Longer Linker Research Articles

A Study of the Interaction between a Family of Gemini Amphiphilic Pseudopeptides and Model Monomolecular Film Membranes Formed with a Cardiolipin.

The interaction between five gemini amphiphilic pseudopeptides (GAPs) differing by the length of the central spacer and a model membrane lipid, 1,3-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (cardiolipin) were studied with the aim to evaluate their possible antimicrobial properties. To this end, monomolecular films were formed at the air/water interface with pure cardiolipin or cardiolipin/GAPs mixtures; film properties were determined using surface pressure and surface potential measurements, as well as polarization-modulation infrared reflection-absorption spectroscopy. Moreover, to better understand the GAPs-phospholipid interaction at the molecular level, molecular dynamics simulations were performed. The results obtained indicate that the length of the central spacer has an effect on the interaction of GAPs with cardiolipin and on the properties of the lipid film. The GAPs with the longer linkers can be expected to be useful for biological membrane modification and for possible antimicrobial applications.

First principle investigation of the linker length effects on the thermodynamics of divalent pseudorotaxanes.

The Gibbs energies of association (Gibbs free (binding) energies) for divalent crown-8/ammonium pseudorotaxanes are determined by investigating the influence of different linkers onto the binding. Calculations are performed with density functional theory including dispersion corrections. The translational, rotational and vibrational contributions are taken into account and solvation effects including counter ions are investigated by applying the COSMO-RS method, which is based on a continuum solvation model. The calculated energies agree well with the experimentally determined ones. The shortest investigated linker shows an enhanced binding strength due to electronic effects, namely the dispersion interaction between the linkers from the guest and the host. For the longer linkers this ideal packing is not possible due to steric hindrance.

Development and characterisation of novel fentanyl-delta opioid receptor antagonist based bivalent ligands

Opioid tolerance is a limiting factor in chronic pain. Delta opioid peptide (DOP)(δ) receptor antagonism has been shown to reduce tolerance. Here, the common clinical mu opioid peptide (MOP)(µ) receptor agonist fentanyl has been linked to the DOP antagonist Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydrisoquinoline-3-carboxylic acid) to create new bivalent compounds. Binding affinities of bivalents(#9, #10, #11, #12 and #13) were measured in Chinese hamster ovary (CHO) cells expressing recombinant human MOP, DOP, Kappa opioid peptide (KOP)(κ) and nociceptin/orphanin FQ opioid peptide (NOP) receptors. Functional studies, measuring GTPγ[(35)S] or β-arrestin recruitment, were performed in membranes or whole cells respectively expressing MOP and DOP. The new bivalents bound to MOP (pKi : #9:7.31; #10:7.58; #11:7.91; #12:7.94; #13:8.03) and DOP (#9:8.03; #10:8.16; #11:8.17; #12:9.67; #13:9.71). In GTPγ[(35)S] functional assays, compounds #9(pEC50:6.74; intrinsic activity:0.05) #10(7.13;0.34) and #11(7.52;0.27) showed weak partial agonist activity at MOP. Compounds #12 and #13, with longer linkers, showed no functional activity at MOP. In antagonist assays at MOP, compounds #9 (pKb:6.87), #10(7.55) #11(7.81) #12(6.91) and #13(7.05) all reversed the effects of fentanyl. At DOP, all compounds showed antagonist affinity (#9:6.85; #10:8.06; #11:8.11; #12:9.42; #13:9.00), reversing the effects of DPDPE ([D-Pen(2,5)]enkephalin). In β-arrestin assays, compared with fentanyl (with response at maximum concentration (RMC):13.62), all compounds showed reduced ability to activate β-arrestin (#9 RMC:1.58; #10:2.72; #11:2.40; #12:1.29; #13:1.58). Compared with fentanyl, the intrinsic activity was: #9:0.12; #10:0.20; #11:0.18; #12:0.09 and #13:0.12. The addition of a linker between fentanyl and Dmt-Tic did not alter the ability to bind to MOP and DOP, however a substantial loss in MOP functional activity was apparent. This highlights the difficulty in multifunctional opioid development.

