Preparation of 6,6,1',1',6',6'-hexadeutero sucrose

The preparation of 6,6,1',1',6',6'-hexadeutero sucrose is reported. The synthesis is based on a triple oxidation of a protected sucrose 6,1',6'-triol to the corresponding 6,1',6'-tricarboxylic acid or ester, followed by reduction with lithium aluminium deuteride. This triple oxidation could be achieved either using cat. TEMPO-NaOCl (to the acid) or PDC-Ac(2)O-t-BuOH (to the t-butyl carboxylic ester).     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 5: Exploration of pyridazinones containing 6-amino-substituents

The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.     

Determination of the P1', P2' and P3' subsite-specificity of factor Xa

Factor Xa is a central protease in the coagulation cascade and the target for many anticoagulant compounds currently under development. The preferences of the enzyme for substrates incorporating residues N-terminal to the cleavage site (P1, P2, etc.) have been elucidated, but little is known of its preferences for residues C-terminal to the cleavage site (P1', P2', etc.). The preferences of bovine factor Xa for substrate residues in the P1', P2' and P3' positions were mapped using fluorescence-quenched substrates. Bovine factor Xa, often used as a model for factor Xa, was most selective for the P2' position, less selective at the P1' position and almost completely non-selective at the P3' position. It appears that while the prime side subsites of factor Xa impose some selectivity towards substrates, the influence of these sites on factor Xa cleavage specificity is relatively low in comparison to related enzymes such as thrombin.     

Studies towards the large scale chemical synthesis of the precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5 from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-alpha,beta-D-allofuranose-3,4-d2 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 16 (precursor of ribonucleosides-3',4',5',5'-2H4) or to 1-O-methyl-3,5-di-O-benzyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 18 (precursor of ribonucleosides-3',4',5',5'-2H4).     

S(1)' and S(2)' subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. In conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.     

The effect of 2',4',7-trihydroxyisoflavone on ultraviolet-induced matrix metalloproteinases-1 expression in human skin fibroblasts

UV-induced matrix metalloproteinases (MMPs) cause connective tissue damage and the skin to become wrinkled and aged. Here, we investigated the effect of 2',4',7-trihydroxyisoflavone (THF) on UV-induced MMP-1 expression in human skin fibroblasts (HSFs). We found that UV irradiation increases MMP-1 expression and that this is mediated by ERK and JNK activation, but not by p38 activation. Pretreatment of HSFs with 2',4',7-THF inhibited UV-induced MMP-1 expression in a dose-dependent manner, and also inhibited the UV-induced activations of ERK and JNK by inhibiting MEK1 and SEK1 activation, respectively. Moreover, inhibitions of ERK and JNK by 2',4',7-THF resulted in the decrease of c-Fos expression and c-Jun phosphorylation/expression induced by UV, respectively, which led to the inhibition of UV-induced AP-1 DNA binding activity. This inhibitory effect of 2',4',7-THF on MMP-1 was not mediated by an antioxidant effect. In conclusion, our results demonstrate that 2',4',7-THF can inhibit UV-induced MMP-1 expression by inhibiting the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways. Therefore, 2',4',7-THF is a potential agent for the prevention and treatment of skin aging.     

Structural analysis of 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine and 2',3'-dideoxycytidine by 500-MHz 1H-NMR spectroscopy and ab-initio molecular orbital calculations.

