Solid-phase synthesis of an oxalic acid amide library

Monoamides of oxalic acid are of interest as bioisosteric replacements for phosphate groups in the design of new enzyme inhibitors. Here, we have demonstrated the use of oxalic acid as a linker to the Wang resin to synthesize individual or libraries of phosphate biosteres. The highly reactive resin-bound acid chloride reacts with arylamines to yield resin-bound N-aryloxamic acids (oxanilic acids). This methodology is especially useful for the rapid synthesis of 2-(oxalylamino)benzoic acids (OBAs), because it can be utilized for library synthesis and eliminates the intermediate purification step necessary in solution-phase reactions. The products are cleaved off the resin with trifluoroacetic acid in dichloromethane in good yields.     

Deazapurine solid-phase synthesis: construction of 3-substituted pyrrolo[3,2-d]pyrimidine-6-carboxylates on cross-linked polystyrene bearing a cysteamine linker

The first solid-phase methodology for the preparation of pyrrolo[3,2-d]pyrimidines is presented. Merrifield resin bearing a cysteamine "traceless" linker was treated with 4-oxo-N-(PhF)proline benzyl ester (10; PhF = 9-(9-phenylfluorenyl)) to provide resin-bound aminopyrrole 20, which was treated with ethyl, phenyl, 4-phenoxyphenyl, and 2,4-dimethoxyphenyl isocyanates to furnish resin-bound ureidopyrroles 21a-d. Resin-bound pyrrolo[3,2-d]pyrimidines 22a-d were then obtained by acylation of 21 using trichloroacetyl chloride in dioxane followed by treatment with Cs2CO3 in DMF. Cleavage of pyrrolo[3,2-d]pyrimidines 22a-d from the resin was achieved in two steps, by oxidation of the sulfur to the sulfone followed by beta-elimination in the presence of t-BuONa. Four pyrrolo[3,2-d]pyrimidines, 24a-d, with different alkyl and aryl substituents at the N3 pyrimidine nitrogen, were thus obtained in overall yields of 42-50% and purities of 90-100%.     

The synthesis of acid- and base-labile lipopeptides on solid support

Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68 % yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.     

Synthesis of a S-(Carboxymethyl)cysteine Analog of (L-2-Amino-6-adipyl)-L-cysteinyl-D-valine and its Cell Free Biosynthetic Conversion into 6-[2-(D-2-Amino-2-carboxyethyl)thio) acetamido]penicillanic Acid

A new tripeptide (dimer), bis[(L -cysteine-S-acetyl)-L -hemicystinyl(S² → S²)-D -valine] ( 6 ) was synthesized by coupling N-(tert-butoxycarbonyl)-S-carboxymethyl-L -cysteine benzyl ester ( 1 ) with S-trityl-L -cysteinyl-D -valine benzyl ester and subsequent removal of the protecting groups. After reduction of the disulfide, the free tripeptide Cys (CH₂CO-Cys-D -Val) ( Ib ) was used as a substrate of isopenicillin-N synthetase in a cell-free conversion to 6-[2-((D -2-amino-2-carboxyethyl)thio)acetamido]penicillanic acid ( IIa ).     

Partial purification, and some properties and reactivities of cetraxate benzyl ester hydrochloride-hydrolyzing enzyme

Debenzylating enzyme from Aspergillus niger enzyme (commercial crude cellulase) catalyzes the hydrolysis of cetraxate benzyl ester hydrochloride (2), a precursor of the antiulcer agent (1). The enzyme was highly purified by three kinds of chromatographies (hydrophobic, ion exchange, gel filtration) with a recovery of 36%. The content of the debenzylating enzyme was about 0.1% in the crude cellulase, but the enzyme showed no cellulase activity. The purified enzyme was inactivated by Hg2+, and diisopropyl phosphorofluoridate (DFP). It was a monomer with a molecular weight of about 35,000, and its isoelectric point was estimated to be 5.3. It showed a debenzylating activity for the phenylpropionic acid benzyl ester moiety of various benzyl ester derivatives, and the benzyl ester of phenylalanine or that of tyrosine was also well hydrolyzed.     