Synthesis and bioactivity of β-substituted fosmidomycin analogues targeting 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the β-position of the hydroxamate analogue of 2. While direct addition of a β-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth.

Enantioselective ring opening of epoxides with TMSN3 by macrocyclic oligomer-supported Cr(III)–salen catalysts

Three Cr(III)–salen catalysts supported onto macrocyclic oligomeric cyclooctene through linkers of different lengths were synthesized. The influences of the linker length and the oligomeric support on the activity and the enantioselectivity of catalysts were investigated in the asymmetric ring opening of a meso-epoxide and the kinetic resolution of terminal epoxides. For both reactions all three catalysts exhibited significantly higher catalytic efficiency than the unsupported monomeric Cr(III)–salen catalyst. The catalyst with the shortest linker 1 showed the highest reaction rate while the catalyst with the longest linker 3 afforded the highest enantioselectivity in both reactions. The recoverability of catalyst 1 was also studied.

Mechanistic evaluation of motion in redox-driven rotaxanes reveals longer linkers hasten forward escapes and hinder backward translations.

Mechanistic understanding of the translational movements in molecular switches is essential for designing machine-like prototypes capable of following set pathways of motion. To this end, we demonstrated that increasing the station-to-station distance will speed up the linear movements forward and slow down the movements backward in a homologous series of bistable rotaxanes. Four redox-active rotaxanes, which drove a cyclobis(paraquat-p-phenylene) (CBPQT(4+)) mobile ring between a tetrathiafulvalene (TTF) station and an oxyphenylene station, were synthesized with only variations to the lengths of the glycol linker connecting the two stations (n = 5, 8, 11, and 23 atoms). We undertook the first mechanistic study of the full cycle of motion in this class of molecular switch using cyclic voltammetry. The kinetics parameters (k, ΔG(‡)) of switching were determined at different temperatures to provide activation enthalpies (ΔH(‡)) and entropies (ΔS(‡)). Longer glycol linkers led to modest increases in the forward escape (t(1/2) = 60 to <7 ms). The rate-limiting step involves movement of the tetracationic CBPQT(4+) ring away from the singly oxidized TTF(+) unit by overcoming one of the thiomethyl (SMe) speed bumps before proceeding on to the secondary oxyphenylene station. Upon reduction, however, the return translational movement of the CBPQT(4+) ring from the oxyphenylene station back to the neutral TTF station was slowed considerably by the longer linkers (t(1/2) = 1.4 to >69 s); though not because of a diffusive walk. The reduced rate of motion backward depended on folded structures that were only present with longer linkers.

Immobilization of gamma globulins and polyclonal antibodies of class IgG onto carbon-encapsulated iron nanoparticles functionalized with various surface linkers

The immobilization of gamma-globulins from human and bovine blood and human polyclonal antibody (IgG) onto carbon-encapsulated iron nanoparticles (CEINs) is reported. First, the CEINs were functionalized to introduce the surface acidic linkers with a different length. The shorter linker was introduced by a typical oxidation treatment in concentrated acids. The longer linker was anchored by a free radical addition route, in which the radicals were generated by the thermal decomposition of succinic acid acyl peroxide. Both functionalization routes caused a partial perforation of the carbon coating. This structural degradation was determined quantitatively by evaluation the Fe content in the functionalized CEINs. Gamma-globulins and human polyclonal antibody (IgG) were immobilized onto the linker-functionalized surface using the carbodiimide–amine type reaction. The success of immobilization was confirmed by infrared spectroscopy, thermogravimetry and protein assay, respectively. The immune reactivity of the immobilized primary human polyclonal antibody (IgG) was also confirmed in a reaction with a FITC-labeled goat secondary antibody (anti-IgG) recognizing the primary human antibody in CEINs. It has been found that the immobilization yield is primarily dependent on the length of the acidic linker, whilst the content of carboxylic groups is of secondary importance.

