Swaminathan et al. 10.1073/pnas.0507656103. |
Fig. 5. (A) Raw data obtained from 30 automatic injections, first and second ones of 1-ml aliquots, and the remaining 10-ml aliquots of 4.26 mM Dap solution into 0.09 mM human PGRP-IaC solution. (B) Incremental heat per mole of added ligand (open squares) for the titration in A. (C) Raw data obtained from 50 automatic injections of 5-ml aliquots of 1.42 mM CL-PGN solution into 0.12 mM human PGRP-S solution. (D) Incremental heat per mole of added ligand (open squares) for the titration in C.
Fig. 6. Synthesis of branched PGN derivatives. (a) 20% piperidine in dimethylformamide (DMF). (b) PyBOP, HOBt, DIPEA, and AA (each 5´) followed by 20% piperidine in DMF. AA is one of the following compounds: Fmoc-d-Ala, Fmoc-l-Lys(ivDde), Fmoc-d-isoGln, Fmoc-l-Ala, and MurNAc. (c) 2% hydrazine in DMF. (d) Five cycles of HBTU, NMP, and Fmoc-l-Gly followed by 20% piperidine in DMF. (e) PyBOP, HOBt, DIPEA and AA (each 5´) followed by 20% piperidine in DMF. AA is one of the following compounds: Fmoc-d-Ala, Fmoc-l-Lys(Mtt), Fmoc-d-isoGln, Fmoc-l-Ala, and MurNAc. (f) 2% TFA, 1% TIS, DCM. (g) Pd/C, EtOH:H2O:1(N)HCl (2:1:0.01).
Supporting Materials and Methods
Peptidoglycan Recognition Protein
(PGRP) Production. Drosophila PGRP-LCx (residues 325–500) and PGRP-LCa (residues 343–520) were prepared similar to the human proteins C-terminal PGN-binding domain of PGRP-Ia (PGRP-IaC) (residues 177–341) and PGRP-S (residues 1–175) (1, 2). Briefly, Escherichia coli BL21(DE3) cells (Stratagene) were transformed with pT7-7 plasmids (Novagen) containing the respective PGRP gene. Seed cultures (300 ml) were grown overnight in Luria–Bertani (LB) medium containing 100 mg/ml ampicillin and used to inoculate 6 liters of LB containing identical antibiotic concentration. At an optical density of 0.6–0.8 at 600 nm, the culture was induced by addition of isopropyl-b-d-thiogalactoside to a final concentration of 1 mM. The cells were harvested by centrifugation after a 3-h induction period. Cell pellets were resuspended in 50 mM Tris·HCl (pH 8.5)/2 mM EDTA/100 mM NaCl/1 mM DTT/5% Triton X-100. Cells were lysed by sonication. Inclusion bodies were washed three times with 50 mM Tris·HCl (pH 8.5)/2 mM EDTA/100 mM NaCl/1 mM DTT/5% Triton X-100, and three times in the same buffer without surfactant. Washed inclusion bodies were solubilized overnight in 50 mM Tris·HCl (pH 8.5)/8 M urea/5 mM DTT. Insoluble aggregates were removed by ultracentrifugation. The PGRPs were folded by slow dilution into 100 mM Tris·HCl (pH 8.5)/0.9 M l-arginine-HCl/0.1 M l-arginine/2 mM EDTA/6.3 mM cysteamine/3.7 mM cystamine to a final concentration <60 mg/liter. After 5 days at 4°C, the folding mixture was concentrated 20-fold and dialyzed against 50 mM Hepes (pH 7.0). Folded proteins were purified by using a MonoS cation exchange column (Amersham Pharmacia Biosciences), followed by a Superdex 75 gel filtration column (Amersham Pharmacia Biosciences).Chemical Synthesis of PGN Analogs.
