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The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine RANTES (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders-of-magnitude higher than that of RANTES itself. It has been hypothesized that the ultra-high potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy (FCCS) assay for saturation and competition binding experiments. The FCCS assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES, and about twenty-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site; whereas, the native chemokines (RANTES and MIP1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.Molecular Therapy (2016); doi:10.1038/mt.2016.134.

Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac and renal diseases, and in the progression of tumour growth in cancer, but current diagnosis and treatment remain inadequate. Peptides derived from the 212 amino acid precursor preproendothelin-1 (ppET-1) may have utility as biomarkers, or cause biological effects that are unaffected by endothelin receptor antagonists. Here, we used specific immunoassays and LC-MS/MS to identify NT-proET-1 (ppET-1[18–50]), Endothelin-Like Domain Peptide (ELDP, ppET-1[93–166]) and CT-proET-1 (ppET-1[169–212]) in conditioned media from cultured endothelial cells. Synthesis of these peptides correlated with ET-1, and plasma ELDP and CT-proET-1 were elevated in patients with chronic heart failure. Clearance rates of NT-proET-1, ELDP and CT-proET-1 were determined after i.v. injection in anaesthetised rats. CT-proET-1 had the slowest systemic clearance, hence providing a biological basis for it being a better biomarker of ET-1 synthesis. ELDP contains the evolutionary conserved endothelin-like domain sequence, which potentially confers biological activity. On isolated arteries ELDP lacked direct vasoconstrictor effects. However, it enhanced ET-1 vasoconstriction and prolonged the increase in blood pressure in anaesthetised rats. ELDP may therefore contribute to disease pathogenesis by augmenting ET-1 responses.

Background: Autoantibodies to apoA-I can be considered markers and mediators of cardiovascular disease. Results: Methodologies for identification of human apoA-I epitopes are described. Two novel immunoreactive peptides were recognized by autoantibodies from cardiac patients. Conclusion: We developed a new procedure to isolate, purify, and identify apoA-I epitopes. Significance: Developing new epitope mapping approaches opens new avenues for biomarkers and therapy., Autoantibodies to apolipoprotein A-I (anti-apoA-I IgG) have been shown to be both markers and mediators of cardiovascular disease, promoting atherogenesis and unstable atherosclerotic plaque. Previous studies have shown that high levels of anti-apoA-I IgGs are independently associated with major adverse cardiovascular events in patients with myocardial infarction. Autoantibody responses to apoA-I can be polyclonal and it is likely that more than one epitope may exist. To identify the specific immunoreactive peptides in apoA-I, we have developed a set of methodologies and procedures to isolate, purify, and identify novel apoA-I endogenous epitopes. First, we generated high purity apoA-I from human plasma, using thiophilic interaction chromatography followed by enzymatic digestion specifically at lysine or arginine residues. Immunoreactivity to the different peptides generated was tested by ELISA using serum obtained from patients with acute myocardial infarction and high titers of autoantibodies to native apoA-I. The immunoreactive peptides were further sequenced by mass spectrometry. Our approach successfully identified two novel immunoreactive peptides, recognized by autoantibodies from patients suffering from myocardial infarction, who contain a high titer of anti-apoA-I IgG. The discovery of these epitopes may open innovative prognostic and therapeutic opportunities potentially suitable to improve current cardiovascular risk stratification.

BACKGROUND AND PURPOSE The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC50= 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked NaV1.4 (IC50= 1.3 nM) and NaV1.2 channels in a long-lasting manner. Cardiac NaV1.5 and DRG-specific NaV1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3β2 nAChR subtype (IC50= 450 nM) and, to a lesser extent, on the α7 and α4β2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

