Design, structural, and immuno-analytical properties of antigenic bioconjugates comprising a beta-amyloid-plaque specific epitope

Immunotherapeutic approaches are investigated for treatment of neurodegenerative diseases of the Alzheimer's dementia (AD) type. The identification of a beta-amyloid-plaque specific epitope, Abeta(4-10) (4FRHDSGY10), recognized by therapeutically active antibodies from transgenic AD mice could provide the basis for the development of AD vaccines. Here we report on the synthesis, structural and immuno-analytical characterization of bioconjugates comprising the beta-amyloid(4-10) epitope as new vaccine lead structures against Alzheimer's disease. To produce antigenic bioconjugates, potential immunogens, the epitope peptide elongated by a cysteine residue or a cysteinyl-pentaglycine hexapeptide unit either at the N- or C-terminus was attached via a thioether bond to synthetic oligopeptide carriers, such as oligotuftsin derivatives, sequential oligopeptide carrier, or lysine dendrimer. The antigenic properties of these constructs were determined by enzyme-linked immunosorbent assay (ELISA) using an anti-Abeta(1-17) monoclonal antibody. Our results indicate that the major factors which influence the antibody binding of the Abeta(4-10) epitope are (i) the epitope topology and (ii) the presence of a spacer moiety between the carrier and the epitope peptide. Interestingly, the carrier type had no marked effect on the binding of the antibody to the epitope-conjugates. The conformational preferences of the conjugates were examined by circular dichroism spectroscopy in water and in trifluoroethanol. In water, the conjugates adopt random coil conformation independently on their primary structure. However, differences related to the attachment site of the epitope to the carriers were determined in TFE, conjugates in which the epitope was attached to the carrier through the N-terminus exhibiting more ordered secondary structure.     

Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII

Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by modification in the flanking regions. However, no systematic studies have been reported to describe the effect of different carrier macromolecules in epitope conjugates. To this end, the influence of carrier structure and topology on antibody recognition of attached epitope has been studied by comparing the antibody binding properties of a new set of conjugates with tetratuftsin analogue (H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2), T20) sequential oligopeptide carrier (SOC(n)), branched chain polypeptide, poly[Lys(Ser(i)-DL-Ala(m))] (SAK), multiple antigenic peptide (MAP), and keyhole limpet hemocyanine (KLH). In these novel constructs, peptide (9)LKNleADPNRFRGKDL(22) ([Nle(11)]-9-22) representing an immunodominant B cell epitope of herpes simplex virus type 1 glycoprotein D (HSV-1 gD) was conjugated to polypeptides through a thioether or amide bond. Here we report on the preparation of sequential and polymeric polypeptides possessing chloroacetyl groups in multiple copies at the alpha- and/or epsilon-amino group of the polypeptides and its use for the conjugation of epitope peptides possessing Cys at C-terminal position. We have performed binding studies (direct and competitive ELISA) with monoclonal antibody (Mab) A16, recognizing the HSV gD-related epitope, [Nle(11)]-9-22, and conjugates containing identical and uniformly oriented epitope peptide in multiple copies attached to five different macromolecules as carrier. Data suggest that the chemical nature of the carrier and the degree of substitution have marked influence on the strength of antibody binding.     

Polypeptide conjugates comprising a beta-amyloid plaque-specific epitope as new vaccine structures against Alzheimer's disease

Immunotherapeutic approaches designed to induce a humoral immune response have recently been developed for possible vaccination to the treatment of Alzheimer's disease (AD). Based on the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, branched polypeptide conjugates with this epitope peptide were synthesized and characterized. In order to produce immunogenic constructs, the Abeta(4-10) epitope alone or together with a promiscuous T-helper cell epitope peptide (FFLLTRILTIPQSLD) were attached via thioether linkage to different branched chain polymeric polypeptides with Ser or Glu in the side chains. A single peptide containing both an Abeta(4-10) and T-helper cell epitope, joined by a dipeptide Cys-Acp spacer, was also attached through the thiol function to chloroacetylated poly[Lys(Seri-DL-Alax)] (SAK). Comparative binding studies of the conjugates with a monoclonal antibody against the beta-amyloid(1-17) peptide in mice were performed by direct ELISA. The conformational preferences of carriers and conjugates in water and in a 9:1 trifluoroethanol:water mixture (v/v) was analyzed by CD spectroscopy. Experimental data showed that the chemical nature of the carrier macromolecule, and the attachment site of the epitope to the carrier, have significant effects on antibody recognition, but have no marked influence on the solution conformation of the conjugates.     

