80H156N12O11S + H]+ 1494.18100, found 1494.18200 UPam-Abu, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Abu-Lys-Lys-Lys-Lys-NH2 (4) 3.22 mg (2.13 µmol, 21%); LC/MS: Rt = 8.31 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1508.2 [M+H]+; HRMS Calcd for [C81H158N12O11S + H]+ 1508.19665, found 1508.19725. UPam-Nva, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Nva-Lys-Lys-Lys-Lys-NH2 (5) 3.65 mg (2.40 µmol, 24%); LC/MS: Rt = 8.32 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1522.2 [M+H]+; HRMS Calcd for [C82H160N12O11S + H]+ 1522.20230, found 1522.21619. UPam-Nle, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Nle-Lys-Lys-Lys-Lys-NH2 (6) 2.97 mg (1.93µmol, 19%); LC/MS: Rt = 8.48 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1536.2 [M+H]+; HRMS Calcd for [C83H162N12O11S + H]+ 1536.22795, found 1536.22914. UPam-Agl, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-(S)-Agl-Lys-Lys-Lys-Lys-NH2 (7) 2.40 mg (1.58 µmol, 16%); LC/MS: Rt = 8.61 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1520.2 [M+H]+; HRMS Calcd for [C82H158N12O11S + H]+ 153, 1520.19665 found 1520.19748. UPam-D-Agl, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-(R)-Agl-Lys-Lys-Lys-Lys-NH2 (8) 1.52 mg (1.0 µmol, 10%); LC/MS: Rt = 8.57 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1520.2

[M+H]+; HRMS Calcd for [C82H158N12O11S + H]+ 1520.19665 found 1520.19760. UPam-Pra, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Pra-Lys-Lys-Lys-Lys-NH2 (9) 4.10 mg (1.70 µmol, 27%); LC/MS: Rt = 8.22 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1518.2 [M+H]+; HRMS Calcd for [C82H156N12O11S + H]+ 1518.18100, found 1518.18214. 1-tetradecylcarbamyl-Cys((RS)-2,3UPam-CNA, di(palimitoyloxy)-propyl)-CNA-Lys-Lys-Lys-Lys-NH2 (10) 3.3 mg (2.17 µmol, 22%); LC/MS: Rt = 8.10 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1519.18 [M+H]+; HRMS Calcd for [C81H155N13O11S + H]+ 1519.17625, found 1519.17775. UPam-Dap, 1-tetradecylcarbamyl -Cys((RS)-2,3di(palimitoyloxy)-propyl)-Dap-Lys-Lys-Lys-Lys-NH2 (11) 0.42mg (0.28 µmol, 3%); LC/MS: Rt = 7.29 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1509.19 [M+H]+; HRMS Calcd for [C80H157N13O11S + 2H]2+ 755.09959, found 755.10017. UPam-Dab, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Dab-Lys-Lys-Lys-Lys-NH2 (12) 2.37 mg (1,56 µmol, 16%); LC/MS: Rt = 10.75 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1523.2 [M+H]+; HRMS Calcd for [C81H159N13O11S + H]+ 1523.20755, found 1523.21037. UPam-Asn, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Asn-Lys-Lys-Lys-Lys-NH2 (13) 2.12 mg (1.38 µmol, 14%); LC/MS: Rt = 10.82 min (C4 Alltima, 10 - 90% MeCN, 15 min run); ESI-MS: m/z 1537.19 [M+H]+; HRMS Calcd for [C81H157N13O12S + H]+ 769.09705, found 769.09771. UPam-L-Cys, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-(R)-Cys-Lys-Lys-Lys-Lys-NH2 (14) 2.68 mg (1.76 µmol, 18%); LC/MS: Rt = 8.48 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1526.2 [M+H]+; HRMS Calcd for [C80H156N12O11S2 + H]+ 1526.15307, found 1526.15434. UPam-D-Cys, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-(S)-Cys-Lys-Lys-Lys-Lys-NH2 (15) 4.04 mg (2.56 µmol, 26%); LC/MS: Rt = 8.36 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1526.2 [M+H]+; HRMS Calcd for [C80H156N12O11S2 + H]+ 1526.15307, found 1526.15387. UPam-Pen, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Pen-Lys-Lys-Lys-Lys-NH2 (16) 2.84 mg (1.83 µmol, 18%); LC/MS: Rt = 8.53 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1555.19 [M+H]+; HRMS Calcd for [C82H160N12O11S2 + H]+ 1554.18437, found 1554.18555. UPam-Tgly, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Tgly-Lys-Lys-Lys-Lys-NH2 (17) 3.12 mg (2.00 µmol, 20%); LC/MS: Rt = 8.78 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1562.2 [M+H]+; HRMS Calcd for [C83H156N12O11S2 + H]+ 1562.15307, found 1562.15408. UPam-Thi, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Thi-Lys-Lys-Lys-Lys-NH2 (18) 2.06 mg (1.31 µmol, 13%); LC/MS: Rt = 8.78 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1576.2