Solid-phase synthesis of 4(1H)-quinolone and pyrimidine derivatives based on a new scaffold-polymer-bound cyclic malonic acid ester

An efficient method for the preparation of polymer-bound cyclic malonic acid ester starting from Merrifield resin has been developed. Reaction of the resin-bound cyclic malonic acid ester with triethyl orthoformate and subsequent double substitution with nucleophilic reagents, such as arylamine, urea, thiourea, 2-aminobenzothiazoles, or isothiosemicarbazones, afforded the corresponding polymer-bound substituted aminomethylene cyclic malonic acid esters, which upon thermal treament led to 4(1H)-quinolones, 3-substituted uracils and thiouracils, 4H-pyrimido[2,1-b]benzothiazol-4-ones, and 1-(N-alkylidene or benzylideneamino)-1,6-dihydro-2-methylthio-6-oxo-pyrimidines, depending on the structures of the nucleophilic reagents.     

Silicon-based metalloprotease inhibitors: synthesis and evaluation of silanol and silanediol peptide analogues as inhibitors of angiotensin-converting enzyme

Silanols are best known as unstable precursors of siloxane (silicone) polymers, substances generally considered stable and inert, but have the potential to mimic a hydrated carbonyl and inhibit protease enzymes. While previous testing of simple silanediol and silanetriol species as inhibitors of hydrolase enzymes found them ineffective, this study reports polypeptide mimics with a central methylsilanol [SiMeOH] or silanediol [Si(OH)(2)] group and their assessment as effective transition state analogue inhibitors of the well-studied metalloprotease angiotensin-converting enzyme (ACE). Central to the synthesis strategy, phenylsilanes were employed as acid-hydrolyzable precursors of the silanol group. The N-benzoyl Leu-[SiMeOH]-Gly benzyl amides proved to be stable and readily characterized. In contrast, the Leu-[Si(OH)(2)]-Gly structure was difficult to characterize, possibly because of self-association. Capping the silanediol with chlorotrimethylsilane gave a well-defined trisiloxane, demonstrating that the silanediol was monomeric. The Leu-[Si]-Gly structures were converted to Leu-[Si]-Ala analogues by enolate alkylation. Coupling of the silanol precursors with proline tert-butyl ester gave N-benzoyl Leu-[Si]-Gly-Pro and N-benzoyl Leu-[Si]-Ala-Pro tripeptide analogues. Treatment of these with triflic acid formed the corresponding methylsilanols and silanediols, all of which were monomeric. The silanediol tripeptide mimics inhibited ACE with IC(50) values as low as 14 nM. Methylsilanols, in contrast, were poor inhibitors, with IC(50) values above 3000 nM. These data, including comparisons with inhibition data from carbon analogues, are consistent with binding of the silanediols by chelation of the ACE active site zinc, whereas the methylsilanols ligate poorly.     

A new resin-bound universal isonitrile for the Ugi 4CC reaction: preparation and applications to the synthesis of 2,5-diketopiperazines and 1,4-benzodiazepine-2,5-diones

[reaction: see text] The preparation and synthetic applications of a novel resin-bound isonitrile are described. The resin is an example of a novel convertible isonitrile that can be utilized in the Ugi multicomponent reaction. Base-activation of the resin-bound Ugi product results in cleavage via formation of a N-acyloxazolidone that is then trapped as a carboxylic acid ester. This resin and the methodology described are suitable for synthesizing diversity libraries of 2,5-diketopiperazines and 1,4-benzodiazepine-2,5-diones.     

A traceless approach for the parallel solid-phase synthesis of 2-(arylamino)quinazolinones

A traceless approach for the parallel solid-phase synthesis of 2-arylamino-substituted quinazolinones is described. Acylation of MBHA resin with o-nitrobenzoic acid derivatives, followed by reduction of the nitro group with tin chloride, generated a resin-bound o-anilino derivative. Reaction of resin-bound o-anilino derivative with arylisothiocyanates yielded resin-bound thioureas, which reacted with amines in the present of Mukaiyama's reagent (2-chloro-1-methylpyridinium iodide) to afford resin-bound guanidines. Following intramolecular cyclization of the resin-bound guanidines during cleavage from the resin by HF/anisole (95/5) for 1.5 h at 0 degrees C, the desired products were obtained in good yield and purity.     