CC chemokine receptor 5 (CCR5), the major HIV coreceptor, is a G protein-coupled receptor (GPCR) involved in cell activation and migration in response to chemokines. Blockade of CCR5 is an effective anti-HIV strategy, and potent anti-HIV chemokine analogs such as PSC-RANTES have been developed. These inhibitors act by interfering with receptor trafficking, thereby inducing prolonged intracellular sequestration of CCR5. Like many GPCRs, CCR5 is desensitized following agonist activation. The initial steps in this process are well understood, but later stages, including where CCR5 is sequestered during desensitization, and how anti-HIV chemokine analogs intervene to achieve prolonged sequestration, have yet to be elucidated in detail. In this study we demonstrate that CCR5 cycles to and from the cell surface via the endosome recycling compartment and the trans-Golgi network during desensitization, accumulating in the trans-Golgi network following internalization by both PSC-RANTES and CCL5, the native ligand from which it was derived. In addition, we show that unlike CCR5 sequestered by CCL5, CCR5 sequestered by PSC-RANTES cannot be induced to return to the cell surface by addition of the small molecule CCR5 inhibitor, TAK-779, and that association of PSC-RANTES with CCR5 is more durable than that of native CCL5 during desensitization. Our findings reconcile the previously conflicting descriptions of the location of sequestered CCR5 during desensitization, as well as providing more general insights into potential trafficking routes for endocytosed GPCRs and further elucidation of the unusual inhibitory mechanism of chemokine analogs with potent anti-HIV activity.

The HIV coreceptor CCR5 is a validated target for both the prevention and therapy of HIV infection. PSC-RANTES, an N-terminally modified analogue of one of the natural chemokine ligands of CCR5 (RANTES/CCL5), is a potent inhibitor of HIV entry into target cells. Here, we set out to engineer the anti-HIV activity of PSC-RANTES into another natural CCR5 ligand (MIP-1beta/CCL4), by grafting into it the key N-terminal pharmacophore region from PSC-RANTES. We were able to identify MIP-1beta/CCL4 analogues that retain the receptor binding profile of MIP-1beta/CCL4, but acquire the very high anti-HIV potency and characteristic inhibitory mechanism of PSC-RANTES. Unexpectedly, we discovered that in addition to N-terminal structures from PSC-RANTES, the side chain of Lys33 is also necessary for full anti-HIV potency.

[reaction: see text] A novel strategy to generate thioester peptides compatible with Fmoc chemistry is presented. Peptide-C(alpha)oxy-(2-mercapto-1-carboxyamide)ethyl ester undergoes an O to S acyl shift during ligation and the newly formed thioester intermediate reacts with an N-terminal cysteine fragment generating a product with native amide bond at the ligation site.

We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl 1 ]RANTES(2-68), known as AOP–RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure–activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES { N -nonanoyl, des-Ser 1 [ l -thioproline 2 , l -cyclohexylglycine 3 ]-RANTES(2–68)}, which is 50 times more potent than AOP–RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.

A new side reaction during Native Chemical Ligation (NCL) at aspartic and glutamic acid/cysteine ligation sites is reported. To exploit NCL at these sites, test peptides having either a C-terminal unprotected aspartyl or glutamyl thioester were ligated with a peptide containing an N-terminal free cysteine generating, in each case, two major compounds having equal masses but different retention times by HPLC analysis. Comparison of these ligation products with reference peptides of identical sequence synthesized by total SPPS (solid phase peptide synthesis) with both natural and unnatural backbones at the cysteinyl residue is in agreement with the hypothesis of migration of the thioester moiety on the side chain carboxyl group of both C-terminal Asp and Glu residues. Enzymatic digestion of the purified ligation products with a V8-protease further confirmed the nature of the side reaction, with a total resistance of the non-natural backbone peptide to the protease. To overcome this problem the use of a protecting group stable to both HF cleavage and ligation conditions, but removable after ligation, became a necessity. A series of side-chain protected derivatives were selected, specifically 9-fluorenylmethyl ester (OFm), (phenylsulfonyl)ethyl ester (OPse), 2,2,2-trichloroethyl ester (OTce), and phenacyl esters (OPac). For the glutamic acid, OPse gave the best results, generating the correct peptide and avoiding the isomerization at the γ-carboxyl group. In the case of the aspartic residue, all the protecting groups were removed under basic conditions, worsening the problem and generating, for example, 70% yield of the β-isomer after OFm deprotection. This prompted us to propose a protecting group derived from the phenacyl ester 1-methyl-2-oxo-2-phenylethyl ester (OMop). This protecting group showed greater stability during the ligation reaction than OPac and OTce, and was easily removed by Zn/acetic acid reduction after the ligation step. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