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[M+H]+; HRMS Calcd for [C84H158N12O11S2 + H]+ 1576.16872, found 1576.16883. UPam-Phe, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Phe-Lys-Lys-Lys-Lys-NH2 (19) 3.1 mg (1.53 µmol, 15 %); LC/MS: Rt = 9.19 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1570.8 [M+H]+; HRMS Calcd for [C86H160N12O11S + H]+ 1570.21230, found 1570.21360 1-tetradecylcarbamyl-Cys((RS)-2,3UPam-Nal, di(palimitoyloxy)-propyl)-Nal-Lys-Lys-Lys-Lys-NH2 (20) 2.26 mg (1.39 µmol, 14%); LC/MS: Rt = 9.02 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1621.3 [M + H]+; HRMS Calcd for [C90H162N12O11S + H]+ 1621.23106, found 1621.23175. UPam-Bal, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Bal-Lys-Lys-Lys-Lys-NH2 (21) 3.76 mg (2.52 µmol, 25%); LC/MS: Rt = 8.61 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1494.2 [M+H]+; HRMS Calcd for [C80H156N12O11S + H]+ 1494.18100, found 1494.18130. UPam-Bly, 1-tetradecylcarbamyl-Cys((RS)-2,3di(palimitoyloxy)-propyl)-Bly-Lys-Lys-Lys-Lys-NH2 (22) 2.31 mg (1.48 µmol, 15%); LC/MS: Rt = 7.38 min (Vidac C4, 50 - 90% MeCN, 15 min run); ESI-MS: m/z 1565.3 [M+H] +; HRMS calcd for [C84H165N13O11S + H]+ 1565.25450, found 1565.25377.

ASSOCIATED CONTENT

In vitro DC maturation assay. The D1 dendritic cell line is a growth factor-dependent immature spleen-derived DC line from C57BL/6 (H-2b) mice. D1 cells were cultured as described.24 Test compounds were titrated in a 96-wells plate (Corning, Amsterdam, The Netherlands) in complete IMDM medium. Next, D1 cells were harvested, counted, and subsequently transferred to the 96-wells plates containing the test compound titrations, using approximately 40.000 cells per well. After 24 hours of incubation at 37°C, supernatant was taken from the wells for ELISA analysis (BioLegend, San Diego, USA) in which the amount of produced IL-12p40 was measured. After 48 hours, the DCs were stained with a fluorescent antibody directed against CD86 (eBioscience, San Diego, USA), and analyzed by flow cytometry acquiring 50,000 cells per sample.

Notes

HEK293-TLR2 activation. The HEK-TLR2 reporter cell line, HEK293 cells stably transfected with human TLR2 were obtained from Invivogen, Toulouse, France. HEK-TLR2 cells were cultured in complete IMDM medium, supplemented with 500 ug/ml geneticin. Test compounds were titrated in a flatbottom 96-wells plate (Corning, Amsterdam, The Netherlands) in complete IMDM medium to which 20.000 HEK-TLR2 cells per well were added. After 48 hours of incubation at 37°C, the supernatant was taken from the wells for ELISA analysis (BioLegend, San Diego, USA) to determine the amount of IL8 that was produced as a measure for TLR2-mediated activation.

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Supporting Information Available:

Detailed description of the solid-phase synthesis of UPam derivatives, supplementary figures depicting the DC maturation and the HEK293 cell activation data. This material is available free of charge via the Internet at http://pubs.acs.org.

Accession codes: PDB code for crystal structure of TLR1/2 cocrystallized with Pam3CysSK4 is 2Z7X.

AUTHOR INFORMATION Corresponding Author * [email protected]. Phone: +31-71-5273571.

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

Funding Sources The project has been funded by Dutch Cancer Society (KWF) grant UL2007-3906 (GZ) and TI Pharma project (MW, SK).

