Heterobifunctionalized tetraethylene glycol: A spacer for surface attachment of viral peptide epitopes for ELISA and derivatization of membrane modifying peptides

Summary New hydrophilic linkers of the formula Fmoc-NHCH₂CH₂COO(CH₂CH₂O)₄X (X=COCH₂CH₂COOH Fmoc-Ats (2), X=CONHCH₂COOH Fmoc-Atg (4) and X=CONHCH₂CH₂COOH Fmoc-Ata (5) have been prepared by heterobifunctional modification of tetraethylene glycol as starting material. These linkers represent a useful tool for solid phase peptide synthesis according to Fmoc/tBu strategy. Two examples are presented to illustrate the applicability of these building blocks: (i) spacing between biotin and a peptide epitope of the hepatitis C virus and evaluation in a biotin-streptavidin ELISA, and (ii) coupling of the new linker to the N- and C-terminus of the peptide antibiotic alamethicin to show eventual influences on the peptide's α-helical conformation.     

Mechanism of inactivation of gamma-aminobutyrate aminotransferase by 4-amino-5-fluoropentanoic acid. First example of an enamine mechanism for a gamma-amino acid with a partition ratio of 0

The mechanism of inactivation of pig brain gamma-aminobutyric acid aminotransferase (GABA-T) by (S)-4-amino-5-fluoropentanoic acid (1, R = CH2CH2COOH, X = F) previously proposed [Silverman, R. B., & Levy, M. A. (1981) Biochemistry 20, 1197-1203] is revised. apo-GABA-T is reconstituted with [4-3H]pyridoxal 5'-phosphate and inactivated with 1 (R = CH2CH2COOH, X = F). Treatment of inactivated enzyme with base followed by acid denaturation leads to the complete release of radioactivity as 6-[2-hydroxy-3-methyl-6-(phosphonoxymethyl)-4-pyridinyl]-4-oxo-5-+ ++hexenoic acid (4, R = CH2CH2COOH). Alkaline phosphatase treatment of this compound produces dephosphorylated 4 (R = CH2CH2COOH). These results support a mechanism that was suggested by Metzler and co-workers [Likos, J. J., Ueno, H., Feldhaus, R. W., & Metzler, D. E. (1982) Biochemistry 21, 4377-4386] for the inactivation of glutamate decarboxylase by serine O-sulfate (Scheme I, pathway b, R = COOH, X = OSO3-).     

Radiochemical investigations of (99m)Tc-N(3)S-X-BBN[7-14]NH(2): an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop (99m)Tc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N(3)S-BBN (N(3)S = dimethylglycyl-l-seryl-l-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = 0 (no spacer), omega-NH(2)(CH(2))(2)COOH, omega-NH(2)(CH(2))(4)COOH, omega-NH(2)(CH(2))(7)COOH, or omega-NH(2)-(CH(2))(10)COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N(3)S-X-BBN[7-14]NH(2) (X = 0 carbons, beta-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) with gentle heating. Re-N(3)S-5-Ava-BBN[7-14]NH(2) was also prepared by the reaction of [Re(V)dimethylglycyl-l-seryl-l-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH(2). ES-MS was used to determine the molecular constitution of the new Re(V) conjugates. The (99m)Tc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[(99m)TcO(4)] with excess SnCl(2), sodium gluconate, and corresponding ligand. The (99m)Tc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.     

Extended binding inhibitors of chymotrypsin that interact with leaving group subsites S1'-S3'

We have synthesized inhibitors of chymotrypsin, based on fluoromethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-ambo-Phe-CF2H (1) (Ki = 25 X 10(-6) M). This inhibitor was modified by successively replacing H of the -CF2H group by -CH2CH2CONHCH3, (4), -CH2CH2CONH-Leu-NHMe (5), -CH2CH2CONH-Leu-Val-OEt (6), and -CH2CH2CONH-Leu-Arg-OMe (7). Corresponding Ki values are 7.8 (4), 0.23 (5), 0.21 (6), and 0.014 (7) microM. Extending 5 to 6 by addition of Val-OEt at P3' does not decrease Ki. In contrast, extension of 5 to 7 by incorporating Arg-OMe at P3' decreases Ki approximately 15-fold, suggesting interaction between Arg and the S3' subsite but no corresponding interaction at that subsite with Val. These results are in accordance with results obtained with the homologous family of avian ovomucoid third domain proteins. Proteins with Arg at the P3' position show highly favorable interactions with the protease at the S3' subsite [Park, S. J. (1985) Ph.D. Thesis, Purdue University; M. Laskowski, Jr., personal communication]. These results establish that incorporation of residues which interact with S' subsites significantly increases the efficacy of inhibitors and that valuable information concerning the most effective amino acid composition of small inhibitors can be obtained from the amino acid sequence of protein inhibitors.     