Solution structure of anti-AIDS drug, 2',3'-dideoxyinosine (ddI) has been assessed by NMR spectroscopy and pseudorotational analysis in conjunction with its analogues: 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxycytidine (ddC). The absence of 3'-hydroxyl groups in these compounds has prompted us to establish the relationship between proton-proton and corresponding endocyclic torsion angles in the 2',3'-dideoxyribofuranose moiety on the basis of five available crystal structures of 2',3'-dideoxynucleosides. A subsequent pseudorotational analysis on ddI (1), ddA (2), ddG (3) and ddC (4) shows that the twist C2'exo-C3'-endo forms of sugar are overwhelmingly preferred (75-80%) over the C2'-endo envelope forms. The phase angles (P) for North and South conformers with the corresponding puckering amplitude (psi m) for ddI (1), ddA (2) and ddG (3) are as follows: PN = 0.1 degrees, PS = 161 degrees and psi m = 34.1 degrees for ddI (1); PN = 1.4 degrees, PS = 160 degrees and psi m = 34.2 degrees for ddA (2) and PN = 2.4 degrees, PS = 163 degrees and psi m = 33.6 degrees for ddG (3). The predominant North conformer of ddC (4) is intermediate between twist C2'-exo-C3'-endo and C3'-endo envelope (P = 10.9 degrees) with a psi m of 34.7 degrees. Note that these preponderant North-sugar structures (approx. 75-80%) found in the solution studies of ddI (1), ddA (2), dG (3) and ddC (4) are not reflected in the X-ray crystal structures of 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures denosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures are found to be in the South-type geometry (ddA crystalizes in C3'-exo envelope form, while ddC adopts the form intermediate between the C3'-exo envelope and C3'-endo-C4'-exo twist form). This means that X-ray structures of ddA (2) and ddC (4) only represent the minor conformer of the overall pseudorotamer population in solution. An assumption that the structure of the pentofuranose sugar (i.e. P and psi m) participating in conformational equilibrium described by the two-state model remains unchanged at different temperatures has been experimentally validated by assessing five unknown pseudorotational parameters with eight unique observables (3J1'2', 3J1'2", 3J2'3', 3J2'3", 3J2"3', 3J2"3", 3J3'4' and 3J3"4') for 2',3'-dideoxynucleosides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excision of 3' termini by the Trex1 and TREX2 3'-->5' exonucleases. Characterization of the recombinant proteins

The excision of nucleotides from DNA 3' termini is an important step in DNA replication, repair, and recombination pathways to generate correctly base paired termini for subsequent processing. The mammalian TREX1 and TREX2 proteins contain potent 3'-->5' exonucleases capable of functioning in this capacity. To study the activities of these exonucleases we have developed strategies to express and purify the recombinant mouse Trex1 and human TREX2 proteins in Escherichia coli in quantities sufficient for biochemical characterization. The Trex1 and TREX2 proteins are homodimers that exhibit robust 3' excision activities with very similar preferred reaction conditions and preferences for specific DNA substrates. In a steady-state kinetic analysis, oligonucleotide substrates were used to measure 3' nucleotide excision by Trex1 and TREX2. The Michaelis constants derived from these data indicate similar apparent kcat values of 22 s(-1) for Trex1 and 16 s(-1) for TREX2 using single-stranded oligonucleotides. The apparent KM values of 19 nm for Trex1 and 190 nm for TREX2 suggest relatively high affinities for DNA for both Trex1 and TREX2. An exonuclease competition assay was designed using heparin as a nonsubstrate inhibitor with a series of partial duplex DNAs to delineate the substrate structure preferences for 3' nucleotide excision by Trex1 and TREX2. The catalytic properties of the TREX proteins suggest roles for these enzymes in the 3' end-trimming processes necessary for producing correctly base paired 3' termini.     

Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1' and P3' residues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-cyclohexylalanine)-(D-Glu)-OH, in which nonpolar P1' residue Xaa or P3' residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3' residue with D-phenylglycine or D-Phe improved the K(i) value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine lowered the K(i) values to (8.2 +/- 0.6) x 10(-14) or (5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement of the K(i) values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/- 0.4) x 10(-14) M, respectively. These K(i) values are equivalent to that of natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L-norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.     

Structure-activity relationships for 1',1'-dimethylalkyl-Delta8-tetrahydrocannabinols

A series of 1',1'-dimethylalkyl-Delta(8)-tetrahydrocannabinol analogues with C-3 side chains of 2-12 carbon atoms has been synthesized and their in vitro and in vivo pharmacology has been evaluated. The lowest member of the series, 1',1'-dimethylethyl-Delta(8)-THC (8, n=0) has good affinity for the CB(1) receptor, but is inactive in vivo. The dimethylpropyl (8, n=1) through dimethyldecyl (8, n=8) all have high affinity for the CB(1) receptor and are full agonists in vivo. 1',1'-Dimethylundecyl-Delta(8)-THC (8, n=9) has significant affinity for the receptor (K(i)=25.8+/-5.8 nM), but has reduced potency in vivo. The dodecyl analogue (8, n=10) has little affinity for the CB(1) receptor and is inactive in vivo. A quantitative structure-activity relationship study of the side chain region of these compounds is consistent with the concept that for optimum affinity and potency the side chain must be of a length which will permit its terminus to loop back in proximity to the phenolic ring of the cannabinoid.     