Conflict of interest: F. Ossendorp, C. Melief, S. Khan, D.V. Filippov, and G. van der Marel are inventors of a patent application related to the work in this article entitled “Ajuvant compound”, with publication number WO 2013/051936 and filing date October 4, 2012. #Present address of C.J.M.M.: ISA Pharmaceuticals, J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.

ABBREVIATIONS Abu, L-2-aminobutyric acid residue; Agl, L-allylglycyl; Bal, βalanyl; Bly, L-β-lysyl; CD86, Cluster of differentiation 86; CNA, L-3-cyanoalanyl; Dab, L-2,4-diaminobutyric acid residue; Dap, L2,3-diaminopropionic acid residue; DC, dendritic cell; HEK, human embryonic kidney; Nal, L-β-(2-naphthyl)-alanyl; Nle, Lnorleucyl; Nva, L-norvalyl; Pen, L-Penicillamine residue; Pra, Lpropargylglycyl; Tgly, L-2-thiophenylglycyl; Thi, β-(2-Thienyl)alanyl; TLR, Toll-like receptor.

REFERENCES (1) Braun, V. Covalent lipoprotein from outer membrane of Escherichia coli. Biochim. Biophys. Acta 1975, 415, 335-377. (2) Bessler, W.; Resch, K.; Hancock, E.; Hantke, K. Induction of lymphocyte-proliferation and membrane changes by lipopeptide derivatives of lipoprotein from outer membrane of Escherichia coli. Z. Immunitatsforsch. Immunobiol. 1977, 153, 1122. (3) Bessler, W. G.; Cox, M.; Lex, A.; Suhr, B.; Wiesmuller, K. H.; Jung, G. J. Synthetic lipopeptide analogs of bacterial lipoprotein are potent polyclonal activators for murine B lymphocytes. J. Immunol. 1985, 135, 1900-1905.


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C. J. M.; Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V.; van der Burg, S. H.; Ossendorp, F. Distinct uptake mechanisms but similar intracellular processing of two different Toll-like receptor ligand-peptide conjugates in dendritic cells. J. Biol. Chem. 2007, 282, 21145-21159. (19) Weterings, J. J.; Khan, S.; van der Heden, G. J.; Drijfhout, J. W.; Melief, C. J. M.; Overkleeft, H. S.; van der Burg, O. H.; Ossendorp, F.; van der Marel, G. A.; Filippov, D. V. Synthesis of 2-alkoxy-8-hydroxyadenylpeptides: towards synthetic epitope-based vaccines. Bioorg. Med. Chem. Lett. 2006, 16, 3258-3261. (20) Moyle, P. M.; Toth, I. Modern subunit vaccines: development, components, and research opportunities. ChemMedChem 2013, 8, 360-376. (21) Lu, W.; Apostol, I.; Qasim, M. A.; Warne, N.; Wynn, R.; Zhang, W. L.; Anderson, S.; Chiang, Y. W.; Ogin, E.; Rothberg, I.; Ryan, K.; Laskowski, M., Jr. Binding of amino acid side-chains to S-1 cavities of serine proteinases. J. Mol. Biol. 1997, 266, 441-461. (22) Wu, W.; Li, R.; Malladi, S. S.; Warshakoon, H. J.; Kimbrell, M. R.; Amolins, M. W.; Ukani, R.; Datta, A.; David, S. A. Structure-activity relationships in Toll-like receptor2 agonistic diacylthioglycerol lipopeptides. J. Med. Chem. 2010, 53, 3198-3213. (23) Crystal structure of murine TLR1 is not available however the high percentage of identity (84%) and similarity (88%) mouse TLR1 shows to human TLR1 in the area involved in the interaction with the ligand (Glu255-Ser345, human numbering) as well as strict conservation of 16 from 18 residues identified by Jin et al. (see ref 12) as those being involved in binding to the ligand (including the crucial Phe312) justifies this conclusion. (24) Winzler, C.; Rovere, P.; Rescigno, M.; Granucci, F.; Penna, G.; Adorini, L.; Zimmermann, V. S.; Davoust, J.; Ricciardi-Castagnoli, P. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J. Exp. Med. 1997, 185, 317-328.

H N

H N 13

O

S

R

O N H

H N Lys-Lys-Lys-Lys-NH 2 O

O 14 O

UPam derivatives

O O 14

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N-Tetradecylcarbamyl Lipopeptides as Novel Agonists for Toll-like

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