Production of antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor coupled to protein carriers in different orientations

The antibodies to nicotinic acetylcholine receptor alpha(181-192) synthetic peptides were elicited in rabbits and mice using the peptides conjugated to protein carriers in different orientations, either through C-terminal Cys (S-conjugates), or through amino groups (N-conjugates). S-conjugated peptides were less potent in eliciting peptide-specific antibodies compared to N-conjugates and this type of conjugation resulted in antibodies to the coupling reagent. However, the epitopes present in either S- or N-conjugated peptides appeared to be similar, indicating that amino acid residues, which form the epitope, were located in the middle part of the peptide and did not include both N- and C-terminal residues. Peptide conjugation to a protein carrier did not play a role in stabilizing the peptide conformation, but was necessary to concentrate the peptide epitopes on the carrier surface enabling bivalent antibody binding.     

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between na?ve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.     

Crystal structure of cyclic Lys48-linked tetraubiquitin

Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85 Å resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin.     

Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

The nature of the bond between peptide and carrier molecule determines the immunogenicity of the construct.

The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester-, a disulfide-, an amide- and a thioether bond were investigated. As carrier molecules a peptide, an N-palmitoylated peptide or a C(16)-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide > thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide-based vaccine than a bond that is less susceptible to biological degradation.     

Conjugation of epitope peptides with SH group to branched chain polymeric polypeptides via Cys(Npys)

Since bioconjugates may play an important role as therapeutics in the future, the development of new and effective conjugation strategies is necessary. For the attachment of peptide-like molecules to carriers, there are two main coupling methods involving amide or disulfide bonds. Conjugation through an amide bond can be achieved in several well-defined ways known from peptide chemistry. However, the formation of disulfide bridges between cysteine-containing peptides and carrier molecules still has some problems. In this paper, we describe a novel approach in which the carrier polypeptide is modified by 3-nitro-2-pyridinesulfenyl (Npys)-protected cysteine and this derivative has been applied for conjugation of Cys-containing epitope peptides with poly(L-lysine)-based branched polypeptides. Considering the stability of Npys group in the presence of pentafluorophenol, Boc-Cys(Npys)-OPfp dervivative was selected for introduction to the N-terminal of branches of polypeptides backbone. The branches of the polymers were built up from oligo(DL-alanine) (poly[Lys(DL-Ala(m))], AK) and elongated by an optically active amino acid [poly[Lys(X(i)-DL-Ala(m))], XAK]. We found that the nature of X (Glu, Ser, Thr) has great influence on the incorporation of the protected cysteine residue. Herpes simplex virus and adenovirus epitope peptides were conjugated to Boc-Cys(Npys)-modified polypeptides. Results indicate that the incorporation of epitope peptides depends on the number of Npys group on the polymers as well as on the presence/absence of Boc-protecting group on the Cys residue. This new class of Cys(Npys)-derivatized branched polypeptides is stable for a couple of months and suitable for effective preparation of epitope peptide conjugates possessing increased water solubility.     

The molecular basis of thrombin allostery revealed by a 1.8 A structure of the "slow" form

Thrombin participates in its own positive and negative feedback loops, and its allosteric state helps determine the hemostatic balance. Here we present the 1.8 A crystallographic structure of S195A thrombin in two conformational states: active site occupied and active site free. The active site-occupied form shows how thrombin can accommodate substrates, such as protein C. The active site-free form is in a previously unobserved closed conformation of thrombin, which satisfies all the conditions of the so-called "slow" form. A mechanism of allostery is revealed, which relies on the concerted movement of the disulphide bond between Cys168 and 182 and aromatic residues Phe227, Trp215, and Trp60d. These residues constitute an allosteric switch, which is flipped directly through sodium binding, resulting in the fast form with an open active site.     