The hydrolysis of esters of N-hippurylglycine and N-pivaloylglycine by carboxypeptidase A

The kinetics of the hydrolysis of five esters of N-hippurylglycine (C6H5CONHCH2CONHCH2CO2CRR1CO2H (2 approximately) and seven esters of N-pivaloylglycine ((CH3)3CCONHCH2CRR1CO2H (3 approximately)) by bovine pancreatic carboxypeptidase A (Peptidyl-L-amino-acidhydrolase, EC 3.4.12.2) have been studied at pH 7.5, 25 degrees C and ionic strength 0.5. All N-hippurylglycine esters (2: R=H, R1=H, C2H5, 4-ClC6H4, C6H5CH2) display Michaelis-Menten kinetics up to at least 0.1 M substrate. The N-pivaloylglycine esters display either Michaelis-Menten kinetics (3 approximately: R=H, R1=H, C2H5 C6H5), substrate activation (3 approximately: R=H, R1=4-ClC6H4; R=R1=CH3) or substrate inhibition (3 approximately: R=H, R1=(CH3)2CHCH2, C6H5CH2). Kinetic parameters have been evaluated for each ester and compared with those for the corresponding hippuric acid esters (1 approximately). The enzymic specificity is shown to be identical for the alcohol moieties of the esters 1 approximately, 2 approximately and 3 approximately and unrelated to the occurrence of substrate activation or inhibition phenomena. These latter phenomena are shown to be characteristic of the enzymic hydrolysis of N-acyl amino acid esters but unimportant for N-acyl dipeptide ester substrates.     

Interaction of a phosphorylated site of muscle phosphofructokinase with the activation of the enzyme by NH4+ and K+.1

The crystal structure of the cyclic peptide disulfide

has been determined by X-ray diffraction. The peptide crystallizes in the space group P2₁2₁2₁, with a = 8.646(1), b = 18.462(2), c = 19.678(3)Å and Z = 4. The molecules adopt a highly folded compact conformation, stabilized by two intramolecular 4→ 1 hydrogen bonds between the Cys (1) and Pro (2) CO groups and the Cys (4) and methylamide NH groups, respectively. The backbone conformational angles for the peptide lie very close to those expected for a 3₁₀ helix. The S-S bridge adopts a right handed twist with a dihedral angle of 82°. The structure illustrates the role of stereochemically constrained residues, in generating novel peptide conformations.     

Comparing micellar, hemolytic, and antibacterial properties of di- and tricarboxyl dendritic amphiphiles

Homologous dicarboxyl dendritic amphiphiles-RCONHC(CH(3))(CH(2)CH(2)COOH)(2), 4(n); and ROCONHC(CH(3))(CH(2)CH(2)COOH)(2), 5(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were synthesized. Critical micelle concentrations (CMCs) in aqueous triethanolamine solutions and at pH 7.4 were measured along with hemolytic activity (effective concentrations, EC(10)) in phosphate-buffered saline (PBS). LogCMC showed a linear dependence on chain length (n); the longest chain in each series had the lowest CMC-in triethanolamine: 4(21), 180μM and 5(22), 74μM and at pH 7.4: 4(21), 78μM and 5(22), 33μM. These two series, 4(n) and 5(n), and three series of homologous tricarboxyl dendritic amphiphiles-RCONHC(CH(2)CH(2)COOH)(3), 1(n); ROCONHC(CH(2)CH(2)COOH)(3), 2(n); RNHCONHC(CH(2)CH(2)COOH)(3), 3(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were tested for growth inhibition of Staphylococcus aureus strain ATCC 6358 and methicillin-resistant S. aureus (MRSA) strain ATCC 43330 by microdilution in 0.1-strength brain heart infusion broth (BHIB). Amphiphiles 4(19), 4(21), 5(18), and 5(20) showed the strongest antibacterial activity (2.2-3.4μg/mL) against S. aureus (vancomycin, MIC=0.25μg/mL). These four plus 1(21), 2(20), 2(22), and 3(20) exhibited the strongest antibacterial activity (1.7-6.8μg/mL) against MRSA (vancomycin, MIC=0.25μg/mL). The MICs of these amphiphiles against six clinical MRSA were similar to those against the ATCC strain. In PBS, EC(10)s of the most active homologues ranged from 7 to 18μg/mL and 18 to 220μg/mL for di- and tricarboxyl dendritic amphiphiles, respectively. To assess the potential safety of using dendritic amphiphiles as drugs, measurements of micellar and hemolytic properties were conducted in the same medium (full-strength BHIB) that was used for antibacterial activity. The CMCs (9-36μg/mL, ～18-72μM) of ten amphiphiles were measured by microdilution (log2 progression) with dye-covered beads. The EC(10)s were similar to those in PBS. The MICs of most amphiphiles (14-72μg/mL) and vancomycin (1.1-2.2μg/mL) against both S. aureus and MRSA increased significantly compared to the MICs measured in 0.1-strength BHIB. The one exception, 5(18), had an MIC against S. aureus of 1.1μg/mL compared to vancomycin (2.2μg/mL). With CMC (9-18μg/mL) and EC(10) (16μg/mL) values higher than the MIC, 5(18) was discovered as a lead for further development.     

