Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues.

Histone deacetylases (HDACs) play essential roles in transcription regulation and are valuable theranostic targets. However, there are no activatable fluorescent probes for imaging of HDAC activity in live cells. Here, we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues. The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker. Upon deacetylation by HDAC, the probe undergoes a rapid self-immolative intramolecular cyclization reaction, producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore. The probe is shown to exhibit high sensitivity, high specificity, and fast response for HDAC detection in vitro. Imaging studies reveal that the probe is able to directly visualize and monitor HDAC activity in living cells. Moreover, the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to 130 µm. This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.

N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of the Glutamine Antagonist 6-Diazo-5-oxo-l-norleucine (DON) as a Potential Treatment for HIV Associated Neurocognitive Disorders.

Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.

Synthesis of quaternary α-amino acid-based arginase inhibitors via the Ugi reaction.

The Ugi reaction has been successfully applied to the synthesis of novel arginase inhibitors. In an effort to decrease conformational flexibility of the previously reported series of 2-amino-6-boronohexanoic acid (ABH) analogs 1, we designed and synthesized a series of compounds, 2, in which a piperidine ring is linked directly to a quaternary amino acid center. Further improvement of in vitro activity was achieved by adding two carbon bridge in the piperidine ring, that is, tropane analogs 11. These improvements in activity are rationalized by X-ray crystallography analysis, which show that the tropane ring nitrogen atom moves into direct contact with Asp202 (arginase II numbering). The synthetic routes described here enabled the design of novel arginase inhibitors with improved potency and markedly different physico-chemical properties compared to ABH. Compound 11c represents the most in vitro active arginase inhibitor reported to date.

Formation mechanism and characterization of dilysyl-dipyrrolones, the Maillard-type yellow pigments.

Our group has recently isolated and identified novel yellow compounds named dilysyl-dipyrrolones A (DPL A; 1) and B (DPL B; 2) in a heated aqueous solution containing xylose and lysine under weakly acidic conditions. In this study, we isolated and identified a novel DPL derivative (DPL C; 3), which has the same structure as DPL B, except for containing a hydroxymethyl group. To estimate the formation mechanism of DPL derivatives, (13)C-labeled DPLs were prepared and analyzed with LC/MS and NMR. (13)C-labeling experiments using [1-(13)C] ribose showed that the formation pathway of DPL C was different from those of DPLs A and B. In addition, (13)C-labeling experiments using [u-(13)C5] ribose and [1-(13)C] lysine showed that C-6 of a methine moiety in DPL C was derived from C-5 of ribose or acetic acid in buffer. Based on these results, we postulated the formation mechanism of DPLs. We then showed that DPLs A and B had potent antioxidant activities.

2-Substituted-2-amino-6-boronohexanoic acids as arginase inhibitors.

Substitution at the alpha center of the known human arginase inhibitor 2-amino-6-boronohexanoic acid (ABH) is acceptable in the active site pockets of both human arginase I and arginase II. In particular, substituents with a tertiary amine linked via a two carbon chain show improved inhibitory potency for both enzyme isoforms. This potency improvement can be rationalized by X-ray crystallography, which shows a water-mediated contact between the basic nitrogen and the carboxylic acid side chain of Asp200, which is situated at the mouth of the active site pocket of arginase II (Asp181 in arginase I). We believe that this is the first literature report of compounds with improved arginase inhibitory activity, relative to ABH, and represents a promising starting point for further optimization of in vitro potency and the identification of better tool molecules for in vivo investigations of the potential pathophysiological roles of arginases.

Studies toward the synthesis of potent anti-inflammatory peptides solomonamides A and B: synthesis of a macrocyclic skeleton and key fragment 4-amino-6-(2'-amino-4'-hydroxyphenyl)-3-hydroxy-2-methyl-6-oxohexanoic acid (AHMOA).

A first synthetic effort toward total synthesis of highly potent solomonamides is disclosed. An efficient strategy to synthesize this class of compounds, along with the synthesis of a core macrocycle (shown in red) and the key fragment AHMOA, is described.

Synthesis and antibacterial activity of alaremycin derivatives for the porphobilinogen synthase.

The preparation and the antibacterial activity of alaremycin derivatives such as their CF(3)-derivatives and (R)- and (S)-4-oxo-5-acetylaminohexanoic acid for the porphobilinogen synthase (PBGS), were described. The IC(50) values of the antibacterial activity of the prepared materials for the inhibitor of PBGS, were determined using PBGS assay.

alpha-Substituted norstatines as the transition-state mimic in inhibitors of multiple digestive vacuole malaria aspartic proteases.

The impact of moving the P1 side-chain from the beta-position to the alpha-position in norstatine-containing plasmepsin inhibitors was investigated, generating two new classes of tertiary alcohol-comprising alpha-benzylnorstatines and alpha-phenylnorstatines. Twelve alpha-substituted norstatines were designed, synthesized and evaluated for their inhibitory potencies against plasmepsin II and the plasmepsin IV orthologues (PM4) present in the digestive vacuole of all four Plasmodium species causing malaria in man. New synthetic routes were developed for producing the desired alpha-substituted norstatines as pure stereoisomers. The best compounds provided K(i) values in the nanomolar range for all PM4, with a best value of 110nM in PM4 from Plasmodium ovale. In addition, excellent selectivity over the closely related human aspartic protease Cathepsin D was achieved. The loss of affinity to Plasmodium falciparum PM4, which was experienced upon the move of the P1 substituent, was rationalized by the calculation of inhibitor-protein binding affinities using the linear interaction energy method (LIE).