Stereoselective Di-pi-Methane Rearrangement of a BINOL-Substituted Dibenzobarrelene Derivative

Abstract Two dibenzobarrelene derivatives were synthesized that are covalently linked to a (S)-BINOL unit through an oxymethylene (1b) or ethylenedioxymethylene (1c) unit, and their photoreactivity was examined. Irradiation of 1b in acetone resulted in a di-π-methane rearrangement to give diastereoselectively one dibenzosemibullvalene isomer. Experimental and theoretical data point to a directing effect of the BINOL auxiliary based on steric repulsion. The derivative 1c with a longer linker between the BINOL unit and the dibenzobarrelene is photoinert, presumably because of fast competing relaxation processes in the excited state.

Preparation and in Vitro Photodynamic Activities of Folate-Conjugated Distyryl Boron Dipyrromethene Based Photosensitizers

Two folate-conjugated diiododistyryl boron dipyrromethenes have been prepared and characterized with various spectroscopic methods. These conjugates exhibit higher photocytotoxicity toward the KB human nasopharyngeal carcinoma cells, which have high expression of folate receptors when compared with the MCF-7 human breast adenocarcinoma cells, which have low expression of folate receptors. The difference in photocytotoxicity for these two cell lines is particularly large for the conjugate with a shorter oligoethylene glycol linker (compound 11a) as a result of its higher cellular uptake and slightly lower aggregation tendency. Its IC50 value toward KB cells (0.06 μM) is 43-fold lower than that for MCF-7 cells, while the difference is only 6-fold for the analogue with a longer linker (compound 11b). The length of the spacer also affects their subcellular localization. While compound 11a shows high affinity toward the endoplasmic reticulum of KB cells, conjugate 11b is mainly localized in the lysosomes.

How the Substitution Faraway from NHCs Affects the Structural Features and Catalytic Activity of Dicarbene Dipalladium Complexes

AbstractA series of [PdPyCl2]2(di‐NHC) complexes were prepared (di‐NHC are two 1‐(2,6‐dimethylphenyl)imidazolidene molecules bridged by an aliphatic –(CH2)n– linker (n = 3, 4, 5, 6, and 10)). All complexes were fully characterized by NMR spectroscopy and elemental analyses. The crystal structures of four complexes (n = 3, 4, 5 and 6) were determined by X‐ray diffraction. The influence of the distant methyl group on the structural features and catalytic activity with increasing of length of linker was investigated by comparing the results of these 2,6‐dimethylphenyl palladium complexes with those of their known mesityl analogues. X‐ray studies show the distant methyl substitution has big impact on the structure feature of the complexes with the shorter linker between two NHC (ethylene and propylene), but has a little or no effect on that of the complexes with longer linker (butylene and hexylene). Catalytic results of the arylation of styrene show that the remote substitute has big effect on the regioselectivity of the product in all complexes with shorter and longer linkers, but has a limited effect on the yield.

Synthesis and evaluation of bifunctional sGC regulators

Background Our previous studies identified dicyanocobinamide as a novel sGC regulator that targets the catalytic region and synergistically enhances activation of sGC by NO-independent regulators. As proof-of-concept study, we designed and synthesized a set of bifunctional sGC regulators, which consist of cobinamide analog conjugated to a protoporphyrin IX derivative through linkers of varying length and composition (Figure 1). Results The length and composition of the linker was proved to be crucial for sGC activation. Our results indicate that only hybrid molecules containing a 13-16 atom chain linker benefited from synergistic engagement of both heme-binding region and catalytic domain. The hybrid compounds containing linkers connected through an ester bond were not only more stable, but also were more effective than those connected though an amide. Hybrids with shorter or longer linkers, or with different linker composition, were much less potent and were no more active than the cobyrinic acid component alone. The most effective compound was conjugated through an ester bond, contained a 13 atom chain linker and a triazole group close to the cobyrinate moiety. This compound displayed more than 60 fold activation of purified sGC.

