Synthesis and biological activities of N-acetyl-1-thiomuramoyl-L-alanyl-D-isoglutamine and some of its lipophilic derivatives

N-Acetyl-1-thiomuramoyl-L-alanyl-D-isoglutamine and some lipophilic analogs were synthesized from benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[D-1-(methoxycarbonyl)ethyl ]- alpha-D-glucopyranoside (1). O-Debenzoylation of 2, derived from 1 by oxidation, gave 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[D-1-(methoxycarbonyl)ethyl ]-D-glucopyranose (3). Condensation of the alkoxy-tris(dimethylamino)phosphonium chloride (4), formed from 3 by the action of carbon tetrachloride and tris(dimethylamino)phosphine, with potassium thioacetate afforded 2-acetamido-1-S-acetyl-2-deoxy-4,6-O-isopropylidene-3-O-[ D-1-(methoxycarbonyl)ethyl]-1-thio-beta-D-glucopyranose (8). Coupling of the acid 9, obtained from 8 by hydrolysis and subsequent S-acetylation, with the methyl ester of L-alanyl-D-isoglutamine gave N-[2-O-(2-acetamido-1-S-acetyl-2,3-dideoxy-4,6-O- isopropylidene-1-thio-beta-D-glucopyranose-3-yl)-D-lactoyl]-L-alan yl-D- isoglutamine methyl ester (10), which was converted, via O-deisopropylidenation, S-deacetylation, and de-esterification, into the N-acetyl-1-thiomuramoyl dipeptide. Condensation of 11 (derived from 10 by S-deacetylation) and of 12 (obtained from 10 by S-deacetylation and de-esterification) with various acyl chlorides yielded the corresponding 1-S-acyl-N-acetylmuramoyl-L-alanyl-D-isoglutamine derivatives, which were converted into the desired, lipophilic 1-thiomuramoyl dipeptides by cleavage of the isopropylidene group. Condensation of 11 with the alkyl bromides yielded the 1-S-alkyl derivatives, which were also converted, via O-deisopropylidenation and de-esterification, into the corresponding 1-S-alkylmuramoyl dipeptides. The biological activities were examined in guinea-pigs and mice.     

The reaction of choline acetyltransferase with sulfhydryl reagents. Methoxycarbonyl-CoA disulfide as an active site-directed reagent.

The reaction of choline acetyltransferase with methoxycarbonyl alkyl disulfides leads to a progressive loss in enzyme activity as the size of the alkyl group increases from methyl to n-butyl. Reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or methoxycarbonyl coenzyme A (CoA) disulfide, leads to a total loss of enzyme activity. DTNB inactivation is biphasic (k1 = approximately 9 x 10(2) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1) with the slow phase being diminished by acetyl-CoA. Methoxycarbonyl-CoA disulfide inactivation is also biphasic (k1 = approximately 2.1 x 10(3) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1), with the rapid phase being diminished in the presence of acetyl-CoA. Inactivation by methoxycarbonyl methyl disulfide, ethyl disulfide, or hydroxyethyl disulfide, or by methyl methanethiosulfonate is not biphasic. Pretreatment of the enzyme with methyl methanethiosulfonate, which leads to a 25% loss in enzyme activity, abolishes the fast phase of DTNB inactivation, the slow phase of methoxycarbonyl-CoA disulfide inactivation, and any further inactivation by methoxycarbonyl ethyl disulfide. These results are interpreted to suggest that choline acetyltransferase contains two classes of reactive sulfhydryl groups, neither of which are required for enzyme activity.     

Efficient, Mg(2+)-dependent photochemical DNA cleavage by the antitumor quinobenzoxazine (S)-A-62176

The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg(2+) self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg(2+). Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg(2+) complex and serve to define the position of this complex on the minor groove of DNA.     

The aspartimide problem in Fmoc-based SPPS. Part I.

