Dihydrofolate

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO): methanol and acetonitrile (MS grade), ammonium acetate, ascorbic acid, β-mercaptoethanol, formic acid and standards, 5-adenosyl-methionine, 5-adenosyl-homocysteine, ATP, betaine, choline, cyanocobalamin, cystathionine, cysteine, dihydrofolate, dimethylglycine, dUMP, folic acid, folinic acid, glycine, homocysteine, methionine, methylcobalamine, methyl-tetrahydrofolate, NADPH, pyridoxal 5-phosphate, riboflavin, serine, taurine, tetrahydrofolate, thymidine 5-phosphate and Dulbecco's modified Eagle's medium mixed 1:1 with Ham's F-12 (DMEM/F12). Ultrapure type 1 water was obtained from a Milli Q water system (Merck Millipore, Darmstadt, Germany). Matrigel (growth factor-reduced) was purchased from BD Biosciences (San Jose, CA). Recombinant basic fibroblast growth factor (bFGF) was purchased from Mylteni (San Diego, USA).

Methotrexate, human serum albumin, hexametylenediamine-2HCl, 1.3 diaminopropate-2HCl, 6-aminocaproic-acid, ethylenediamine, dihydrofolate (DHF), N,N dicyclohexylcarbodiimide (DCC), β-nicotinamide adenine dinucleotide 2’ phosphate (NADPH) were purchased from Sigma. Fmoc-Glutamic acid (Fmoc-Glu-OH) and Fmoc-Glutamic acid amide were obtained from DgPeptides Co. LTD (China). PEG₄₀₀₀₀-OSu (mPEG2-N-hydroxysuccinimide ester) was obtained from Shearwater (Huntsville, AL). PEG₂₀₀₀₀-SH (ME-200SH sunbright) and PEG₃₀₀₀₀-SH were purchased from RAPP Polymere (Tuebingen, Germany). All other materials used in this study were of analytical grade.

We bought from Sigma Aldrich, St Quentin Fallavier, France: dihydrofolate, Leu-enkephalin, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), naproxen, indomethacin and Tris, NaCl, the oligonucleotides with or without TEG biotin tags were purchased with HPLC purification. The sequence of the 48 mer DNA was: 5′ATA TAT ATC TAT GTC CAT ATA TAT ATA AAA CAC AGC GTG TGT GTG TAA 3′. The sequence of TAR-polyA was: 5′ (A)₂₁ GAA AGG AGC CUG GGA GCU CC 3′. Substance P (1-7) was synthesized by Genecust, Boynes, France. The plasmids for NTD, mutants and the full-length N were obtained from Genecust, Boynes, France.