The chemokine receptors CCR5 and CXCR4 are promising non-virus-encoded targets for human immunodeficiency virus (HIV) therapy. We describe a selection procedure to isolate mutant forms of RANTES (CCL5) with antiviral activity considerably in excess of that of the native chemokine. The phage-displayed library of randomly mutated and N-terminally extended variants was screened by using live CCR5-expressing cells, and two of the selected mutants, P1 and P2, were further characterized. Both were significantly more potent HIV inhibitors than RANTES, with P2 being the most active (50% inhibitory concentration of 600 pM in a viral coat-mediated cell fusion assay, complete protection of target cells against primary HIV type 1 strains at a concentration of 10 nM). P2 resembles AOP-RANTES in that it is a superagonist of CCR5 and potently induces receptor sequestration. P1, while less potent than P2, has the advantage of significantly reduced signaling activity via CCR5 (30% of that of RANTES). Additionally, both P1 and P2 exhibit not only significantly increased affinity for CCR5 but also enhanced receptor selectivity, retaining only trace levels of signaling activity via CCR1 and CCR3. The phage chemokine approach that was successfully applied here could be adapted to other chemokine-chemokine receptor systems and used to further improve the first-generation mutants reported in this paper.

G protein-coupled receptors (GPCRs) are the largest superfamily of membrane receptors in the human genome and they are targets for a quarter of all prescription drugs. Activation of a GPCR by an agonist ligand results in G protein-mediated downstream signaling, followed by kinase action and arrestin-mediated desensitization, internalization/sequestration, and recycling. Selective manipulation of these individual steps of the GPCR activation cycle is often desired when creating drugs targeting a given receptor. We are interested in the C-C chemokine receptor CCR5 that is the major HIV coreceptor used in person-to-person transmission. Globally, the HIV/AIDS pandemic has caused nearly 30 million deaths and a similar number of people are currently infected. Certain analogues of the chemokine RANTES/CCL5 are highly potent entry inhibitors against R5-tropic HIV-1 strains, in vitro and in vivo. Three such analogues, 5P12-, 5P14-, and 6P4-RANTES, are particularly interesting because while they differ only slightly in structure they show strikingly different pharmacological profiles (G protein-linked signaling activity, stimulation of receptor internalization). We have recently developed a general, simple, and robust method for stoichiometric, site-specific fluorescence labeling of expressed GPCRs. The method is based on bioorthogonal conjugation of a fluorescent reporter group to a genetically encoded azido group introduced into expressed GPCRs using amber codon suppression.[1] We have adopted a similar strategy for fluorescent labeling of chemokines with azido groups introduced by chemical synthesis. Here we present our progress towards automated, multi-color, single-molecule fluorescence studies of the compositional and conformational dynamics of GPCR signaling complexes ("signalosomes") using fluorescently labeled chemokines and receptors in biochemically defined systems.[1] H Tian, TP Sakmar, & T Huber (2013) Site-specific labeling of genetically encoded azido groups for multi-color, single-molecule fluorescence imaging of GPCRs. Methods in Cell Biology, 117, in press.

A general procedure to prepare peptide thioacids by solid-phase peptide synthesis is presented. The method involves the synthesis of 4-[α-( S -acetyl)mercaptobenzyl]phenoxyacetic acid as general precursor. This reagent once attached to a solid support is derivatized with the Boc-amino acid of choice after deprotection of the thiol.