Preparation of Resin-Bound Bismethylene Cyclic Malonic Acid Ester and Facile Solid-Phase Synthesis of 2-Alkylthio-4(1H)-quinolone and 2-Alkyl-4(1H)-quinolone

Abstract

The resin-bound bismethylthiomethylene cyclic malonic acid ester 3 was built up efficiently. Resin 3 can react with arylamines and subsequent thermal cyclizations give 2-alkylthio-4-(1H)-quinolones. Furthermore, conjugate addition of Grignard reagents to the resin 3 forms the resin 6 which was reacted with aryl amines and subsequent thermal-cyclization-cleavages afford the 2-alkyl-4-(1H)-quinolones.     

Deazapurine solid-phase synthesis: combinatorial synthesis of a library of N3,N5,C6-trisubstituted pyrrolo[3,2-d]pyrimidine derivatives on cross-linked polystyrene bearing a cysteamine linker

Solid-phase methodology for the preparation of pyrrolo[3,2-d]pyrimidine-6-carboxylates with diversity at the N3 pyrmidine nitrogen has now been elaborated to allow for the generation of pyrrolopyrimidine libraries with members possessing diversity at the N3, N5, and C6 positions. The diversification of the N5 position was achieved by treating the parent resin-bound pyrrolo[3,2-d]pyrimidines 3 with an alkyl halide in the presence of Cs2CO3 in DMF. Modification of the C6 carboxylate of resin-bound pyrrolopyrimidines 3-5 was first achieved by hydrolysis of the benzyl ester using LiOH in a mixture of THF/H2O/MeOH. Further alteration of the C6 position of resin-bound pyrrolo[3,2-d]pyrimidine-6-carboxylic acids 6-8 was then performed by activation with triphosgene and treatment with an amine to furnish resin-bound pyrrolo[3,2-d]pyrimidine-6-amides. Twenty-two pyrrolo[3,2-d]pyrimidines 1a-v with different substituents at the N3, N5, and C6 positions were obtained in yields of 21-83% and purities of 61-98% after cleavage from the solid support.     

A nickel tripeptide as a metallodithiolate ligand anchor for resin-bound organometallics

The molecular structure of the acetyl CoA synthase enzyme has clarified the role of individual nickel atoms in the dinickel active site which mediates C-C and C-S coupling reactions. The NiN2S2 portion of the biocatalyst (N2S2 = a cysteine-glycine-cysteine or CGC4- tripeptide ligand) serves as an S-donor ligand comparable to classical bidentate ligands operative in organometallic chemistry, ligating the second nickel which is redox and catalytically active. Inspired by this biological catalyst, the synthesis of NiN2S2 metalloligands, including the solid-phase synthesis of resin-bound Ni(CGC)2-, and sulfur-based derivatization with W(CO)5 and Rh(CO)2+ have been carried out. Through comparison to analogous well-characterized, solution-phase complexes, Attenuated Total Reflectance FTIR spectroscopy establishes the presence of unique heterobimetallic complexes, of the form [Ni(CGC)]M(CO)x, both in solution and immobilized on resin beads. This work provides the initial step toward exploitation of such an evolutionarily optimized nickel peptide as a solid support anchor for hybrid bioinorganic-organometallic catalysts.     

Liquid-phase combinatorial synthesis of 1,4-benzodiazepine-2,5-diones as the candidates of endothelin receptor antagonism

A library of 1,4-benzodiazepine-2,5-dione dicarboxylate derivatives containing aryl substituents at N(1)- and N(4)-positions to mimic the amino acid residues of Try-13, Phe-14, and Asp-18 in endothelin-1 is established by using the starting materials of alpha-amino esters, hydroxybenzaldehydes, nitrobenzoyl chlorides, and benzyl bromides in a polyethylene resin-bound liquid-phase synthesis. All of the six synthetic steps are conducted under mild conditions to give the desired products with reasonable yields and purity. The poly(ethylene glycol) support plays as a part of ester linkage that is released at the final step.     