Reactions of thioether carboxylic acids with mercury(II). Formation and X-ray crystal structure of mercury(II) mercaptoacetate

Reactions of Hg(II) salts with thioether carboxylic acids o-C₆H₄[CH(SCH₂COOH)₂]₂ (1a) and PhCH(SCH₂COOH)₂ (3) in water were found to lead to the decomposition of these ligands with the formation of mercury(II) mercaptoacetate Hg(SCH₂COOH)₂ (2) and aldehydes o-C₆H₄(CHO)₂ and PhCHO, respectively. A similar reaction was observed between Hg(NO₃)₂ and CH₃(CH₂)₂CH(SCH₂COOH)₂ (4). The X-ray structure of Hg(SCH₂COOH)₂ (2) shows a linear -S-Hg-S- moiety. The mechanism of the formation of 2 in the reactions between Hg²⁺ and thioether carboxylic acids in water is discussed.     

N,N-bis(2-mercaptoethyl)methylamine: a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the [M(NNC5H4NH(x))2] core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M[CH3N(CH2CH2S)2](NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re[C5H3N-2,6-(CH2S)2](NNC5H4N)(NHNC5H4N)].CH2Cl2.0.5MeOH (7), [Re[SCH2CH2)2O] (NNC5H4N)(NHNC5H4N)] (8), and [Re[(SCH2CH2)2S](NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3[NNC5H3NH(CO2R)] [NHNC5H3N(CO2R)]] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3[NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)] [NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)]].2HCl (14.2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2[NNC5H3N(CO2Me)][NHNC5H3N(CO2Me)]] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.     

Ring conformation of cyclic octapeptide cyclo (L-Pro-Sar)4 in the crystalline state

A single-crystal X-ray diffraction analysis has been made of the structure of the cyclic octapeptide cyclo(L-Pro-Sar)4. The material [C32H48O8N8 X (21/4) H2O X (1/2) CH3OH, Mr = 799.43] crystallizes in the monoclinic space group C2 with cell dimensions a = 14.544 (3), b = 11.902 (2), c = 14.064 (3), and beta = 122.26 (2) degrees (lambda = 1.54178 A, T = 293 K). The final R value for the 1980 observed reflections is 0.079. The ring conformation has the peptide bond sequences of cis-cis-trans-trans-cis-cis-trans-trans (Pro-Sar-Pro peptide bond linkages are cis-cis- or trans-trans). The pyrrolidine rings in the four proline residues take an envelope form in which the gamma-carbon atom deviates from the plane of the remaining four atoms in the ring.     

Peroxyl radical is produced upon the interaction of hypochlorite with tert-butyl hydroperoxide

As we reported previously, hypochlorite interacting with organic hydroperoxides causes their decomposition ((1995) Biochemistry (Moscow), 60, 1079-1086). This interaction was supposed to be a free-radical process and serve as a source of free radicals initiating lipid peroxidation (LP). The present study is the first attempt to detect and identify free radicals produced in the reaction of hypochlorite with tert-butyl hydroperoxide, (CH₃)₃COOH, which we have used as an example of organic hydroperoxides. We have used a direct method for free radical detection, EPR of spin trapping, and the following spin traps: N-tert-butyl--phenylnitrone (PBN) and -(4-pyridyl-1-oxyl)-N-tert-butylnitrone (4-POBN). When hypochlorite was added to (CH₃)₃COOH in the presence of a spin trap, an EPR spectrum appeared representing a superposition of two signals. One of them belonged to a spin adduct formed as a result of direct interaction of hypochlorite with the spin trap (hyperfine splitting constants were: _H H = 0.148 mT; a_N = 1.537 mT; and H_PP = 0.042 mT for 4-POBN and _H = 0.190 mT; a_N = 1.558 mT; and H_PP = 0.074 mT for PBN). The other signal was produced by hypochlorite interactions with (CH3)3COOH itself (hyperfine splitting constants were: _H = 0.233 mT; aN = 1.484 mT; H_PP = 0.063 mT and _H = 0.360 mT; a_N = 1.547 mT; H_PP = 0.063 mT for 4-POBN and PBN, respectively). Comparison of spectral characteristics of this spin adduct with those of tert-butoxyl or tert-butyl peroxyl radicals produced in known reactions of (CH₃)₃COOH with Fe²⁺ and Ce⁴⁺, respectively, showed that the radical (CH₃)₃COO. is produced from the interaction of hypochlorite with (CH₃)₃COOH^. Like Ce⁴⁺ but not Fe²⁺, hypochlorite addition to (CH₃)₃COOH was accompanied by a bright flash of chemiluminescence characteristic of the reactions in which peroxyl radicals are produced. Thus, all these results suggest peroxyl radical production in the reaction of hypochlorite with hydroperoxide. This reaction is one of the most possible ways for the initiation of free-radical LP that occurs in vivo, when hypochlorite interacts with unsaturated lipids comprising natural protein–lipid complexes, such as lipoproteins and biological membranes.     

On the appearance and role of a spacer group in the protein amino acids

Summary Of the 20 protein amino acids, 16 have a methylene group at the position, and a further three bear a methine group. No aromatic, carboxamido, carboxylic carbon, or hetero atoms are attached directly to the carbon, but they are separated by this methylene or occasionally by a longern-alkylene spacer group. Therefore, the structure of the protein amino acids should rather be formulated as H₂N–CH((CH₂)_n–R)–COOH instead of the generally accepted H₂N–CH(R)–COOH. The appearance of and the role played by the spacer group are discussed in an evolutionary context. It is suggested that the spacer group appeared as a result of prebiotic selection, based on the relative abundance, racemization rate, and suitability for thermal polymerization of the protein amino acids and their homologs with various spacer group lengths. At the biotic level of evolution the requirements for ribosomal polymerization, as well as the abilities of polypeptides to maintain a stable and flexible threedimensional structure and to bind ligands are considered and are proposed to have been responsible for the possible exclusion of longer spacer groups. It is concluded that the general role of the spacer group is to ensure the uniformity of the constant regions H₂N–CH(-)–COOH and the individuality of the R contact groups by spatially separating them.     

Interaction of tert-butyl hydroperoxide with hypochlorite induces peroxyl radicals. A chemiluminescence study

The interaction of hypochlorite (HOCl/OCl-) with tert-butyl hydroperoxide ((CH3)3COOH) was investigated by chemiluminescence. It was shown that the addition of HOCl/OCl- to (CH3)3COOH induces a fast chemiluminescent flash. The intensity of this flash increases with the increase in both HOCl/OCl- and (CH3)3COOH concentration. The chemiluminescence is quenched in a concentration-dependent manner in the presence of free radical spin traps N-tert-butyl nitrone and alpha-(4-pyridyl-1-oxyl)-N-tert-butyl nitrone. This fact proves that free radicals take part in the interaction of HOCl/OCl- and (CH3)3COOH. Hypochlorite yielded a very similar chemiluminescence spectrum in its reaction with (CH3)3COOH as Ce4+. It differed considerably from the spectrum in the system H2O2 and HOCl/OCl-. It is well known that the interaction of Ce4+ and (CH3)3COOH produces peroxyl radicals. These results confirm the hyothesis that the interaction of HOCl/OCl- and (CH3)3COOH is mediated by peroxyl radicals. Thus, organic hydroperoxides always present in unsaturated lipids can induce lipid peroxidation processes in the reaction with HOCl/OCl-.     

Hyperpolarizabilities of substituted benzene molecules: MNDO calculations

The values for a series of substituted benzene molecules of the H-C6H4-R and R-C6H4-R types (R, R=H, CH3, OCH3, CHO, CN, COOH, F, Cl, Br, I, OH, NO2, NO, NH2, NHMe, N(Me)2, Li, CF3, NHNH2, NHOH, C2H, COCN, COF, SO3H, phenyl, vinyl, cyclopropenyl, cyclo-pentadienyl) have been computed using the finite perturbation method at the MNDO level. Our results are compared to experimental data and to other theoretical values. A classification of the various substituting groups is set up so as to help in the design of new optically interesting molecules. It has been shown that the pCCB (para-Cyclopropenyl, Cyclopentadienyl Benzene) presents a large hyperpolarizability value (61.54 10^–36 esu) compared with (C6H6)=0.72 10^–36 esu.     

Reversible covalent binding of peptide nitriles to papain

The dissociation constants for reversible covalent binding of twelve peptide nitrile inhibitors to the active site of papain have been measured by means of fluorescence titration. The binding constants generally parallel the kinetic specificity constants (kcat/Km) for related papain substrates, supporting earlier suggestions that peptide nitriles behave as transition state analog inhibitors of papain. In ten cases the temperature dependence of binding was analyzed to determine the enthalpic and entropic contributions to the binding energy. A compensation plot of delta H vs. T delta S resulted in two parallel lines, one for 'specific' nitriles (i.e., N-Ac-L-aa-NHCH2CN; aa = Phe, Leu, Met) and the other for 'non-specific' nitriles (e.g., N-Ac-D-Phe-NHCH2CN, PhCH2CH2CONHCH2CN hippurylnitrile, etc.). For both specific and nonspecific nitriles representing an 1800-fold range of Kd values (0.27 microM-490 microM), the solvent deuterium isotope effect on binding (Kd(H2O)/Kd(D2O) = DKd) was very close to 2.0. This isotope effect could be accounted for entirely by the simple protonic change which occurs upon the reversible addition of the active site sulfhydryl of papain to the nitrile group of the peptide derivative to form a covalent thioimidate linkage. In contrast, six closely related non-nitrile ligands containing identical peptide side chains but having C-terminal groups incapable of binding covalently to papain had unmeasureably high dissociation constants. Collectively, these results indicate that strong binding of peptide nitrile substrate analogs to papain requires a combination of (1) hydrophobic interaction (especially at the P2 position), (2) specific intermolecular hydrogen bonding and (3) covalent interaction of the nitrile with the active site sulfhydryl group.     

Synthesis of 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes: "thymine replacement" analogs of thymidine for evaluation as anticancer and antiviral agents

A group of unnatural 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluorobenzenes having a variety of C-5 two-carbon substituents [-C...C-X, X = I, Br; -C...CH; (E)-CH=CH-X, X = I, Br; -CH=CH2; -CH2CH3; -CH(N3) CH2Br], designed as nucleoside mimics, were synthesized for evaluation as anticancer and antiviral agents. The 5-substituted (E)-CH=CH-I and -CH2CH3 compounds exhibited negligible cytotoxicity in a MTT assay (CC50 = 10(-3) to 10(-4)M range), relative to thymidine (CC50 = 10(-3) to 10(-5)M range), against a variety of cancer cell lines. In contrast, the C-5 substituted -C...C-I and -CH(N3)CH2Br compounds were more cytotoxic (CC50 = 10(-5) to 10(-6)M range). The -C...C-I and -CH2CH3 compounds exhibited similar cytotoxicity against non-transfected (KBALB, 143B) and HSV-1 TK+ gene transfected (KBALB-STK, 143B-LTK) cancer cell lines expressing the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (TK+). This observation indicates that expression of the viral TK enzyme did not provide a gene therapeutic effect. The parent group of 5-substituted compounds, that were evaluated using a wide variety of antiviral assay systems [HSV-1, HSV-2, varicella-zoster virus (VZV), vaccinia virus, vesicular stomatitis, cytomegalovirus (CMV), and human immunodeficiency (HIV-1, HIV-2) viruses], showed that this class of unnatural C-aryl nucleoside mimics are inactive and/or weakly active antiviral agents.     

Exploiting the Peptide — MHC Water Interface in the Computer-Aided Design of Non-Natural Peptides that Bind to the Class I MHC Molecule HLA-A2