A variety of Asp beta-carboxy protecting groups, Hmb backbone protection and a range of Fmoc cleavage protocols have been employed in syntheses of the model hexapeptide H-VKDGYI-OH to investigate the aspartimide problem in more detail. The extent of formation of aspartimide and aspartimide-related by-products was determined by RP-HPLC. This study included three new Fmoc-Asp-OH derivatives: the beta-(4-pyridyl-diphenylmethyl) and beta-(9-phenyl-fluoren-9-yl) esters and also the orthoester Fmoc-beta-(4-methyl-2,6,7-trioxabicyclo[2.2.2]-oct-1-yl)-alanine. 3-Methylpent-3-yl protection of the Asp side chain resulted in significant improvements with respect to aspartimide formation. Complete suppression was achieved using the combination OtBu side chain protection and Hmb backbone protection for the preceding Gly residue.     

High pressure mediated Diels-Alder reaction using 2-iminomalonates as dienophiles: novel synthesis of carbocyclic nucleosides.

9-[c-4-(1,2-Dihydroxyethyl)cyclopent-2-en-r-1-yl]-9H-adenine , an isomer of BCA, has been synthesized from Diels-Alder adducts prepared by the reaction of 2-iminomalonates with cyclopentadiene under high pressure. The synthetic method involves a novel C-N bond cleavage reaction by NaBH4-K2CO3 (RRA reaction conditions) and has wide applicability for the synthesis of various carbocyclic nucleosides.     

Studies on the enzymatic hydrolysis of amino acid carbamates

Several commercially available enzymes were tested for their ability to hydrolyze amino acid carbamates. No activity was found with pig liver esterase, the hydantoinase from Pseudomonas fluorescens DSM 84, or the urease from jack beans. A stereoselective cleavage of the carbamyl group yielding L-amino acids was observed by acylase and acetylcholinesterases from bovine and human erythrocytes. Racemic mixtures of N-(methoxycarbonyl)-DL-alanine, N-(ethoxycarbonyl)-DL-alanine, and the corresponding valine carbamates are hydrolyzed to L-alanine and L-valine, respectively, by acylases leaving the D-amino acid carbamates unchanged. The lysine carbamates were not hydrolyzed by acylases. In contrast only the methoxycarbonyl amino acids were split by acetylcholinesterases, which, however, also cleave alpha, epsilon-(N-methoxycarbonyl)-DL-lysine stereoselectively at the alpha position, yielding epsilon-N-methoxycarbonyl-L-lysine. The optimum pH for enzymatic activity of hog kidney acylase was 7.5 and a Km value of 8.2 mM for N-(methoxycarbonyl)-DL-alanine was determined. For the acetylcholinesterases the reaction rate reaches an optimum between pH 7.5 and 8. The Km value was 68 mM for N-(methoxycarbonyl)-DL-alanine.     

Photolytic cleavage of DNA by nitrobenzamido ligands linked to 9-aminoacridines gives DNA polymerase substrates in a wavelength-dependent reaction.

A series of reagents containing 3- or 4-nitrobenzamido ligands tethered to 9-aminoacridine via variable-length linkers have been prepared and their properties as photochemical DNA cleavers (photonucleases) examined. When irradiated with approximately 300-nm light, where the nitrobenzamido ligand can absorb, they cleave DNA in an oxygen-independent reaction presumably involving oxygen transfer from the nitro group to the deoxyribose units of the DNA backbone (Nielsen et al., 1988b). This reaction is pH independent and only slightly affected by the linker length, and the DNA fragments are not substrates for DNA polymerase. When approximately 420-nm light is used, were only the 9-aminoacridinyl ligands absorb, the DNA cleavage is also oxygen-independent but pH dependent, requires DNA saturation with the reagent (base pair:reagent less than or equal to 2), and is most efficient with the longer linkers. The cleavage is specific for guanine residues and results in 5'-phosphate termini and heterogeneous (more than four products) 3'-termini. One of the products is presumably 3'-hydroxy since DNA photocleaved with nitrobenzamido acridine reagents and 420-nm radiation are substrates for DNA polymerase in a nick translation assay as well as for the Klenow fragment. An electron-transfer mechanism is suggested.     

Synthesis of carbocyclic nucleosides bearing a cyclopropane ring.

A carbocyclic cyclopropane fused nucleoside, 9-(c-4-hydroxymethylbicyclo[3.1.0]hex-r-2-yl)-9H-adenine, has been efficiently synthesized from 2-azabicyclo-[2.2.1]hex-5-en-3-one (ABH) in 6 steps, namely cyclopropanation, -reductive amide cleavage (RAC) reaction and adenine ring construction.     

