Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides

Although the sequence specificity, biostability, and low toxicity of PMO (phosphorodiamidate morpholino oligomers) make them good antisense agents to study gene function, their limited ability to cross cell membranes limits their use in cell culture. In this paper we show that conjugation to arginine-rich peptides significantly enhanced the cellular uptake of PMO. The factors that affect the conjugate's cellular uptake and its antisense activity toward a targeted mRNA were investigated. Factors studied include the number of arginines in the peptide, the choice of cross-linker, the peptide conjugation position, the length of the PMO, and the cell culture conditions. Delivery of PMO to the cell nucleus and cytosol required conjugation rather than complexation of peptides to PMO. R(9)F(2)C was best suited to deliver a PMO to its target RNA resulting in the strongest antisense effect. By simply adding the R(9)F(2)C-PMO conjugate into the cell culture medium at low microM concentration, missplicing of pre-mRNA was corrected. This particular peptide-conjugated PMO was more effective than the PMO conjugated to the transmembrane transport peptides of HIV-1 Tat protein, Drosophila antennapedia protein, or to peptides with fewer arginines. Length of PMO did not affect a peptide's delivery efficacy, but all other factors were important. R(9)F(2)C peptide provided a simple and efficient delivery of PMO to a RNA target. Conjugation of peptide to PMO enhances the opportunities to evaluate gene functions in cell cultures.     

Stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells

Cell penetrating peptides (CPPs) have been shown to enhance the cellular uptake of antisense oligonucleotides (AOs). However, the effectiveness of the CPPs for cytoplasmic or nuclear delivery of therapeutic AOs must take into account the possible entrapment of the CPP-AO conjugates in endosomes/lysosomes and the overall stability of the CPP-AO conjugates to enzymes. This includes the stabilities of the CPPs and AOs themselves as well as the linkage between them. In this study, we investigated the effects of several structural features of arginine-rich CPPs on the metabolic stability of CPP conjugated to phosphorodiamidate morpholino oligomers (PMOs) in human serum and in cells. Those structural features include amino acid configurations (d or l), incorporation of non-alpha-amino acids, peptide sequences, and types of linkages between CPPs and PMOs. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that the stability of the CPP portion was varied although the PMO portion of the conjugate was completely stable both in cells and in human serum. d-Configuration CPPs were completely stable, while l-CPPs were degraded in both serum and HeLa cells. Insertions of 6-aminohexanoic acid residues (X) into an R8 peptide increased the corresponding CPP's serum stability with the degree of stability being dependent upon the positions of X. However, X-containing CPPs were degraded rapidly intracellularly. Insertions of beta-alanines (B) into the R8 peptide increased its serum stability and intracellular stability. An amide or a maleimide linkage was stable in both serum and cells; however, an unhindered disulfide linkage was not stable in either. By using fluorescent microscopy, flow cytometry, and an antisense splice correction assay, the cellular uptakes of an X-containing conjugate and its fragments were compared to their antisense activities. We found that a large fraction of the conjugate was trapped within vesicles and the degraded fragments cannot escape from the vesicles. This study indicates that the incorporation of non-alpha-amino acids into l-CPPs can increase the metabolic stability of CPP-PMOs without using costly d-CPPs. However, the position and type of non-alpha-amino acids affect the degree of stability extracellularly and intracellularly. In addition, this study reveals that the degradation of an X-containing CPP-PMO conjugate is a more rapid process than degradation of a B-containing conjugate. Last, the endosomal/lysosomal trapping limits the effectiveness of a CPP-PMO conjugate, and the stability of the CPP is one of the factors affecting the ability of the conjugate to escape the endosomes/lysosomes.     

Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication

RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5'- and 3'-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5'-terminal 20 nucleotides (5'End) or targeting the 3'-terminal element involved in a potential genome cyclizing interaction (3'CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5'End or 3'CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 muM concentration without apparent cytotoxicity. The 3'CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3'CSI sequences of specific viruses. Mode-of-action analyses showed that the 5'End and 3'CSI PMOs suppressed viral infection through two distinct mechanisms. The 5'End PMO inhibited viral translation, whereas the 3'CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3' untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle.     

Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.     