We present the concept of chemo-enzymic backbone engineering of proteins. Recombinant DNA techniques are used to produce appropriate proteins that are enzymically fragmented to give the starting materials. These fragments are modified specifically at their chain termini either enzymically (coupling of a hydrazide to the C terminus) or chemically (periodate oxidation of N-terminal serine to a glyoxylyl function). The modified fragments, which need no side protection whatever, are mixed together and religate themselves spontaneously under mild conditions. The hydrazone bond thus formed can be reduced if desired, which stabilizes the linkage and enhances the flexibility of the local conformation. In this way biologically or chemically derived structures can be incorporated into the protein, and the choice of the chemical ones is free of all of the constraints of the genetic code. We believe that this combined approach gives access to constructions that could not be derived by either recombinant or chemical methods alone. We illustrate the particularity of this concept by the engineered modifications of the 64-74 disulfide loop region of human granulocyte colony-stimulating factor. Analogs constructed include one which, in spite of having a nonpeptide link in its backbone, has full biological activity.

New HIV prevention methods are needed, and among those currently being explored are "microbicides", substances applied topically to prevent HIV acquisition during sexual intercourse. The chemokine analogue PSC-RANTES (N(alpha)(n-nonanoyl)-des-Ser(1)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)]-RANTES(4-68)) is a highly potent HIV entry inhibitor which has shown promising efficacy in its initial evaluation as a candidate microbicide. However, a way must be found to produce the molecule by cheaper means than total chemical synthesis. Since the only noncoded structures are located at the N-terminus, a possible solution would be to produce a protein fragment representing all but the N-terminal region using low-cost recombinant production methods and then to attach, site specifically, a short synthetic fragment containing the noncoded N-terminal structures. Here, we describe the evaluation of a range of different conjugation chemistries in order to identify those with potential for development as economical routes to production of a PSC-RANTES analogue with antiviral activity as close as possible to that of the parent protein. The strategies tested involved linkage through oxime, hydrazone/hydrazide, and Psi[CH2-NH] bonds, as well as through a peptide bond obtained either by a thiazolidine rearrangement or by direct alpha-amino acylation of a protein fragment in which 4 of the 5 lysine residues of the native sequence were replaced by arginine (the fifth lysine is essential for activity). Where conjugation involved replacement of one or more residues with a linker moiety, the point in the main chain at which the linker was introduced was varied. The resulting panel of 22 PSC-RANTES analogues was evaluated for anti-HIV activity in an entry inhibition assay. The [Arg (25,45,56,57)] PSC-RANTES analogue has comparable potency to PSC-RANTES, and one of the oxime linked analogues, 4L-57, has potency only 5-fold lower, with scope for improvement. Both represent promising leads for development as microbicide compounds that could be produced at low cost via semisynthesis.

We investigated venoms from members of the genus Atheris (Serpentes, Viperidae), namely the rough scale bush viper (Atheris squamigera), the green bush viper (A. chlorechis) and the great lakes bush viper (A. nitschei), using mass spectrometry-based strategies, relying on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with de novo peptide sequencing. We discovered a set of novel peptides with masses in the 2-3 kDa range and containing poly-His and poly-Gly segments (pHpG). Complete primary structural elucidation and confirmation of two sequences by Edman degradation indicated the consensus sequence EDDH(9)GVG(10). Bioinformatic investigations in protein sequence databanks did not show relevant homology with known peptides or proteins. However, a more extensive investigation of data in nucleic acid databases revealed some similarities to the precursor sequences of bradykinin potentiating peptides (BPP) and C-type natriuretic peptides (CNP), agents that are known to affect the cardiovascular system by acting on specific metalloproteases and receptors. The novel pHpG peptides found in Atheris venoms might also act on the cardiovascular system by inhibiting particular metalloproteases, which however remain to be identified.

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.