Synthesis of enantiopure trans-N-Boc-3-aminobicyclo[2.2.2]octane-2-carboxylic acids and their bicyclic 1,3-amino alcohol derivatives via the [4+2] cycloaddition of 1,3-cyclohexadiene to a chiral β-nitroacrylate

The chiral β-nitroacrylate 2 derived from the (R)- or (S)-4-(3-hydroxy-4,4-dimethyl-2-oxopyrrolidin-1-yl) benzoic acid 1 acts as a reactive dienophile in a diastereoselective Diels-Alder reaction with 1,3-cyclohexadiene. The major cycloadducts have been isolated and transformed into enantiopure trans(2S,3S)- or (2R,3R)-N-Boc-3-aminobicyclic[2,2,2]octane-2-carboxylic acids 5. The trans-(2S,3S)- or (2R,3R)-N-Boc 3-(hydoxymethyl)-2-aminobicyclic[2,2,2]octane 6 derivatives were also obtained.     

Synthesis and in vitro antitumor activity of thiophene analogues of 5-chloro-5,8-dideazafolic acid and 2-methyl-2-desamino-5-chloro-5,8-dideazafolic acid

N-[5-[N-(2-Amino-5-chloro-3,4-dihydro-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (6) and N-[5-[N-(5-chloro-3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (7), the first reported thiophene analogues of 5-chloro-5,8-dideazafolic acid, were synthesized and tested as inhibitors of tumor cell growth in culture. 4-Chloro-5-methylisatin (10) was converted stepwise to methyl 2-amino-5-methyl-6-chlorobenzoate (22) and 2-amino-5-chloro-3,4-dihydro-6-methyl-4-oxoquinazoline (19). Pivaloylation of the 2-amino group, followed by NBS bromination, condensation with di-tert-butyl N-(5-amino-2-thenoyl)-L-glutamate (28), and stepwise cleavage of the protecting groups with ammonia and TFA yielded. Treatment of 9 with acetic anhydride afforded 2,6-dimethyl-5-chlorobenz[1,3-d]oxazin-4-one (31), which on reaction with ammonia, NaOH was converted to 2,6-dimethyl-5-chloro-3,4-dihydroquinazolin-4-one (33). Bromination of, followed by condensation with and ester cleavage with TFA, yielded. The IC(50) of and against CCRF-CEM human leukemic lymphoblasts was 1.8+/-0.1 and 2.1+/-0.8 microM, respectively.     

Studies on the synthesis of the hypermodified base isolated from rat liver phenylalanine transfer ribonucleic acid

Oxidation of methyl (S,E)-4-[4,9-dihydro-4,6-dimethyl-9-oxo-1- (phenylmethyl)-1H-imidazo[1,2-alpha]purine-7-yl]-2-[(methoxycarbonyl) amino]-3-butenoate (3) with osmium tetroxide/N-methylmorpholine N-oxide provided a mixture of diastereomers 4 and 7. Hydrogenolysis of the major dihydroxy compound 4 over Pd-C gave beta-hydroxywybutine [[R-(R*,S*)]-1]. The minor isomer 7 was transformed into [S-(R*,R*)]-1 through the cyclic carbonate 8.     

Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site

We have used nucleobase substitution and kinetic analysis to test the hypothesis that hammerhead catalysis occurs by a general acid-base mechanism, in which nucleobases are directly involved in deprotonation of the attacking 2'-hydroxyl group and protonation of the 5'-oxygen that serves as the leaving group in the cleavage reaction. We demonstrate that simultaneous substitution of two important nucleobases, G8 and G12, with 2,6-diaminopurine shifts the pH optimum of the cleavage reaction from greater than 9.5 to approximately 6.8 in two different hammerhead constructs. Controls involving substitution with other nucleobases and combinations of nucleobases at G5, G8, and/or G12 do not show this behavior. The observed changes in the pH-rate behavior are consistent with a mechanism in which N1 protonation-deprotonation events of guanine or 2,6-diaminopurine at positions 8 and 12 are essential for catalysis. Further support for the participation of G8 and G12 comes from photochemical cross-linking experiments, which show that G8 and G12 can stack upon the two substrate nucleobases at the reactive linkage, G(or U)1.1 and C17 (Heckman, J. E., Lambert, D., and Burke, J. M. (2005) Photocrosslinking detects a compact active structure of the hammerhead ribozyme, Biochemistry 44, 4148-4156). Together, these results support a model in which the hammerhead undergoes a transient conformational change into a catalytically active structure, in which stacking of G8 and G12 upon the nucleobases spanning the cleavage site provides an appropriate architecture for general acid-base catalysis. The hammerhead and hairpin ribozymes may share similarities in the organization of their active sites and their catalytic mechanism.     

Photochemical Preparation of a Novel Low Molecular Weight Heparin

Commercial low molecular weight heparins (LMWHs) are prepared by several methods including peroxidative cleavage, nitrous acid cleavage, chemical ?-elimination, and enzymatic β-elimination. The disadvantages of these methods are that strong reaction conditions or harsh chemicals are used and these can result in decomposition or modification of saccharide units within the polysaccharide backbone. These side-reactions reduce product quality and yield. Here we show the partial photolysis of unfractionated heparin can be performed in distillated water using titanium dioxide (TiO(2)). TiO(2) is a catalyst that can be easily removed by centrifugation or filtration after the photochemical reaction takes place, resulting in highly pure products. The anticoagulant activity of photodegraded LMWH (pLMWH) is comparable to the most common commercially available LMWHs (i.e., Enoxaparin and Dalteparin). (1)H NMR spectra obtained show that pLMWH maintains the same core structure as unfractionated heparin. This photochemical reaction was investigated using liquid chromatography/mass spectrometry (LC/MS) and unlike other processes commonly used to prepare LMWHs, photochemically preparation affords polysaccharide chains of reduced length having both odd and even of saccharide residues.     

Novel carboranyl amino acids and peptides: reagents for antibody modification and subsequent neutron-capture studies.

A new alpha-amino acid derivative incorporating the 1,2-dicarba-closo- dodecarborane(12) cage, namely 5-(2-methyl-1,2-dicarba-closo-dodecarborane(12)-1-yl)- 2-aminopentanoic acid (2), was synthesized by the alkylation of the benzophenone Schiff's base of glycine methyl ester with 3-(2-methyl-1,2-dicarba-closo-dodecaborane(12)-1-yl)pr opyl iodide (8). This amino acid was employed in the synthesis of peptide derivatives such as 19-21 using solid-phase Merrifield methods. Dipeptide 19 was converted to a water-soluble ionic derivative by the pyrrolidine-mediated carborane cage degradation reaction followed by cation exchange to afford sodium salt 22. Dansylation of 22 with dansyl chloride yielded fluorescence-labeled dipeptide 23. Undecapeptide 21 was dansylated while still anchored to the Merrifield resin. Following its cleavage from the resin with hydrogen fluoride, product 25 was acetylated to block the free amino group on the lysine residue and then converted to water-soluble derivative 27. Trial conjugations of dipeptide 23 and undecapeptide 27 to T84.66, an anti-CEA antibody, were carried out by means of carboxyl activation with N-hydroxysulfosuccinimide and N,N-diisopropylcarbodiimide. Studies of the chemical syntheses of these and other peptide derivatives and the conjugation of 23 and 27 to the antibody are described.     

Synthesis and DNA cleavage reaction characteristics of enediyne prodrugs activated via an allylic rearrangement by base or UV irradiation

A number of enediyne prodrugs 1-5 possessing an (E)-3-hydroxy-4-(2'-hydroxy-1'-phenylethylidene)cyclodeca-1,5-diyne scaffold have been synthesized via the Sonogashira coupling and an intramolecular Nozaki-Hiyama-Kishi reaction as the key steps. Upon incubation with enediyne prodrugs 4 and 5 possessing a free hydroxymethyl group on the exocyclic double bond, circular supercoiled DNA (Form I) underwent single strand cleavage into circular relaxed DNA (Form II) in buffer solution at pH 8.5, while the silylated analogs 1-3 showed very weak DNA cleavage activity. Alternatively, the silylated analogs 1-3 could be activated by UV irradiation via a photochemical alkene isomerization followed by an allylic rearrangement to form the putative epoxy enediyne, resulting in efficient DNA cleavage similar to the level observed with the prodrugs 4 and 5.     