Atom Transfer Radical Polymerization Preparation and Photophysical Properties of Polypyridylruthenium Derivatized Polystyrenes

A ruthenium containing polymer featuring a short carbonyl-amino-methylene linker has been prepared by atom transfer radical polymerization (ATRP). The polymer was derived from ATRP of the N-hydroxysuccinimide (NHS) derivative of p-vinylbenzoic acid, followed by an amide coupling reaction of the NHS-polystyrene with Ru(II) complexes derivatized with aminomethyl groups (i.e., [Ru(bpy)2(CH3-bpy-CH2NH2)](2+) where bpy is 2,2'-bipyridine, and CH3-bpy-CH2NH2 is 4-methyl-4'-aminomethyl-2,2'-bipyridine). The Ru-functionalized polymer structure was confirmed by using nuclear magnetic resonance and infrared spectroscopy, and the results suggest that a high loading ratio of polypyridylruthenium chromophores on the polystyrene backbone was achieved. The photophysical properties of the polymer were characterized in solution and in rigid ethylene glycol glasses. In solution, emission quantum yield and lifetime studies reveal that the polymer's metal-to-ligand charge transfer (MLCT) excited states are quenched relative to a model Ru complex chromophore. In rigid media, the MLCT-ground state band gap and lifetime are both increased relative to solution with time-resolved emission measurements revealing fast energy transfer hopping within the polymer. Molecular dynamics studies of the polymer synthesized here as well as similar model systems with various spatial arrangements of the pendant Ru complex chromophores suggest that the carbonyl-amino-methylene linker probed in our target polymer provides shorter Ru-Ru nearest-neighbor distances leading to an increased Ru*-Ru energy hopping rate, compared to those with longer linkers in counterpart polymers.

Highly sensitive nanomechanical assay for the stress transmission of carbon chain

Here, we report the first quantitative experimental study into the molecular basis of the transmission of mechanical signal that originates from biochemical reaction focusing on the length of carbon chain. We designed an experiment by using n-alkanethiols with a same carboxyl group and different chain lengths (n=1, 5, 10 and 15) to immobilize a same receptor molecule on the gold surface of a microcantilever, and detected the nanomechanical response of biochemical reaction. The sensitivity of the microcantilever was found to be greatly influenced by the chain length of linker that is between the receptor molecule and the microcantilever surface. The efficiency of stress transmission increases significantly with decreasing length of carbon chain. At the same time, we develop a label-free microcantilever sensor for highly sensitive detection of Glycyrrhizic acid (GL). The detection limit of the microcantilever sensor for GL is found to be as low as 20pg/mL for the shortest linker (n=1), which is 500 times lower than the longest linker (n=15) and 50 times lower than that of the corresponding icELISA. These findings will provide new insights into the fundamental mechanisms of stress transmission, which may be exploited for biochemical sensor and nanoactuation applications.

Photoaffinity labeling the lipid binding site of mammalian diacylglycerol kinase

We are taking a photoaffinity labeling approach to identifying the lipid binding site on mammalian diacylglycerol kinase (DGK). The probe includes a diacylglycerol(DAG)‐like moiety, except with ether linkages instead of acyl linkages to prevent chain migration, in order to target the probe to the lipid binding site on DGK. The probe also includes a benzophenone moiety, in order to covalently crosslink to whatever amino acids happen to be near it when excited with 360 nm light. In this way, the probe is expected to covalently modify the enzyme near the DAG‐binding site(s). When the probe is incorporated into liposomes, DGK's enzymatic activity is reduced in a probe‐ and UV‐dependent way, but only when the linker connecting the benzophenone moiety and the DAG‐like moiety on the probe is sufficiently short. Probes with longer linkers are able to be used as substrates by the enzyme, suggesting that they are able to access the active site but not to crosslink to the protein. Mass spectrometry studies are currently underway to map the site of crosslinking. Preliminary results show reduced amino acid coverage for in‐gel trypsin‐digested crosslinked enzyme as compared to control: these missing peptides are candidates for sites of crosslinking. This research is supported by Grant GM059251 from the National Institutes of Health.

Synthesis, structure and reactivity of NHC–nickel complexes containing carbonato ligands

Reactions of different alkane-bridged (n = 1–3) di-NHC precursors, bisimidazolium chlorides with the NiCl2 in presents of K2CO3 in acetonitrile give the corresponding 16-electron binuclear complexes of the type Ni(di-NHC)(μ-O)2CO in moderate yield. The molecular structures of 1–3 have been determined by X-ray crystallography, which revealed a cis-chelating di-NHC on a slightly distorted square-planar nickel center with κ2-cabonato ligand. The influence of the different di-NHC ligands on the structure and reactivity of the complexes has been studied. The catalytic properties of the complexes were investigated in the Kumada reaction for cross-coupling of phenylmagnesium bromide with a variety of aryl halides. The complex 3 which has the longest linker between two NHCs shows the best catalytic activity in these reactions.

Effect of interaction with coesite silica on the conformation of Cecropin P1 using explicit solvent molecular dynamics simulation

Explicit solvent molecular dynamics (MD) simulation was carried out for the antimicrobial peptides (i) Cecropin P1 and C-terminus cysteine modified Cecropin P1 (Cecropin P1 C) in solution, (ii) Cecropin P1 and Cecropin P1 C adsorbed onto coesite -Si - O - and Si - O - H surfaces, and (iii) Cecropin P1 C tethered to coesite -Si - O - surface with either (PEO)(3) or (PEO)(6) linker. Low energy structures for Cecropin P1 and Cecropin P1 C in solution consists of two regions of high α helix probability with a sharp bend, consistent with the available structures of other antimicrobial peptides. The structure of Cecropin P1 C at low ionic strength of 0.02 M exhibits two regions of high α helix probability (residues AKKLEN and EGI) whereas at higher ionic strength of 0.12 M, the molecule was more compact and had three regions of higher α helix probability (residues TAKKLENSA, ISE, and AIQG) with an increase in α helical content from 15.6% to 18.7% as a result of shielding of electrostatic interactions. In the presence of Cecropin P1 C in the vicinity of -Si - O - surface, there is a shift in the location of two peaks in H - O - H density profile to larger distances (2.95 Å and 7.38 Å compared to 2.82 Å and 4.88 Å in the absence of peptide) with attenuated peak intensity. This attenuation is found to be more pronounced for the first peak. H-bond density profile in the vicinity of -Si - O - surface exhibited a single peak in the presence of Cecropin P1 C (at 2.9 Å) which was only slightly different from the profile in the absence of polypeptide (2.82 Å) thus indicating that Cecropin P1 C is not able to break the H-bond formed by the silica surface. The α helix probability for different residues of adsorbed Cecropin P1 C on -Si - O - surface is not significantly different from that of Cecropin P1 C in solution at low ionic strength of 0.02 M whereas there is a decrease in the probability in the second (residues ISE) and third (residues AIQG) α helical regions at higher ionic strength of 0.12 M. Though the total α helical content of adsorbed and tethered Cecropin P1 C was lower for hydrophilic Si - O - H surface compared to hydrophobic -Si - O -, hydrophobicity of the surface did not significantly affect the α helix probability of different residues. The conformation of Cecropin P1 C in solution is closer to that of tethered to -Si - O - with (PEO)(6) than that tethered with (PEO)(3) as a result of less surface interaction of tethered polypeptide with a longer linker. At low ionic strength of 0.02 M, tethered Cecropin P1 C to -Si - O - is found to exhibit lower α helix (13.0%) compared to adsorbed (15.6%) for (PEO)(3) linker with this difference being insignificant for larger (PEO)(6) linker molecule. Experimental values of % α helix inferred from circular dichroism spectra of Cecropin P1 in solution as well as in adsorbed state on silica surface compared well with the corresponding values obtained from MD simulation thereby validating the simulation procedure.

Toxicity in tumor cells, DNA binding mode, and resistance to decomposition by sulfur nucleophiles of new dinuclear bifunctional trans-PtII complexes containing long alkane linkers

In an effort to design dinuclear PtII compounds that maintain the target (DNA) binding profile of the trans-oriented dinuclear bifunctional PtII complexes containing aliphatic linker chains but are less susceptible to metabolic decomposition, the new, long-chain dinuclear PtII complexes—[{trans-PtCl(dien)}2-μ-(CH2) n ]2+ (n = 7,10,12, dien = diethylenetriamine)—were synthesized. The toxicity of these metallodrugs was examined in ovarian tumor cell lines. The results showed that the activity of these complexes increased with growing length of the linker; the activity of complex containing the longest linker (n = 12) was comparable with that of cis-diamminedichloridoplatinum(II) (cisplatin). This observation correlated with the results of DNA binding studies performed in cell-free media. The results of these studies demonstrated that the growing length of the aliphatic bridge promoted more distorting conformational alterations induced in DNA. Attention was also paid to the reactivity of {[Pt(dien)Cl]2-alkane} compounds with glutathione (GSH). The results of these experiments support the thesis that the dinuclear structure of {[Pt(dien)Cl]2-alkane} complexes remains stable in the presence of S-containing compounds without undergoing chemical degradation as previously observed for some di/trinuclear bifunctional PtII complexes. This enhanced stability represents a favorable property which may contribute to reduce side effects and increase therapeutic efficacy of the dinuclear {[Pt(dien)Cl]2-alkane} compounds.

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions.

Influence of Support and Linker Parameters on the Activity of Silica‐Supported Catalysts for Cyclic Carbonate Synthesis

AbstractSilica‐supported, bimetallic aluminium(salen) complexes bearing pendant quaternary ammonium groups catalyse the synthesis of cyclic carbonates from epoxides and carbon dioxide in both batch and gas‐phase flow reactors. The influence of particle and pore size of the silica support on catalyst activity was investigated with smaller particle sizes (&lt;80 μm) being advantageous to catalyst activity, whereas the silica pore size had little effect on catalyst activity. The nature of the silica support also affected catalyst stability and the Fluorochem LC301 silica gave a catalyst with both the highest intrinsic activity and the lowest rate of deactivation. The catalysts are attached to the silica through a linker containing three or eleven carbon atoms. The longer linker was found give a catalyst loading three times higher than the shorter linker, but this was offset by a three times lower activity for the catalyst with the longer linker.

Atrial Natriuretic Peptide-Fc, ANP-Fc, Fusion Proteins: Semisynthesis, In Vitro Activity and Pharmacokinetics in Rats

Atrial natriuretic peptide (ANP) may be a useful molecule for the treatment of cardiovascular diseases due to its potent natriuretic effects. In an effort to prolong the short in vivo half-life of ANP, fusions of the peptide to the Fc domain of IgG were generated using a semisynthetic methodology. Synthetic ANP peptides were synthesized with thioesters at either the N- or C-termini of the peptide and subsequently linked to the N-terminus of recombinantly expressed Fc using native chemical ligation. The linker length between the ANP and Fc moieties was varied among 2, 11, or 16 amino acids. In addition, either one ("monomeric") or two ("dimeric") ANP peptides were linked to Fc to study whether this modification had an effect on in vitro activity and/or in vivo half-life. The various constructs were studied for in vitro activity using a cell-based cGMP assay. The ANP-Fc fusion constructs were between 16- and ∼375-fold weaker than unconjugated ANP in this assay, and a trend was observed where the most potent conjugates were those with longer linkers and in the dimeric configuration. The pharmacokinetics of several constructs were assessed in rats, and the half-life of the ANP-Fc's were found to be approximately 2 orders of magnitude longer than that of the unconjugated peptide. There was no significant difference in terminal half-life between the monomeric and dimeric constructs (2.8-5.5 h), but a trend was observed where the C(max) of the monomeric constructs was approximately 3-fold higher than that of the dimeric constructs, although the origin of this effect is not understood. These novel ANP-Fc fusion constructs hold promise for future therapeutic application in the treatment of cardiovascular diseases.