PBMC activation via the ERK and STAT signaling pathways enhances the anti-tumor activity of <Emphasis Type="Italic">Staphylococcal</Emphasis> enterotoxin A

In a recent study, we highlighted the importance of cationic charge and arginine residues for the neuroprotective properties of poly-arginine and arginine-rich peptides. In this study, using cortical neuronal cultures and an in vitro glutamic acid excitotoxicity model, we examined the neuroprotective efficacy of different modifications to the poly-arginine-9 peptide (R9). We compared an unmodified R9 peptide with R9 peptides containing the following modifications: (i) C-terminal amidation (R9-NH2); (ii) N-terminal acetylation (Ac-R9); (iii) C-terminal amidation with N-terminal acetylation (Ac-R9-NH2); and (iv) C-terminal amidation with d-amino acids (R9D-NH2). The three C-terminal amidated peptides (R9-NH2, Ac-R9-NH2, and R9D-NH2) displayed neuroprotective effects greater than the unmodified R9 peptide, while the N-terminal acetylated peptide (Ac-R9) had reduced efficacy. Using the R9-NH2 peptide, neuroprotection could be induced with a 10 min peptide pre-treatment, 1–6 h before glutamic acid insult, or when added to neuronal cultures up to 45 min post-insult. In addition, all peptides were capable of reducing glutamic acid-mediated neuronal intracellular calcium influx, in a manner that reflected their neuroprotective efficacy. This study further highlights the neuroprotective properties of poly-arginine peptides and provides insight into peptide modifications that affect efficacy.     

HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers

Phosphorodiamidate morpholino oligomers (PMO) are uncharged antisense molecules that bind complementary sequences of RNA, inhibiting gene expression by preventing translation or by interfering with pre-mRNA splicing. The techniques used to deliver PMO into cultured cells have been mostly mechanical methods. These delivery methods, although useful, have limitations. We investigated the ability of the HIV Tat peptide (pTat) and other cationic peptides to deliver PMO into cultured cells. Fluorescence was seen in 100% of HeLa cells treated with pTat-PMO-fluorescein conjugate. pTat-PMO conjugate targeted to c-myc mRNA downregulated c-myc reporter gene expression with an IC50 of 25 microM and achieved nearly 100% inhibition. pTat-PMO conjugate targeted to a mutant splice site of beta-globin pre-mRNA dose-dependently corrected splicing and upregulated expression of the functional reporter gene. Neither unconjugated PMO nor unconjugated pTat caused antisense activities. However, compared with mechanically mediated delivery, pTat-mediated PMO delivery required higher concentrations of PMO (>10 microM) to cause antisense activity and caused some toxicity. Most pTat-PMO conjugate was associated with cell membranes, and internalized conjugate was localized in vesicles, cytosol, and nucleus. The other three cationic peptides are much less effective than pTat. pTat significantly enhances delivery of PMO in 100% of cells assayed. pTat-mediated delivery is a much simpler procedure to perform than other delivery methods.     

Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate

OBJECTIVE: Conjugation of arginine-rich cell-penetrating peptide (CPP) to phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance cytosolic and nuclear delivery of PMO. However, the in vivo disposition of CPP-PMO is largely unknown. In this study, we investigated the pharmacokinetics, tissue distribution, stability, and safety profile of an anti-c-myc PMO conjugated to the CPP, (RXR)4 (X = 6-aminohexanoic acid) in rats. METHODS: The PMO and CPP-PMO were administrated intravenously into rats. The concentrations of the PMO and the CPP-PMO in plasma and tissues were monitored by HPLC. The stability of the CPP portion of the CPP-PMO conjugate in rat plasma and tissue lysates was determined by mass spectrometry. The safety profile of the CPP-PMO was assessed by body weight changes, serum chemistry, and animal behavior. RESULTS: CPP conjugation improved the kinetic behavior of PMO with a 2-fold increase in the estimated elimination half-life, a 4-fold increase in volume of distribution, and increased area under the plasma concentration vs time curve. Consistent with the improved pharmacokinetic profile, conjugation to CPP increased the uptake of PMO in all tissues except brain, varied between organ type with greater uptake enhancement occurring in liver, spleen, and lungs. The CPP-PMO conjugate had greater tissue retention than the corresponding PMO. Mass spectrometry data indicated no observable degradation of the PMO portion, while there was identifiable degradation of the CPP portion. Time-dependent CPP degradation was observed in plasma and tissue lysates, with the degradation in plasma being more rapid. The pattern of degraded products differed between the plasma and lysates. Safety evaluation data showed that the CPP-PMO was well-tolerated at the dose of 15 mg/kg with no apparent signs of toxicity. In contrast, at the dose of 150 mg/kg, adverse events such as lethargy, weight loss, and elevated BUN (p < 0.01) and serum creatinine (p < 0.001) levels were recorded. Supplementation with free L-arginine ad libitum showed improved clearance of serum creatinine (p < 0.05) and BUN (p < 0.01) at the toxicological dose, suggesting that the CPP caused toxicity in kidney. CONCLUSION: This study demonstrates that conjugation of CPP to PMO enhances the PMO pharmacokinetic profile, tissue uptake, and subsequent retention. Therefore, when dosed at < or = 15 mg/kg, CPP is a promising transporter for enhancing PMO delivery in therapeutic settings.     

Morpholino oligonucleotides do not participate perfectly in standard Watson-Crick complexes with RNA

RNase P from E. coli will cleave a RNA at a site designated in a complex with an external guide sequence (EGS). The location of the site is determined by the Watson-Crick complementary sequence that can be formed between the RNA and the EGS. Morpholino oligonucleotides (PMOs) that have the same base sequences as any particular EGS will not direct cleavage by RNase P of the target RNA at the expected site in three mRNAs. Instead, cleavage occurs at a secondary site that does not correspond exactly to the expected Watson-Crick sequence in the PMO. This cleavage in the mRNA for a drug resistance gene, CAT mRNA, is at least second order in the concentration of the PMOs, but the mechanism is not understood yet and might be more complicated than a simple second-order reaction. EGSs and PMOs inhibit the reactions of each other effectively in a competitive fashion. A basic peptide attached to the PMO (PPMO) is more effective because of its binding properties to the mRNA as a substrate. However, a PMO is just as efficient as a PPMO on a mRNA that is mutated so that the canonical W-C site has been altered. The altered mRNA is not recognizable by effective extensive W-C pairing to an EGS or PMO. The complex of a PMO on a mutated mRNA as a substrate shows that the dimensions of the modified oligonucleotide cannot be the same as a naked piece of single-stranded RNA.     

Hitting bacteria at the heart of the central dogma: sequence-specific inhibition

An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of different human cancers and other human diseases. Applying antisense gene silencing and exploiting RNA interference (RNAi) are highly developed approaches in many eukaryotic systems. But in bacteria RNAi is absent, and gene silencing by antisense compounds is not nearly as well developed, despite its great potential and the intriguing possibility of applying antisense molecules in the fight against multiresistant bacteria. Recent breakthrough and current status on the development of antisense gene silencing in bacteria including especially phosphorothioate oligonucleotides (PS-ODNs), peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) will be presented in this review.     

Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient

Background

Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD_J) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient''s cells.

Methodology/Principal Findings

We converted fibroblasts of CXMD_J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD_J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.

Conclusion/Significance

Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.     

Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis

Arginine-rich peptides, including octaarginine (R8), HIV-1 Tat, and branched-chain arginine-rich peptides, belong to one of the major classes of cell-permeable peptides which deliver various proteins and macromolecules to cells. The importance of the endocytic pathways has recently been demonstrated in the cellular uptake of these peptides. We have previously shown that macropinocytosis is one of the major pathways for cellular uptake and that organization of the F-actin accompanies this process. In this study, using proteoglycan-deficient CHO cells, we have demonstrated that the membrane-associated proteoglycans are indispensable for the induction of the actin organization and the macropinocytic uptake of the arginine-rich peptides. We have also demonstrated that the cellular uptake of the Tat peptide is highly dependent on heparan sulfate proteoglycan (HSPG), whereas the R8 peptide uptake is less dependent on HSPG. This suggests that the structure of the peptides may determine the specificity for HSPG, and that HSPG is not the sole receptor for macropinocytosis. Comparison of the HSPG specificity of the branched-chain arginine-rich peptides in cellular uptake has suggested that the charge density of the peptides may determine the specificity. The activation of the Rac protein and organization of the actin were observed within a few minutes after the peptide treatment. These data strongly suggest the possibility that the interaction of the arginine-rich peptides with the membrane-associated proteoglycans quickly activates the intracellular signals and induces actin organization and macropinocytotis.     

Achieving specificity in selected and wild-type N peptide-RNA complexes: the importance of discrimination against noncognate RNA targets

The boxB RNA pentaloops from the P22 and lambda phages each adopt a GNRA tetraloop fold upon binding their cognate arginine-rich N peptides. The third loop base in P22 boxB (3-out) and the fourth in lambda boxB (4-out) are excluded to accommodate this structure. Previously, we selected a pool of lambda N sequences with random amino acids at loop contacting positions 13-22 for binding to either of these two GNRA-folded pentaloops or a canonical GNRA tetraloop and isolated a class of peptides with a new conserved arginine (R15). Here, we characterize the binding of lambda N and these R15 peptides using fluorescent titrations with 2-aminopurine labeled versions of the three GNRA-folded loops and circular dichroism spectrometry. All peptides preferentially bind the lambda boxB RNA loop. lambda N and R15 peptide specificity against the P22 loop arises from the cost of rearranging its loop into the 4-out GNRA structure. Modeling indicates that the interaction of R8 with an additional loop phosphate in the 4-out GNRA pentaloop selectively stabilizes this complex relative to the tetraloop. R15 peptides gain additional discrimination against the tetraloop because their arginine also preferentially interacts with the 4-out GNRA pentaloop phosphate backbone, whereas K14 and W18 of lambda N contribute equal affinity when binding the tetraloop. Nonspecific electrostatic interactions by basic residues near the C-termini of these peptides create significantly steeper salt dependencies in association constants for noncognate loops, aiding discrimination at high salt concentrations. Our results emphasize the importance of considering specificity against noncognate as well as nonspecific targets in the combinatorial and rational design of biopolymers capable of macromolecular recognition.     

Arginine-rich cell penetrating peptides: design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides.

Rerouting the splicing machinery with steric-block oligonucleotides (ON) might lead to new therapeutic strategies in the treatment of diseases such as beta-thalassemia, Duchenne muscular dystrophy, or cancers. Interfering with splicing requires the sequence-specific and stable hybridization of RNase H-incompetent ON as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO). Unfortunately, these uncharged DNA mimics are poorly taken up by most cell types and conventional delivery strategies that rely on electrostatic interaction do not apply. Likewise, conjugation to cell penetrating peptides (CPPs) as Tat, Arg9, Lys8, or Pen leads to poor splicing correction efficiency at low concentration essentially because PNA- and PMO-CPP conjugates remain entrapped within endocytotic vesicles. Recently, we have designed an arginine-rich peptide (R-Ahx-R)4 (with Ahx for aminohexanoic acid) and an arginine-tailed Penetratin derivative which allow sequence-specific and efficient splicing correction at low concentration in the absence of endosomolytic agents. Both CPPs are undergoing structure-activity relationship studies for further optimization as steric-block ON delivery vectors.     

Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy

The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage (‘click chemistry’) in the other. The most active bi-specific CPP–PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP–PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.     

Interactions of antisense peptides with ovine prolactin

Two antisense peptides were synthesized to a sense peptide corresponding to amino acid residues 23-35 of ovine prolactin. Both of the antisense peptides formed a saturable complex with the sense peptide and ovine prolactin. The sense peptide inhibited the interaction of ovine prolactin with the antisense peptides. Both of the antisense peptides have a common core sequence VMNV which can bind to ovine prolactin. The lactogenic hormones, rat prolactin and human growth hormone, compete with the binding of ovine prolactin to an antisense peptide whereas a nonlactogen, ovine growth hormone, does not compete indicating a degree of specificity in the interaction.     

Peptide Conjugates of Oligonucleotides As Enhanced Antisense Agents

The use of synthetic oligonucleotides and their analogs to block gene expression by binding the complementary RNA sequences in cells, the antisense principle, has been limited by poor uptake of the agents by cells in culture. This review describes attempts to harness by chemical conjugation the ability of certain peptides that may cross membranes to enhance the cellular uptake of oligonucleotides. These include fusogenic and hydrophobic peptides, nuclear localization signals, receptor targeting and translocating peptides, and various combinations. We also outline briefly some popular methods of peptide–oligonucleotide conjugation. Finally, we review the use of noncovalent peptide additives and the recent studies of conjugates of peptide nucleic acid (PNA) with peptides.     

A concise method for the preparation of peptide and arginine-rich peptide-conjugated antisense oligonucleotide

Peptide-oligonucleotide conjugates were synthesized using two strategies: a mimetic signal peptide-conjugated oligonucleotide was assembled stepwise on CPG by using 2,2-dimethyl-3-hydroxypropionic acid as a linker. To solve the precipitation problem in the coupling reaction caused by the electrostatic interaction of arginine-rich peptides and oligonucleotide, oligonucleotides were absorbed on an anion-exchange resin, and then the on-resin fragment was applied for the conjugation with arginine-rich peptide. The peptide-antisense oligonucleotides showed permeability to the cell membrane of HepG-2 cells.     

A novel prosthetic group for site-selective labeling of peptides for positron emission tomography

Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.     

Translocation of branched-chain arginine peptides through cell membranes: flexibility in the spatial disposition of positive charges in membrane-permeable peptides

A basic peptide derived from HIV-1 Tat has been reported to have the ability to translocate through cell membranes and to bring exogenous proteins into cells. We have demonstrated that these features could be observed among many arginine-rich peptides, and the presence of a ubiquitous internalization mechanism for arginine-rich oligopeptides has been suggested. In this report, we report that these features are also applicable to the peptides having branched-chain structures. Peptides that have arginine residues on four branched chains (R(n))(4) [n (number of arginine residues)= 0-6] were prepared. Fluorescence microscopic observation revealed that the (R(2))(4) peptide exhibited the most efficient translocation. The dependence on the number of arginine residues of the translocation efficiency and cellular localization was also observed for the branched-chain peptides as was seen in the linear peptides. Quite interestingly, efficient translocation was also recognized in the (RG(3)R)(4) peptide, where three glycine residues intervened between two arginine residues on each chain of (R(2))(4). The results strongly suggested that a linear structure was not indispensable for the translocation of arginine-rich peptides and that there could be considerable flexibility in the location of the arginine residue in the molecules.