Effect of conjugation with polypeptide carrier on the enzymatic degradation of Herpes simplex virus glycoprotein D derived epitope peptide

Two conjugates with epitope peptide (278)LLEDPVGTVA (287) derived from glycoprotein D (gD-1) of Herpes simplex virus (HSV) were synthesized for analysis of the effect of conjugation on protection against enzymatic degradation. In this design, the turn-forming epitope core (281)DPVG (284) was positioned in the central part of the peptide and elongated by three amino acids from the native sequence at both termini. Conjugation was achieved by the introduction of amide bond or thioether linkage between the C-terminal of the HSV peptide and the side chain of four lysine residues of the oligotuftsin derivative used as carrier molecule. We compared the proteolytic stability of the conjugates in diluted human sera as well as in rat liver lysosomal preparation. The data obtained in lysosomal preparation at two pH values (pH 3.5 and 5.0) show that the type of covalent bond between the carrier and the epitope peptide had no significant effect, as compared to the stability of the free, unconjugated peptide. Based on the identification of degradation fragments by mass spectrometry we found marked differences in the lengths and amounts of oligopeptides obtained. In contrast, in 10% and 50% human serum the conjugation provided full protection against enzymatic hydrolysis over 96 h, while the free peptide was decomposed quickly.     

A modular approach to assembly of totally synthetic self-adjuvanting lipopeptide-based vaccines allows conformational epitope building

The technology described here allows the chemical synthesis of vaccines requiring correctly folded epitopes and that contain difficult or long peptide sequences. The final self-adjuvanting product promotes strong humoral and/or cell-mediated immunity. A module containing common components of the vaccine (T helper cell epitope and the adjuvanting lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine) was assembled to enable a plug and play approach to vaccine assembly. The inclusion within the module of a chemical group with chemical properties complementary and orthogonal to a chemical group present in the target epitope allowed chemoselective ligation of the two vaccine components. The heat-stable enterotoxin of enterotoxigenic Escherichia coli that requires strict conformational integrity for biological activity and the reproductive hormone luteinizing hormone-releasing hormone were used as the target epitopes for the antibody vaccines. An epitope from the acid polymerase of influenza virus was used to assemble a CD8(+) T cell vaccine. Evaluation of each vaccine candidate in animals demonstrated the feasibility of the approach and that the type of immune response required, viz. antibody or cytotoxic T lymphocyte, dictates the nature of the chemical linkage between the module and target epitope. The use of a thioether bond between the module and target epitope had little or no adverse effect on antibody responses, whereas the use of a disulfide bond between the module and target epitope almost completely abrogated the antibody response. In contrast, better cytotoxic T lymphocyte responses were obtained when a disulfide bond was used.     

Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.     

Crystal and molecular structure of the serine proteinase inhibitor CI-2 from barley seeds

Chymotrypsin inhibitor 2 (CI-2), a serine proteinase inhibitor from barley seeds, has been crystallized and its three-dimensional structure determined at 2.0-A resolution by the molecular replacement method. The structure has been refined by restrained-parameter least-squares methods to a crystallographic R factor (= sigma parallel Fo magnitude of-Fo parallel/sigma magnitude of Fo) o of 0.198. CI-2 is a member of the potato inhibitor 1 family. It lacks the characteristic stabilizing disulfide bonds of most other members of serine proteinase inhibitor families. The body of CI-2 shows few conformational changes between the free inhibitor and the previously reported structure of CI-2 in complex with subtilisin Novo [McPhalen, C.A., Svendsen, I., Jonassen, I., & James, M.N.G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7242-7246]. However, the reactive site loop has some significant conformational differences between the free inhibitor and its complexed form. The residues in this segment of polypeptide exhibit relatively large thermal motion parameters and some disorder in the uncomplexed form of the inhibitor. The reactive site bond is between Met-59I and Glu-60I in the consecutive sequential numbering of CI-2 (Met-60-Glu-61 according to the alignment of Svendsen et al. [Svendsen, I., Hejgaard, J., & Chavan, J.K. (1984) Carlsberg Res. Commun. 49, 493-502]). The network of hydrogen bonds and electrostatic interactions stabilizing the conformation of the reactive site loop is much less extensive in the free than in the complexed inhibitor.     

Crystal structure of the all-ferrous [4Fe-4S]0 form of the nitrogenase iron protein from Azotobacter vinelandii

The structure of the nitrogenase iron protein from Azotobacter vinelandii in the all-ferrous [4Fe-4S](0) form has been determined to 2.25 A resolution by using the multiwavelength anomalous diffraction (MAD) phasing technique. The structure demonstrates that major conformational changes are not necessary either in the iron protein or in the cluster to accommodate cluster reduction to the [4Fe-4S](0) oxidation state. A survey of [4Fe-4S] clusters coordinated by four cysteine ligands in proteins of known structure reveals that the [4Fe-4S] cluster of the iron protein has the largest accessible surface area, suggesting that solvent exposure may be relevant to the ability of the iron protein to exist in three oxidation states.     