Stereoselective one-pot synthesis of constrained N,O-orthogonally protected C-glycosyl norstatines [C(1')-aminoglycosyl-1,3-dioxolan-4-ones].

A procedure for the synthesis of conformationally constrained C-glycosyl norstatines has been developed. The key step of the reaction is the addition of (S)-N-sulfinyl azomethines to chiral (2S)-enolates of dioxolanones which exploits Seebach's "SRS" principle. The trisubstituted C-glycosyl-alpha-hydroxy-beta-amino acids are formed as N,O-orthogonally protected 1'-glycosyl-sulfinylamino-dioxolan-4-ones, usually with high diastereomeric excesses. Both the sulfinyl group at the nitrogen atom and the acetal moiety of the dioxolanone ring were selectively removed, thus demonstrating the orthogonality of the two protecting groups. In fact, the MeO(-) induced methanolysis of the acetal group gave the corresponding methyl C-glycosyl-sulfinylamino-isoserinates, while the acid induced removal of the sulfinyl group gave the Nu-deprotected 1'-glycosylamino-dioxolan-4-ones, which were in several cases subjected to a one-pot base-induced cyclization yielding the corresponding glycosyl-beta-lactams. This allowed the stereochemical configuration assessment of the parent 1'-glycosyl-sulfinylamino-dioxolan-4-ones by chemical correlation methods or by NOE experiments performed on the beta-lactams.

A comparison of flexible and constrained haptens in eliciting antibody catalysts for paraoxon hydrolysis.

A new amine-oxide hapten was employed as an antigen, producing seven monoclonal antibodies (mAbs) from a panel of 20 that catalyzed paraoxon hydrolysis. The current hapten design differs from that previously described in that the molecule is inherently more flexible than its constrained predecessor. One of the seven antibody catalysts, mAb 1H9, showed the highest activity and was selected for detailed study. At pH = 8.77, the catalytic hydrolysis of paraoxon by mAb 1H9 followed Michaelis Menten kinetics affording a k(cat) = 3.73 x 10(-4) min(-1) and a Km = 1.12 mM with a rate acceleration k(cat)/k(uncat) = 56. The hapten was found to be a competitive inhibitor of antibody-catalyzed paraoxon hydrolysis with a Ki = 0.54 mM. A comparison of both the number and proficiency of antibody catalysts obtained when utilizing a flexible versus constrained hapten indicates that, for paraoxon hydrolysis, constrained haptens elicit superior catalysts, suggesting that further development should begin with the use of constrained haptens in producing more proficient antibody catalysts for paraoxon hydrolysis.

Synthesis and activity of N alpha-(epsilon-aminocaproyl)-alanines and N epsilon-(epsilon-aminocaproyl)-caproic acid.

Four new dipeptides containing epsilon-aminocaproic acid were synthesized. Antibrinolytic, antiacaseinolytic activity and influence on activation of plasminogen by streptokinase were tested.

3-Amino-5,5-dimethylhexanoic acid. Synthesis, resolution, and effects on carnitine acyltransferases.

The selective inhibition of individual carnitine acyltransferases may be useful in the therapy of diabetes and heart disease. Aminocarnitine (3) is a weak competitive inhibitor (K(i) = 4.0 mM) for carnitine acetyltransferase (CAT), although the N-acetyl derivative 4 is about 165 times more potent (K(i) = 0.024 mM) than 3. Compound 3 is also a potent competitive inhibitor for carnitine palmitoyltransferases 1 and 2 (CPT-1 and CPT-2) (IC50 for CPT-2 = 805 nM). We synthesized 3-amino-5,5-dimethylhexanoic acid (7) and its N-acetyl derivative (8) as isosteric analogs of 3 and 4 that lack the quaternary ammonium positive charge. Like 3 and 4, compounds 7 and 8 were competitive inhibitors of CAT with significantly different potencies, but in this case, 8 (K(i) = 25 mM) was 10 times less potent than 7 (K(i) = 2.5 mM). R-(-)-7 and S-(+)-7 were stereoselective inhibitors of CAT (K(i) = 1.9 and 9.2 mM, respectively). Racemic 7 was a weak competitive inhibitor of CPT-2 (K(i) = 20 mM) and had no effect on CPT-1. These results are consistent with differences among the carnitine-binding sites on carnitine acyl-transferases that may be useful in selective inhibitor design. Furthermore, the data suggest that the quaternary ammonium positive charge of carnitine may be important for the proper orientation of carnitine and its analogs in the binding site.

Structure-activity relationships of HIV-1 PR inhibitors containing AHPBA.