Synthesis and biological properties of α-thymidine 5′-aryl phosphonates

Diethyl(N-arylaminocarbonyl)methyl phosphonates have been obtained by the reaction of diethylphosphonoacetic acid imidazolides with methyl-4-aminobenzoate or 3,5-bis(trifluoromethyl)phenylamine. Their treatment with Me₃SiBr in DMF led to a mixture of the corresponding (N-arylaminocarbonylmethyl)phosphonic acids and their monoethyl esters. After separation, they were condensed with 3′-O-acetyl-α-thymidine, which, after the removal of the acetyl protecting group, gave (α-D-thymidine-5′-yl)-[4-aminocarbonyl-, methoxycarbonyl-, or carboxy)phenylaminocarbonyl]methyl phosphonates and (α-D-thymidine-5′-yl)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. It was shown that the compounds are stable under different conditions, low toxic (in Vero and K-562 cell cultures), and capable of penetrating into K-562 cells. Only ethyl (α-D-thymidine-5′-yl)-[4-(methoxycarbonyl)phenylaminocarbonyl]methyl phosphonate at a high concentration (200 μg/mL) inhibited in vitro the growth of the laboratory strain M. tuberculosis H37Rv.     

A novel L-neopentylglycine derivative as auxiliary for copper-catalyzed asymmetric Michael reactions

L-Neopentylglycine diethylamide (4a). was prepared from the new unnatural amino acid L-neopentylglycine (1). The utilization of amide 4a as a chiral auxiliary in the copper(II)-catalyzed asymmetric Michael reaction was investigated in comparison with L-valine diethylamide (4b). Cyclic beta-oxocarboxylates 7 react with 4a and 4b to give the respective enaminoesters 8, which were converted with methyl vinyl ketone (9) in the presence of 10 mol% Cu(OAc)(2). H2O at room temperature in acetone to yield the optically active Michael addition products (R)-10a, b with high selectivity independent of the starting enamine. In the case of the seven-membered beta-oxocarboxylate 7c, however, the valine-derived enamine 8f led to higher enantioselectivity for product 10c. Despite the bulkiness of the neopentyl group, the isopropyl group with an alpha-branch has a better stereoinducing effect.     

Base-selective oxidation and cleavage of DNA by photochemical cosensitized electron transfer.

A photochemical mechanism for single-strand cleavage of DNA is proposed in which a photoexcited intercalator transfers an electron to an externally bound cosensitizer. Once formed, the oxidized intercalator oxidizes an adjacent base, creating a charge-separated complex from which reactions leading to cleavage of the sugar-phosphate backbone occur in competition with back electron transfer. Using ethidium bromide (EB) as the intercalator and methyl viologen (MV) as the externally bound cosensitizer, a 10-fold enhancement in the rate of single-strand break formation was found in pBR322 DNA over that for EB alone using 488-nm excitation. The rate of cleavage correlated with the amount of MV bound to DNA. In accord with the expected redox properties of the one-electron-oxidized EB and the DNA bases, cleavage occurs selectively at guanines. Although the reaction proceeds in nitrogen-purged solutions, the rate of cleavage in air-saturated solutions was enhanced 2-fold. Treatment of irradiated samples with alkali leads to a 2-fold increase in the yield of single-strand breaks. These results support a mechanism in which cleavage occurs by selective oxidation of guanines in DNA, initiated by photochemical cosensitized electron transfer from intercalated EB to externally bound MV, and may provide a basis for the development of light-activated base-selective DNA cleaving agents.     

A-hydroxyphosphonate oligonucleotides: a promising DNA type?

The synthesis of monomers (S)-1, (R)-1 and 2 derived from (5'S)-, (5'R)-2'-deoxythymidine-5'-C-phosphonic acids and 2',5'-dideoxythymidine-5'-C-phosphonic acids was elaborated. The protection of the 5'-hydroxyl by the methoxycarbonyl group was a key step of the synthesis. Prepared monomers were used for the solid-phase assembly of several types oligothymidylate 15-mers (S)-3, (S)-4, (S)-5, (R)-4 and (R)-5 containing the chiral 3'-O-P-CH(OH)-5' internucleotide linkage. Their hybridization properties with dA15 and rA15 were studied as well as their resistance against nuclease cleavage.     

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating arylsulfonylureido and 5-dibenzo-suberenyl/suberyl moieties

Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.