Publisher Summary Chemokines are a rapidly growing family of proinflammatory cytokines that are involved in cell recruitment and activation. This chapter observes the direct binding of chemokines by techniques such as fluorescence activated cell sorting (FACS) analysis and confocal microscopy. Binding with fluorescent ligands has been limited to the commercially available phycoerythrin-IL-8 conjugate, which consists of the addition of a relatively large fluorescent moiety of 240 kDa attached to the 8-kDa protein through its side chains. Such a conjugate is many times larger than the chemokine itself, and because there are so many side chains available for conjugation, the product is likely to be a mixture of structural isomers. Therefore a highly controlled, site-specific reaction is sought to attach fluorophores to the polypeptide chain to ensure that each chain had one fluorophore, always at the same, chosen position in the chain. The chemical reactions involved had to be efficient and, so as not to compromise the biological behavior of the products, had to take place under mild, aqueous conditions.

A convenient method for the construction of site-specifically modified poly(ethylene glycol)-protein conjugates is described. This method relies on the ability to generate a reactive carbonyl group in place of the terminal amino group. If the protein has N-terminal serine or threonine, this can be done by very mild periodate oxidation and generates a glyoxylyl group. A method less restricted by the nature of the N-terminal residue, but which requires somewhat harsher conditions, is metal-catalyzed transamination, which gives a keto group. The N-terminal-introduced reactive carbonyl group specifically reacts, under mild acidic conditions, with an aminooxy-functionalized poly(ethylene glycol) to form a stable oxime bond. Using polymers of different size and shape (linear or multibranched), various conjugates of IL-8, G-CSF, and IL-1ra were constructed and further characterized with respect to their biological activity and pharmacokinetic behavior in rats. Unlike most previous methods, this approach places a single PEG chain at a defined site on the protein. It should therefore be more likely to conserve biological activity when the latter depends on interaction with another macromolecule (unlike enzymic activity which often survives multiple PEGylation).

A new approach is described for linking, through a thioether bond, the C-terminus of one unprotected polypeptide with the N-terminus of another. Homocysteine thiolactone is attached to the C-terminus of one polypeptide by reverse proteolysis and provides through hydroxylamine treatment a free sulfhydryl group. The alpha-amino group of a second polypeptide is selectively iodoacetylated by reaction with iodoacetic anhydride at pH 6.0 or the N-hydroxysuccinimide ester derivative at pH 7.0. Coupling of the two modified fragments occurs in a spontaneous alkylation reaction under mild conditions. After preliminary experiments with small peptides, this approach was extended to large protein fragments derived from recombinant analogs of G-CSF by enzymatic digestion. This approach provides a means of making head-to-tail protein chimeras or introducing noncoded structural elements into a protein.

Nanoparticles carrying biologically active functional sets (e.g., targeting moiety, payload, tracer) have potential use in a wide range of clinical applications. Though complex, such constructions should, as far as possible, have a defined molecular architecture and be monodisperse. However, the existing methods to achieve this goal are unsuitable for the incorporation of peptides and proteins, and those that provide for orthogonal introduction of two different types of functional element are incompatible with the use of commercially available materials. In this study, we have developed approaches for the production of nanoparticles based on commercially available polyamidoamine (PAMAM) dendrimers. First, we identified an optimized oxime conjugation strategy under which complex dendrimers can be fully decorated not only with model peptides, but also with recombinant proteins (insulin was taken as an example). Second, we developed a strategy based on a two-chain covalent heterodendrimer (a "diblock") based on cystamine core PAMAM dendrimers and used it to generate heterodendrimers, into which a peptide array and a mannose array were orthogonally introduced. Finally, by incorporating a functionalized linker into the diblock architecture we were able to site-specifically introduce a third functional element into the nanoparticle. We exemplified this approach using fluorescein, a mannose array, and a peptide array as the three functionalities. We showed that incorporation of a mannose array into a nanoparticle strongly and specifically enhances uptake by sentinel cells of the immune system, an important property for vaccine delivery applications. These PAMAM dendrimer-based approaches represent a robust and versatile platform for the development of bioactive nanoparticles.