Abstract

Class I major histocompatibility complex (MHC) molecules bind peptides derived from intra-cellular proteins and present them to cytotoxic T cells. Certain human immunological diseases are associated with errors in this process. Here we describe an approach to the design of non-natural peptides that could potentially interfere with peptide presentation associated with autoimmune diseases. We have shown previously that the interaction of the peptide GILGFVFTL with the MHC molecule HLA-A2 is mediated by a network of water molecules. In principle, the addition of hydroxyl groups to the peptide could allow for an enhanced interaction of the modified peptide with this water network. Here we illustrate this approach using a peptide having the non-natural amino acid homoserine at position 3, GIhSGFVFTL, and also peptides in which the Cα(F5)—CO—NH1—Cα(V6) peptide bond is replaced by an ether. Cα(F5)—CH(X)—O—Cα(V6), to give the non-natural peptide GILGF—CH(X)—O—VFTL, where X = CH₂OH or CH₃. In a 200 ps solvated molecular dynamics simulation of the HLA-A2 complexes of each peptide for GIhSGFVFTL and GILGF—CH(CH₂OH)—O—VFTL the peptide conformation remained essentially unchanged from that of GILGFVFTL in the X-ray structure of its complex with HLA-A2. In contrast, for GILGF—CH(CH₃)—O—VFTL the peptide conformation deviated from the X-ray conformation, indicating the importance of the hydroxyl group.     

Modulation of fibronectin adhesive functions for fibroblasts and neural cells by chemically derivatized substrata

Adhesion responses of fibroblasts (Balb/c 3T3 cells) and human neuron-derived (Platt neuroblastoma) cells have been examined with plasma fibronectin (pFN) adsorbed to glass surfaces derivatized with an alkyl chain and six chemical end groups interfacing with the bound pFN to test regulation of pFN function. Using new derivatization protocols, the following surfaces have been tested in order of increasing polarity: [CH3], [C = C], [Br], [CN], [Diol], [COOH], and underivatized glass [( SiOH]). For all substrata, pFN bound equivalently using either a supersaturating amount of pFN or a subsaturating amount in competition with bovine albumin. Attachment of both cell types was also equivalent on all substrata. However, spreading/differentiation responses varied considerably. F-actin reorganization was tested in 3T3 cells with rhodamine-phalloidin staining. While stress fibers formed effectively on pFN-coated [SiOH] and [Br] substrata, only small linear bundles of F-actin and a few thin stress fibers were observed on the [COOH], [Diol], and [CN] substrata; the hydrophobic substrata [( CH3] and [C = C]) gave an intermediate response. When a synthetic peptide containing the Arg-Gly-Asp-Ser sequence required for integrin binding to FNs was included in the medium as an inhibitor, additional differences were noted: Stress fiber formation was completely inhibited on [SiOH] but not on [Br] and stress fiber formation was very sensitive to inhibition on the hydrophobic substrata while the F-actin patterns on the [CN] and [COOH] substrata were unaffected. Evaluation of neurite outgrowth by neuroblastoma cells on these substrata revealed both qualitative and quantitative differences as follows: [Diol] = [COOH] greater than [SiOH] much greater than [CN] = [Br] greater than [CH3] = [C = C]. While there was poor cytoplasmic spreading and virtually no neurites formed on the hydrophobic surfaces when pFN alone was adsorbed, neurite formation could be "rescued" if a mixture of pFN with an excess of bovine albumin was adsorbed, demonstrating complex conformational interactions between substratum-bound pFN and adhesion-inert neighboring molecules. In summary, these studies demonstrate that different chemical end groups on the substratum modulate pFN functions for cell adhesion, principally by affecting the conformation of these molecules rather than the amounts bound. Furthermore, these studies confirm multiple-receptor interactions with the FN molecules in cell type-specific adhesion patterns.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and antimicrobial activity of symmetrical two-tailed dendritic tricarboxylato amphiphiles

Two series of water-soluble, symmetrical two-tailed homologous dendritic amphiphiles--R(2)NCONHC((CH(2))(2)COOH)(3), 2(n,n), and R(2)CHNHCONHC((CH(2))(2)COOH)(3), 3(n,n), where R=n-C(n)H(2n+1)--were synthesized and compared to R'NHCONHC((CH(2))(2)COOH)(3), 1(n), R'=n-C(n)H(2n+1), to determine whether antimicrobial activity was influenced by total or individual alkyl chain lengths, and whether antimicrobial activity depends on hydrophobicity or tail topology (one or two). In a broad screen of 11 microorganisms, 2(n,n) and 3(n,n) generally displayed higher minimal inhibitory concentrations (MICs) than 1(n) against growth as measured by broth microdilution assays. Chain-length specificity was observed against Candida albicans as 1(16), 2(8,8), and 3(8,8) showed the lowest MIC in their respective series. The one case where two-tailed compounds displayed the lowest MICs-3(10,10), 15 microM; 3(11,11), 7.2microM; and 3(12,12), 6.9 microM-was against Cryptococcus neoformans.