Annulation of primary amines to piperazines and diazaspirocycles utilizing alpha-methyl benzyl resin

The microwave-assisted solid-phase synthesis of piperazines, 3,9-diazaspiro[5.5]undecanes and 2,9-diazaspiro[5.5]undecanes is reported. The synthesis relies on the direct annulation of primary amines with resin-bound bismesylates. Critical to the success of this chemistry was the development of alpha-methyl benzyl carbamate resin linker. This resin permits the cleavage of the heterocycles under mildly acidic conditions, free of contaminating linker-derived N-alkylated byproducts.     

A differential array of metalated synthetic receptors for the analysis of tripeptide mixtures

A novel library of resin-bound receptors within a cross-reactive differential array for the identification and discrimination of tripeptides and tripeptide mixtures is reported. Pattern recognition using principal component analysis showed complete discrimination of four similar tripeptides and three tripeptide mixtures. The library is comprised of a Cu(II)-centered core with two proximally appended tripeptide arms emanating outward. One tripeptide arm was prepared using combinatorial chemistry to generate the differential nature of the library. Thirty resin-bound receptors were randomly selected from the library and placed within a silicon microchip array that included integrated microfluidics elements, and an indicator-uptake assay was used for colorimetric signaling. The indicator Orange G yielded an accurate measure of the degree of association between receptors and analytes as determined by kinetic analysis of the indicator-uptake assays. Within this paper we detail the method used for differential sensing using a novel receptor library. This work further demonstrates the power and utility of a differential array of synthetic receptors for identification and discrimination of complex bioanalytes.     

Synthesis of spirohydantoins and spiro-2,5-diketopiperazines via resin-bound cyclic alpha,alpha-disubstituted alpha-amino esters

A seven-step solid-phase synthesis of spirohydantoins and an eight-step solid-phase synthesis of spiro-2,5-diketopiperazines is reported. Key intermediate in the synthesis of both compound libraries is the resin-bound cyclic alpha,alpha-disubstituted alpha-amino ester, which can be obtained after selective homogeneous reduction of the aliphatic nitro ester using tin(II) chloride dihydrate. Nitro ester, in turn, is synthesized by a high-pressure-assisted [4 + 2] cycloaddition of resin-bound nitro alkene and butadiene, whereas nitro alkene is obtained by a Knoevenagel condensation of resin-bound nitro acetate with an imine. Novel spirohydantoins are obtained by isocyanate coupling with the resin-bound amino ester 5, followed by cyclization cleavage using a base. Novel spiro-2,5-diketopiperazines are obtained by PyBOP coupling of a Fmoc-protected amino acid with resin-bound amino ester, followed by Fmoc deprotection and an acid-assisted cyclization cleavage. After preparation of seven different resin-bound alpha,alpha-disubstituted alpha-amino esters, a 7 x 8 compound library of spirohydantoins was synthesized using eight different isocyanates, and a 7 x 8 compound library of spiro-2,5-diketopiperazines was synthesized using eight different Fmoc amino acids.     

Screening combinatorial libraries for optimal enzyme substrates by mass spectrometry.

A method has been developed for the rapid identification of optimal enzyme substrates from combinatorial libraries. This methodology was validated by screening a 361-member N-terminally formylated tripeptide library, f-XXR (X = 19 different amino acids), for optimal substrates of Escherichia coli peptide deformylase (PDF). The library was synthesized on a solid phase via the split-pool synthesis method. The N-terminal formyl group was added by treating the resin with a 1:1 (mol/mol) mixture of HCO(2)H and DCO(2)D in the presence of dicyclohexylcarbodiimide. In a mass spectrum, each member of the library produced a doublet peak (separated by 1.0063 Da). Limited treatment of this library with E. coli PDF resulted in the deformylation of those peptides that are the most efficient substrates of the enzyme. The deformylated products, due to loss of the mass-degenerate formyl group, each generated a singlet peak in the mass spectrum. Thus, the PDF product peaks were readily identified and sequenced via tandem mass spectrometry. The results showed that PDF strongly prefers a norleucine and, to a lesser extent, a phenylalanine as the N-terminal residue, whereas it has little selectivity at the penultimate position. This result is in excellent agreement with the literature data and therefore demonstrates the methodology as an effective approach to the identification of optimal enzyme substrates. This method should be generally applicable to other enzymes as well as synthetic catalysts.     

Synthesis of analogs of D-Ala-D-Ala as potential inhibitors of bacterial cell wall biosynthesis

The syntheses of the L,L- and D,D-stereoisomers of N- phenoxyacetyl -X-alanine in which X = Ser, Ala( beta Cl ) or Arg, are described. The antibacterial activity of these peptides and some of their synthetic intermediates has been examined. Four of the intermediates in which X = Ala( beta Cl ) and Arg(NO2), which possess C-terminal benzyl ester groups, were active against viridans streptococci and Streptococcus agalactiae. The D,D-enantiomers were more active than the corresponding L,L-isomers. None of the compounds were active against beta-lactamase producing bacteria or acted as beta-lactamase inhibitors.     

The acetyl CoA synthase paradigm for hybrid bio-organometallics: Quantitative measures for resin-bound Ni-Rh complexes

The active site of Acetyl CoA Synthase utilizes a square planar NiN₂S₂ complex in the form of Ni^II(CGC)²⁻ (CGC = the cysteine-glycine-cysteine tripeptide motif within the protein) to serve as a bidentate sulfur-donor ligand to chelate a second, catalytically active Ni atom responsible for the C-C and C-S coupling reactions for the production of Acetyl CoA. Metalloenzymes, such as this, which house stable catalytic complexes within intricately designed pockets accessible by solvent channels, have inspired design of resin-bound complexes. Through the use of TentaGel S-RAM^® resin beads, the O-Ni(CGC)²⁻ ligand has been synthesized and derivatized with the Rh^I(CO)₂ moiety. The identification of the adduct on these resin beads is afforded by attenuated total reflectance FTIR spectroscopy in the ν(CO) region and compared to solution analogues. The goal of this study is to establish a quantitative measure of the loading of nickel and rhodium on the tripeptide modified resin beads, O-(CGC). The extent of CGC derivatization was determined by Fmoc cleavage of the Fmoc protected O-(CGC). Nickel and rhodium loading were determined by Neutron Activation Analysis. This work provides evidence that the TentaGel S-RAM^® resin beads greatly decrease the air sensitivity of the Ni-Rh complex as compared to the unsupported solution phase analogue. The derivatized beads have also been studied for their ability to withstand a number of physical stresses, i.e., for leaching.     

Synthesis of an oligosaccharide–polylysine dendrimer with reducing sugar terminals leading to acquired immunodeficiency syndrome vaccine preparation

A novel cellobiose–polylysine dendrimer with reducing sugar terminals was synthesized in which the reactive reducing end of a disaccharide cellobiose was directing outward. Hexa‐O‐benzyl‐4′‐(1‐carboxyethyl)‐cellobioside (HBCEC) was synthesized through the reaction of a 4′‐hydroxyl group of benzyl hexa‐O‐benzyl‐cellobioside with methyl 2‐chloropropionate, followed by the removal of the methyl ester group. HBCEC was reacted with polylysine dendrimer generation 3 (G3) to produce benzylated cellobiose–polylysine dendrimer G3. After debenzylation, a cellobiose–polylysine dendrimer G3 was obtained in which the reducing end of the cellobiose was the terminal group of the dendrimer. For the preparation of a dendrimer‐type acquired immunodeficiency syndrome vaccine, the cellobiose–polylysine dendrimer was reacted with a tripeptide (glycyl–prolyl–leucine) and a cyclic oligopeptide from the human immunodeficiency virus by reductive amination; this produced a tripeptide‐bound cellobiose–polylysine dendrimer and an insoluble compound, respectively. The structure analysis was carried out with NMR and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2195–2206, 2005