Investigation of the interactions of polyhedral borane anions with serum albumins

The retention of polyhedral borane anions within tumor cells has been attributed to the possible formation of covalent bonds with nucleophilic protein substituents. In an effort to identify the nature of possible interactions between polyhedral borane anions and proteins, three polyhedral borane anions, [B(20)H(18)](2-), [B(20)H(17)OH](4-), and [B(20)H(17)SH](4-), were allowed to react with either bovine or human serum albumin. Reaction products were analyzed with matrix assisted laser desorption ionization (MALDI) mass spectrometry and gel electrophoresis. Evidence of disulfide bond formation was observed with the [B(20)H(17)SH](4-) anion, whereas no evidence of covalent binding was observed with the [B(20)H(18)](2-) and [B(20)H(17)OH](4-) ions. The potential for disulfide bond formation was confirmed by examining the reactions of the [B(20)H(17)SH](4-) ion with both DTNB and reduced glutathione. An understanding of the nature of the binding will provide a basis for the design and synthesis of boron-containing compounds for application in boron neutron capture therapy.     

Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83–99)

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83–99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.     

CD4 binding partially locks the bridging sheet in gp120 but leaves the beta2/3 strands flexible

The structure of the free form HIV gp120, critical for therapeutic agent development, is unavailable due to its high flexibility. Previous thermodynamic data, structural analysis and simulation results have suggested a large conformational change in the core domain upon CD4 binding. The bridging sheet, which consists of four beta-strands with beta20/21 nestling against the inner/outer domains and beta2/3 facing outward, more exposed to the solvent, was proposed to be unfolded in the native state. In order to test this proposition and to characterize the native conformations, we performed potential mean force (PMF) molecular dynamics (MD) simulations on the CD4-bound crystal structure. We pushed the bridging sheet away from the inner and outer domain to explore the accessible conformational space for the bridging sheet. In addition, we performed conventional MD simulations on structures with the bridging sheet partially unfolded to investigate the stability of the association between the inner and outer domains. Based on the free energy profiles, we find that the whole bridging sheet is unlikely to unfold without other concurrent conformational changes. On the other hand, the partial bridging sheet, beta strands 2/3, can switch its conformation from the folded to the unfolded state. Furthermore, relaxation of conformation with partially unfolded bridging sheet through MD simulations leads to a conformation with beta strands 20/21 quickly re-anchoring against the inner and outer domains. Such a conformation, although lacking some of the hydrophobic interactions present in the CD4-bound structure, displayed high stability as further indicated by other restrained MD simulations. The relevance of this conformation to the free form structure and the pathway for conformational change from the free form to the CD4-bound structure is discussed in detail in light of the available unliganded SIV gp120 crystal structure.     

Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides

Crystals of 5-fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] with the furanose ring in the favorite C2'-endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and -69.3(4) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. The crystal structure is stabilized by a network of N-H...O and O-H...O involving the N3 of the uracil base and the sugar 03' and 02' as donors and the 02 and 04 of the uracil base and 03' oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5-fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi angle 3) a wide variation of conformations of the sugar ranging froni C2'-endo through C3'-endo to C4'-exo 4) the preferred g+ across the exocyclic C4'-C5' bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.     

Changes in the conformation of myo-inositol residues in triphosphoinositides]

Dependency of optical rotation of di- and triphosphoinositide water solutions on pH is investigated. The inversion of myo-inosite ring in triphosphoinositide molecule is found to take place at pH range corresponding to the transition of phosphomonoesters from monoanionic into dianionic form. Stabilization of optical rotation is observed at pH range, where one monoester phosphate group is in monoanionic form and the other--in dianionic form. Probably, triphosphoinositide is in the conformation (distorted boat) at this pH range. Diphosphoinositide does not change its optical rotation under pH changes of water or organic solutions. A contribution is estimated of free conformational energy into standard free energy of splitting triphosphoinositide phosphate group depending on pH value. It is concluded that low energy phosphate bond becomes high energy bond due to the free electrostatic interaction of dianionic phosphate group with other negatively charged group in sin-clynal conformation.