A series of Human Immunodeficiency Virus type-1 protease (HIV-1 PR) inhibitors that contain 3-amino-2-hydroxy-4-phenylbutanoic acid (AHPBA) at the scission site of the substrate were prepared and evaluated for their inhibitory activity. Preliminary studies on the chain length of inhibitors and the hydroxyl configuration of AHPBA indicated that small (2S,3S)-derivatives, composed of the regions between the P3 and P2' sites, showed enough inhibitory activity toward HIV-1 PR to become prototypes for further structural modification. Systematic replacement at the sites from P3 to P2' revealed that some bicyclic heteroarylcarbonyl derivatives possessed strong potency and good enzyme selectivity.

Investigation of the active site of aminopeptidase A using a series of new thiol-containing inhibitors.

Aminopeptidase A (APA) and aminopeptidase N (APN) are two metallopeptidases which have been suggested to be involved in the enzymatic cascade of the renin-angiotensin system. APA liberates angotensin III from angiotensin II by releasing the N-terminal aspartate, and APN participates in the inactivation of angiotensin III. As the role of angiotensin III in the regulation of blood pressure in the central nervous system and at the periphery is controversial, it was of interest to develop selective and efficient inhibitors of APA. Starting from Glu-thiol(1), which was the first efficient APA inhibitor described, but however is equipotent on APA (Ki = 0.14 microM) and APN (Ki = 0.12 microM), beta-amino thiols bearing various carboxyalkyl chains have been synthesized and their inhibitory potencies measured on both purified enzymes. Compounds containing a carboxylated aromatic ring inhibited APA and APN with Ki values in the micromolar range but were slightly more active on APA. Conversely, inhibitors containing a cyclohexyl ring were more efficient on APN. Various modifications of the structure of Glu-thiol decreased inhibitory activity on both enzymes but increased the selectivity for APA, and compound 9d ((S)-4-amino-6-mercaptohexanoic acid) was 23 times more potent on APA (Ki = 2.0 microM) than on APN (Ki = 45 microM).

Use of structural alterations in the synthesis of halothane metabolite antigens to mimic halothane-induced immunogen.

Four hapten-carrier conjugates were synthesized to evaluate any potential antigenic similarities between these synthetic compounds and the immunogens induced in vivo by the anesthetic, halothane and, thus, be used eventually as a more sensitive probe to detect the presence of these halothane-induced antibodies in halothane-exposed individuals. In this study, antibodies from five halothane hepatitis patients were used to evaluate these antigenic alterations since the specificity of these antibodies would most accurately reflect the antigenic structure of halothane-induced immunogens. Quantitation of antibody binding to these synthetic proteins was determined in an enzyme linked immunosorbent assay and immunoblot techniques. Trifluoroacetylated rabbit serum albumin was 5 times more reactive with these antibodies and thus more antigenic than the homologous acetylated moiety confirming the importance of the trifluoromethyl moiety as an epitope in the immunogen in vivo. Insertion of a spacer arm, aminocaproic acid, between the hapten and carrier moieties and an epitope density of 40% acetylation also increased antigenicity. Through these structural alterations produced in vitro, antigenic compounds have been produced which may resemble more closely the immunogen elicited in vivo and which may ultimately serve as more sensitive probes for halothane-induced antibodies from exposed individuals.

[Relationship between structure and activity in antifibrinolytic peptides containing E-aminocaproic acid]. / Zaleznosc miedzy struktura a aktywnoscia antyfibrynolityczna peptydów zawierajacych kwas e-aminokapronowy.

New peptides containing E-aminocaproic acid were synthetized. Some conclusions on structure-activity relationship were made.

Labeling of DNA probes with a photoactivatable hapten.

A photoactivatable reagent for introducing haptens onto DNA probes has been prepared using a commercially available bifunctional linker arm reagent and amino-derivatized 2,4-dinitrophenyl (DNP). The resulting compound (photo-DNP) couples efficiently to DNA using an ordinary sunlamp. Under optimum conditions, about 7-23 DNP molecules per 1000 bases are incorporated into the DNA. Hybridization experiments demonstrate that as little as 1.5 x 10(5) copies of target DNA can be detected by filter hybridization with a photo-DNP-labeled probe and immunochemical detection.

[Synthesis of N alpha-(tosyl-beta-alanyl)- and N alpha-(tosyl-epsilon-aminocapronyl)amidinophenylalanineamides as strongly effective inhibitors of thrombin]. / Synthese von N alpha-(Tosyl-beta-alanyl)- und N alpha-(Tosyl-epsilon-aminocapronyl)amidinophenylalaninamiden als stark wirksame Thrombininhibitoren.

The headline compounds were prepared for further determination of structure-activity-relationships. By conversion of cyanophenylalanines with the acidic chloride of tosyl-beta-alanine resp. tosyl-epsilon-aminocapronic acid and aminolysis of the following prepared p-nitrophenylic esters the cyano compounds would get the desired structure, which were converted over the thioamides and thioimidic esters in the amidines by the common way. At the preparation of epsilon-aminocapronic acid derivatives occurred difficulties because of simultaneous formation of the N-tosyl-epsilon-caprolactame. The preparation of corresponding aminobutyric acid derivatives wasn't possible. The prolongation of the C-chain of the branchless amino acid decreased the antithrombine effect.