Synthesis of Compound 9 (Fig. 6): Sieber Amide resin (238 mg, 99.9 mmol, Novabiochem) was swelled in dry dimethylformamide (DMF, »120 min, 5 ml), treated with 20% piperidine in DMF (3 × 5 min, 3 × 3 ml), washed with freshly distilled DMF (3 × 5 ml), and then reacted with Fmoc-d-Ala-OH (62 mg, 0.39 mmol, Novabiochem) in DMF by using PyBOP (104 mg, 0.39 mmol) (Novabiochem), HOBt (26 mg, 0.39 mmol) (Aldrich), and DIPEA (70 ml, 0.39 mmol) (Alfa Aesar). Progress of the reaction was monitored by the Kaiser test. After completion of the coupling, the resin was washed with (3 × 5 ml) and the Fmoc protecting group was removed with 20% piperidine in DMF (3 × 5 min, 3 × 3 ml). The reaction cycle was repeated by using Fmoc-d-Ala-OH (62 mg, 0.39 mmol), Fmoc-l-Lys(ivDde)-OH (114 mg, 0.39 mmol), Fmoc-d-isoGln (73 mg, 0.39 mmol), Fmoc-l-Ala-OH (62 mg, 0.39 mmol), and, subsequently, 2-N-acetyl-1-b-O-allyl-4,6-benzylidene-3-muramic acid (83 mg, 0.39 mmol). The resin-bound glycopeptide was then treated with 2% hydrazine in DMF (3 × 5 min, 3 × 3 ml) to cleave the ivDde protecting group on the side chain of lysine. The resin was then washed with DMF (3 × 5 ml) and DCM (3 × 5 ml). The wet resin was transferred into a peptide synthesizer to assemble five consecutive Fmoc-Gly-OH (5 × 297 mg, 0.99 mmol), Fmoc-d-Ala (311 mg, 0.99 mmol), and Fmoc-Lys(Mtt)-OH (623 mg, 0.99 mmol) by using HBTU in NMO. The resin-bound branched glycopeptide was then transferred back to the reactor, washed with DMF (3 × 5 ml) and reacted with Fmoc-d-isoGln (73 mg, 0.39 mmol), Fmoc-l-Ala-OH (62 mg, 0.39 mmol), and, finally, 2-N-acetyl-1-b-O-allyl-4,6-benzylidene-3-muramic acid (83 mg, 0.39 mmol). The resulting resin-bound branched glycopeptide was washed with DMF (3 × 5 ml), dichloromethane (7 × 5 ml), and methanol (3 × 5 ml). The resin was dried in vacuo for 4 h, reswelled in DCM (»5 ml), and filtered. The glycopeptide was released by treatment of the resin with 2% trifluoroacetic acid (TFA) in DCM (10 × 2 ml). The combined washings were concentrated under reduced pressure and coevaporated with toluene (3 × 10 ml) to remove traces of TFA. The resulting product was purified by Sephadex G15 size exclusion column (Amersham Pharmacia Biosciences) chromatography to give compound 9 (63.3 mg, 34%) as a white amorphous solid. 1H-NMR (500 MHz, D2O): d 5.76–5.82 (2H, m, OCH2CHCH2), 5.13–5.20 (4H, m, OCH2CHCH2,), 4.40 (2H, d, H1, J = 8.10 Hz), 4.03–4.23 (15H, m, a CH, Ala × 5, a CH, Lys 2, a CH Gln × 2, a CH, Lac × 2, OCH2CHCH2 × 2), 3.64–3.92 (16H, m, H-2 × 2, H-6 × 2, CH2, Gly × 5), 3.59–3.42 (6H, m, H-3 × 2, H-4 × 2, H-5 × 2), 3.04–3.11 (2H, m, e-CH2, Lys(Gly)), 2.89 (2H, t, e-CH2, Lys), 2.27–2.30 (4H, m, g-CH2, Gln), 2.03 (2H, m, b-CH, Gln), 1.86–1.89 (8H, m, b-CH, Gln, NH(COCH3) × 2), 1.40–1.63 (8H, m, b-CH2, Lys, d-CH2, Lys), 1.25–1.32 (25 H, m, CH3 × 5, Ala, CH3 × 2 Lac, g-CH2, Lys). C-13 (HSQC): 118.50 (OCH2CHCH2), 100.44 (C-1), 83.19, 76.16, 69.34 (C-3, C-4, C-5), 78.97 (a-C, Lac), 71.15 (OCH2CHCH2), 61.32 (C-6), 55.10, 53.29, 50.28 (a-C, Ala, a-C, Gln, a-C, Lys), 43.26 (a-C, Gly), 39.45 (e-C, Lys), 32.02 (g-C, Gln), 27.61 (b-C, Gln), 22.79 (NHCOCH3), 30.82, 28.61, 22.59 (b-C, Lys, g-C, Lys, g-C, Lys(Gly)), 19.58, 19.18, 16.97. MALDI-TOF calculated for C75H125N21O30 (M + Na): 1822.89, measured 1822.02.Synthesis of Compound
3 (Fig. 6). Compound 9 (10.6 mg, 5.6 mmol) was dissolved in a mixture of ethanol/acetic acid/water (EtOH/H2O/1(N) HCl, 2:1:0.01, 0.9 ml), and 10% Pd on charcoal (9 mg) was added. After stirring at room temperature for 48 h, the reaction mixture was filtered. The filtrate was concentrated under reduced pressure, and the residue was coevaporated from toluene (3 × 20 ml). The residue was subjected to Sephadex G15 size exclusion column chromatography to give the target compound 10 as a mixture of a/b anomers (8.4 mg, 87%). 1H-NMR (600 MHz, D2O): d 5.16 (1H, d, H-1-a4.51 (1H, d, H-1-bJ = 8.5 Hz), 4.49 (1H, d, H-1-bJ = 8.9 Hz), 4.04–4.36 (11H, m, a CH, Ala × 5, a CH, Lys × 2, a CH Gln × 2, a CH, Lac × 2), 3.69–4.04 (16H, m, H-2 × 2, H-6 × 2, CH2, Gly × 5), 3.50–3.60 (6H, m, H-3 × 2, H-4 × 2, H-5 × 2), 3.17–3.21 (2H, m, e-CH2, Lys(Gly)), 2.99 (2H, t, e-CH2, Lys), 2.38–2.39 (4H, m, g-CH2, Gln), 2.14 (2H, m, b-CH, Gln), 1.98–2.03 (8H, m, b-CH, Gln, NH(COCH3) x 2), 1.52–1.74 (8H, m, b-CH2, Lys, d-CH2, Lys), 1.38–1.42 (25 H, m, CH3 × 5, Ala, CH3 × 2 Lac, g-CH2, Lys). C-13 (HSQC): 101.37 (C-1-b), 91.52 (C-1-a), 83.02, 79.96, 78.60, 76.22, 73.84, 71.80, 70.45, 69.43, 61.27, 60.93, 55.66, 54.81, 54.30, 54.13, 53.62, 53.11, 52.09, 44.28, 43.09, 39.39 (e-C, Lys), 32.04 (g-C, Gln), 30.68, 28.48, 27.29 (b-C, Gln), 27.12, 23.04, 22.87 (NHCOCH3), 19.47, 18.96, 17.26, 17.09. MALDI-TOF calculated for C69H117N21O30 (M + Na): 1742.83, measured 1742.79.Synthesis of Compounds
1 and 2. Compounds 1 and 2 (Fig. 1B) were prepared by using similar methods with either Fmoc-L-Lys(Mtt) or Fmoc-Dap(Boc, tBu), respectively. The NMR data of the l-lysine-type (Lys-type) MurNAc-l-Ala-d-isoGln-l-Lys-d-Ala-d-Ala (MPP) (1) and meso-diaminopimelic acid-type (Dap-type) MPP (2) are as follows: 1: 1H-NMR (500 MHz, D2O): d 5.04 (0.60H, d, H1-a anomer, J = 3.3 Hz), 4.56 (0.39H, d, H-1-b anomer, J = 8.4 Hz), 4.17–4.08 (6H, m, a H-Lys, a H-Gln, a H-Ala × 3, a H-3-propionic acid), 3.36–3.86 (6H, m, H2, H3, H4, H5, H6), 2.87 (2H, t, e CH2-Lys), 2.21–2.29 (2H, m, g CH2-Gln), 2.19–2.03 (1H, m, b CH2-Gln), 1.82-1.87 (4H, m, CH2-Gln, NHAc), 1.54–1.67 (4H, bd-Lys), 1.37–1.45 (14H, m, g CH2-Lys, CH3-lactic acid, CH3-Ala × 3). 13C NMR (75 MHz, D2O) 177.89, 176.14, 175.94, 175.32, 175.21, 174.93, 174.82, 174.38, 174.13, 95.07 (C1-a), 91.13 (C1-b), 82.78, 79.87, 78.24, 77.90, 75.87, 71.65, 69.03, 68.81, 60.87, 60.68, 56.33, 54.30, 53.86, 52.89, 50.13, 49.96, 49.61, 39.30 (e CH2-Lys), 31.42 (g CH2-Gln), 30.26, 27.08, 26.51, 22.38, 22.22, 22.15, 18.81, 16.73, 16.68, 16.37. 2: 1H NMR (500 MHz, D2O): d5.16 (0.41H, bs, H1), 4.40 (0.58 H, d, H1, J = 9.0 Hz), 4.22–4.29 (7H, m, a-H, e-H, Dap, a-H × 3, Ala, a-H, Gln, a-H, Lac), 3.50–3.98 (6H, m, H2, H6, H3, H4, H5), 2.39 (2H, m, g-CH2, Gln), 2.13–2.15 (1H, m, b-CHH, Gln), 1.90–2.03 (8H, m, b, d-CH2, Dap, b-CHH, Gln, NHCOCH3), 1.38–1.57 (14H, m, g-CH2, Dap, CH3, Lac, CH3 × 3, Ala). 13C (HSQC): 102.39 (C-1-b), 91.66 (C-1-a), 83.35, 80.13, 78.52, 78.25, 76.11, 72.08, 69.40, 60.95 (C6), 60.88, 60.60, 57.40, 55.59, 54.61, 54.40, 53.77, 53.01, 50.01, 31.60, 30.48 (C-Dap), 27.05, 22.40 (NHCOCH3), 21.01, 19.06, 16.83.1. Wiseman, T., Williston, S., Brandts, J. F. & Lin, L. N. (1989) Anal. Biochem. 179, 131–137.
2. Turnbull, W. B. & Daranas, A. H. (2003) J. Am. Chem. Soc. 125, 14859–14866.