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS.

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.     

Synthesis and stability studies of 2',3',5'-tri-O-acetyl-2-amino(-N6-cyclopentyl)-1-deazaadenosines

In this article, we report on the synthesis of 2',3',5'-tri-O-acetyl-2-amino-1-deazaadenosine and of 2',3',5'-tri-O-acetyl-2-amino-N(6)-cyclopentyl-1-deazaadenosine, which are very versatile intermediates for the preparation of 2-substituted 1-deazaadenosine derivatives. The two synthesized compounds showed to be quite unstable, with the N(6)-substituted derivatives being less stable than the N(6)-unsubstituted counterpart, according to the calculated HOMO-LUMO energy gap. Stability studies were performed through HPLC-MS analysis.     

Immediate-early mRNA-2 of herpes simplex viruses types 1 and 2 is unspliced: conserved sequences around the 5' and 3' termini correspond to transcription regulatory signals

Nuclease S1 and exonuclease VII analyses of immediate-early (IE) mRNA-2 of herpes simplex viruses types 1 and 2 (HSV-1, HSV-2) show them to be unspliced and of similar length. The DNA sequences around the 5' and 3' termini have been determined. Comparison of the sequences around the 5' ends reveals several common features. (1) Four discrete blocks of upstream homology which are precisely colinear with respect to the 5' termini of the mRNAs; the blocks include the 'TATA' box, a G-C rich sequence and a sequence (AATTAAATACAT) which may be involved in the coordinate induction of the IE class of genes. (2) Several copies of the sequence CCCCGCCC, found in different upstream positions in HSV-1 and HSV-2, which may be important in the expression of a wide variety of eukaryotic genes. (3) Potential hairpin structures in the region of the 5' termini which are present at similar locations in HSV-1 and HSV-2. Sequence comparison around the 3' termini of IEmRNA-2 reveals high homology at the proposed C-terminus of the polypeptide.     

Facile synthesis of 1',2'-cis-beta-pyranosyladenine nucleosides

1',2'-cis-beta-Glycosyladenine nucleosides, such as beta-altroside, beta-mannoside, and beta-idoside, were efficiently synthesized from the corresponding 1',2'-trans-beta-6-chloropurine derivatives, beta-glucoside, and beta-galactoside. Nucleophilic substitution of the O-trifluoromethanesulfonyl groups at the C-2' and/or 3' was carried out using tetrabutylammonium acetate or cesium acetate under mild conditions. Subsequent deprotection and amidation afforded the desired compounds, 1',2'-cis-beta-pyranosyladenine nucleosides.     

Cyclic sulfamide HIV-1 protease inhibitors, with sidechains spanning from P2/P2' to P1/P1'

Previous studies of HIV protease inhibitors have shown that it is possible to elongate the P1/P1' sidechains to reach the S3/S3' binding sites. By analogy, we expected that it would be possible to design inhibitors reaching between the S1/S1' and S2/S2' binding sites. Molecular modeling suggested that this could be achieved with appropriate ortho-substitution of the P2/P2' benzyl groups in our cyclic sulfamide inhibitors. Four different spacer groups were investigated. The compounds were smoothly prepared from tartaric acid in five steps and exhibit low to moderate activity, the most potent inhibitor possessing a Ki value of 0.53 microM.     

The RAD2 domain of human exonuclease 1 exhibits 5' to 3' exonuclease and flap structure-specific endonuclease activities

The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg(2+) being the preferred metal ion. HEX1-N2 is approximately 3-fold less active in Mn(2+)-containing buffers and exhibits <5% activity in the presence of Co(2+), Zn(2+), or Ca(2+). The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (k(cat)) and site of incision, and is as efficient (k(cat)/K(m)) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.     

Structural determinants of RXPA380, a potent and highly selective inhibitor of the angiotensin-converting enzyme C-domain

RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P(1)' and P(2)' positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S(2)' pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S(2)' composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors.