Data Availability StatementAll relevant data are within the paper. with little or no contribution of the p-aminobenzoate-pteridine ring that is linked to the -amino side chain of the glutamate. Carboxylate-linked Fmoc-Glu–CONH-(CH2)6-NH2 undergoes hydrolysis in a nearly indistinguishable fashion. A free carboxylate moiety is essential for this effect. Carboxylate linked Fmoc-glutamic-amide–CONH-(CH2)6-NH2 undergoes no hydrolysis under acidic conditions. Based on these findings, we designed a cysteine specific MTX made up of reagent. Its linkage to bovine serum albumin (BSA) yielded a conjugate with profound antiproliferative efficacy in a MTX-sensitive glioma cell collection. In conclusion, carboxylate linked MTX-amino Tubastatin A HCl manufacturer derivatives in particular, and carboxylate linked R–GLU- amino compounds in general are equipped withbuilt-in chemical machinery that releases them under moderate acidic conditions. Introduction In previous studies we have focused on developing long-acting prodrugs that undergo reactivation in body fluids with desired pharmacokinetic properties (examined in [1, 2]). Our efforts were undertaken predominantly in the fields of diabetes and obesity [3C8]. The prodrug concept was launched in the late 1950s [9] and is relevant also to the field of malignancy therapy, in particular antibody-drug conjugates which have emerged in recent years as encouraging anti-cancer brokers [10]. Low molecular excess weight anticancer drugs covalently linked to proteins are ordinarily inactive conjugates that can be engineered to undergo reactivation, either enzymatically or chemically upon reaching their target tissues [11]. To date, linkage of low molecular-weight anticancer drugs was conducted through the cysteine moiety or Tubastatin A HCl manufacturer the -amino side Tubastatin A HCl manufacturer chains of proteins [12]. In this study we in the beginning linked MTX-amino derivatives to the carboxylate moieties, of a macromolecular model compound, composed of polyethylene glycol of 20-40kDa, made up of a single carboxylate, and then to proteins. We report here that such carboxylate-based macromolecule-MTX conjugates have unique, unexpected chemical and pharmacological features. These are described here in detail. Materials and Methods Materials Methotrexate, human serum albumin, hexametylenediamine-2HCl, 1.3 diaminopropate-2HCl, 6-aminocaproic-acid, ethylenediamine, dihydrofolate (DHF), N,N dicyclohexylcarbodiimide (DCC), -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. Synthesis of MTX-anhydride Methotrexate (45.4 mg, 100moles) was dissolved in 0.9 ml dimethyl sulfoxide (DMSO) and 95 l from a solution of 1M DCC in DMF (95 moles) was then added. The reaction was carried out for 3 hrs at 25C. Dicyclohexylurea was removed by filtration. The MTX-anhydride created was kept at 4C. Synthesis of MTX-hexamethylenamine and other MTX-amino derivatives Hexamethylenediamine-DiHCl (100 moles) was dissolved in 1.0ml DMSO, neutralized with N,N-Diisopropylethylamine (DIPEA) and combined with the solution of MTX-anhydride (100 moles in 1.0ml DMSO). The reaction combination was stirred PTGS2 for 4 hrs, precipitated with ethylacetate, washed 4 occasions with ethylacetate and desiccated. With this procedure, the -carboxylate, rather than the -carboxylate moiety of MTX is usually predominantly derivatized [13]. MTX-CONH-(CH2)6-NH2 was obtained in 60% yield. It has the molar extinction coefficient much like MTX (305 = 22700, 372 = 7200). MTX-CONH-(CH2)2-NH2, MTX-CONH-(CH2)3-NH2 and MTX-CONH-CH-(COOH)-(CH2)4-NH2 were synthesized by the same process applied to MTX-hexamethylene-amine except that hexamethylene-diamine was replaced by ethylenediamine, 1.3 diaminopropane-2HCl, or N-t-BOC-L-lysine respectively. The protecting group was then removed by trifluoroacetic acid. The calculated molecular weights for MTX-CONH-(CH2)2-NH2 is usually 496 Da, found by ESMS M+H = 497.36, that for MTX-CONH-(CH2)3-NH2 is 539, found by ESMS M+H = 540.45 and for MTX-CONH-(CH2)6-NH2 is 552 Da, found for M+H = 553.41 Da and for MTX-CONH-CH-(COOH)-(CH2)4-NH2 is 682 Da, found M+H = 683.37 Da. Preparation of PEG40-CONH-(CH2)6-NH2 Solid PEG40-OSu (280mg ~ 7 moles) was added to 5.0 ml hexamethylenediamine-2HCl (1M in 0.1M Hepes buffer pH 7.4 precooled to 0C). The reaction was carried out for 5 hrs at 0C, with stirring. The derivative obtained was dialyzed against H2O with several changes of H2O over a period of 3 days and then lyophilized. Preparation of PEG40-CONH-(CH2)5-COOH This derivative was prepared by the same process applied for PEG40-CONH-(CH2)5-NH2 except that hexamethylenediamine was replaced by 6-aminocaproic acid. The product thus obtained was extensively dialyzed against H2O and then lyophilized. Synthesis of cysteine-specific-MTX-containing reagents [MAL-(CH2)2-CONH-(CH2)2,3,6-NHCO-MTX] Solutions of MTX-CONH-(CH2)2-NH2, MTX-CONH-(CH2)3-NH2 or MTX-CONH-(CH2)6-NH2 (100 moles of each in 1 ml DMSO) were neutralized with one equivalent of DIPEA (100moles, 17.4 l), and each combined with 120moles of MAL-(CH2)2-COOSu (32mg in 0.3ml DMSO). The reaction was carried out for 3 hrs at 25C with stirring. The products were precipitated with ethylacetate, washed 5 occasions with this solvent,.