Synthesis of different types of dipeptide building units containing N‐ or C‐terminal arginine for the assembly of backbone cyclic peptides*

Abstract: Different types of dipeptide building units containing N‐ or C‐terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg). For cyclization in the N‐terminal sequence N‐carboxyalkyl and N‐aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N‐terminal arginine to the N‐alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z‐Arg(Z)₂‐OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C‐terminal arginine was prepared to synthesize BK‐analogues with backbone cyclization in theC‐terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM^? technology was applied using the Boc‐Arg(Alloc)₂‐OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc‐protected amino aldehyde, derived from Boc‐Phe‐OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid‐phase synthesis in the same manner as amino acid derivatives.     

Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues

Abstract: A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C‐ and N‐terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N‐carboxy alkylated and N‐amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc‐glycine to the N‐alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre‐built in solution. For backbone cyclization at the C‐terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea‐pig ileum (GPI) assays. Additionally, the potentiation of the BK‐induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [d ‐Phe⁷]‐BK. Side chain as well as backbone cyclization in the N‐terminus of [d ‐Phe⁷]‐BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin‐induced contraction of guinea‐pig ileum.     

Chemical synthesis and receptor binding of catfish somatostatin: a disulfide‐bridged β‐d‐Galp‐(1→3)‐α‐d‐GalpNAc O‐glycopeptide*

Abstract: The glycopeptide hormone catfish somatostatin (somatostatin‐22) has the amino acid sequence H‐Asp‐Asn‐Thr‐Val‐Thr‐Ser‐Lys‐Pro‐Leu‐Asn‐Cys‐Met‐Asn‐Tyr‐Phe‐Trp‐Lys‐Ser‐Arg‐Thr‐Ala‐Cys‐OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains d ‐GalNAc and d ‐Gal O‐glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc‐Cys(Trt)‐PAC‐PEG‐PS support, using stepwise Fmoc solid‐phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin‐22 were accessed by incorporating as building blocks, respectively, N^α‐Fmoc‐Thr(Ac₃‐α‐D‐GalNAc)‐OH and N^α‐Fmoc‐Thr(Ac₄‐β‐D‐Gal‐(1→3)‐Ac₂‐α‐D‐GalNAc)‐OH. Acidolytic deprotection/cleavage of these peptidyl‐resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl‐protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N^α‐dithiasuccinoyl (Dts)‐glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2‐fold tighter binding.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N‐backbone cyclic peptide chemistry

Abstract: Protected N^α‐(aminoallyloxycarbonyl) and N^α‐(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of N^α‐backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and α,β‐unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side‐chain, resulting in fully orthogonal trifunctional building units for the solid‐phase peptide synthesis of small cyclic analogs of peptide loops (SCAPLs^?). N^α‐amino groups of building units were protected by Fmoc, functional side‐chains were protected by t‐Bu/Boc/Trt and N‐alkylamino or N‐alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large‐scale solid‐phase peptide synthesis. These building units have significant advantage in the synthesis of peptido‐related drugs.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists

Abstract: A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C‐terminal hexapeptide sequence Tyr‐Phe‐Ser‐Pro‐Arg‐Leu‐NH₂ of PBAN1‐33NH₂; whereas library II (d ‐Phe library) was based on the sequence of the PBAN lead linear antagonist Arg‐Tyr‐Phe‐d ‐Phe‐Pro‐Arg‐Leu‐NH₂. In both libraries the Pro residue was replaced by the BBC building unit N^α‐(ω‐aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N‐terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl₃I for selective deprotection of the Boc group from the building unit prior to on‐resin amino‐end to backbone‐nitrogen (AE‐BN) cyclization, during solid‐phase synthesis with Fmoc chemistry.     

Convenient and efficient synthesis of Boc‐/Z‐/Fmoc‐β‐amino acids employingN‐protected α‐amino acid fluorides

Abstract: A new and efficient method for the synthesis ofN^α‐Fmoc‐/Boc‐/Z‐β‐amino acids using the two‐step Arndt‐Eistert approach is described. Fmoc‐/Boc‐/Z‐α‐Amino acid fluorides were used for the acylation of diazomethane synthesizing Fmoc‐/Boc‐/Z‐α‐aminodiazoketones as crystalline solids with good yield and purity. They were then converted to the corresponding β‐amino acids using PhCOOAg/dioxane/H₂O.     

Solid‐phase synthesis of peptides containing the spin‐labeled 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC)

Abstract: 2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) is a nitroxide spin‐labeled, achiral C^α‐tetrasubstituted amino acid recently shown to be not only an effective β‐turn and 3₁₀/α‐helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid‐phase synthesis in an automated apparatus.     

A solid‐phase synthetic strategy for the preparation of peptide‐based affinity labels: synthesis of dynorphin A analogs

Abstract: Solid‐phase synthetic methodology was developed for the preparation of peptide‐based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p‐X)⁴,d ‐Pro¹⁰]Dyn A(1–11)NH₂ containing isothiocyanate (X = –N=C=S) and bromoacetamide (X = –NHCOCH₂Br) groups. The peptides were assembled on solid supports using Fmoc‐protected amino acids, and the side chain amine to be functionalized, Phe(p‐NH₂), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(0), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol–polystyrene (PEG–PS) resins containing the PAL [peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity‐labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side‐chain and backbone anchoring strategies,and compatible protection for N‐terminal cysteine*,†

Abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C‐terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N^α‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N^α‐t‐butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side‐chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N‐terminal cysteine residue have been developed: (i) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐(N‐methyl‐N‐phenylcarbamoyl)sulfenylcysteine [Fmoc‐Cys(Snm)‐OH], allowing creation of an otherwise fully protected resin‐bound intermediate with N‐terminal free Cys; (ii) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐triphenylmethylcysteine [Fmoc‐Cys(Trt)‐OH], generating a stable Fmoc‐Cys(H)‐peptide upon acidolytic cleavage; and (iii) incorporation of N^α‐t‐butyloxycarbonyl‐S‐fluorenylmethylcysteine [Boc‐Cys(Fm)‐OH], generating a stable H‐Cys(Fm)‐peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C‐termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on‐resin, as well as in solution after cleavage/purification.     

(αMe)Aun: a highly lipophilic,chiral, Cα‐tetrasubstituted α‐amino acid. Incorporation into model peptides and preferred conformation

Abstract: Using a chemo‐enzymatic approach we prepared the highly lipophilic, chiral, C^α‐methylated α‐amino acid (αMe)Aun. Two series of terminally protected model peptides containing either d ‐(αMe)Aun in combination with Aib or l ‐(αMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT‐IR absorption, ¹H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its α‐carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that d ‐(αMe)Aun favors the formation of the left‐handed 3₁₀‐helical conformation.     

A comparison of folding techniques in the chemical synthesis of the epidermal growth factor‐like domain in neu differentiation factor α/β

Abstract: The 52‐residue α/β chimera of the epidermal growth factor‐like domain in neu differentiation factor (NDF_eα/β) has been synthesized and folded to form a three disulfide bridge (Cys¹⁸²–Cys¹⁹⁶, Cys¹⁹⁰–Cys²¹⁰, Cys²¹²–Cys²²¹) containing peptide. We investigated two general strategies for the formation of the intramolecular disulfide bridges including, the single‐step approach, which used fully deprotected and reduced peptide, and a sequential approach that relied on orthogonal cysteine protection in which specific pairs are excluded from the first oxidation step. Because there are 15 possible disulfide bridge arrangements in a peptide with six cysteines, the one‐step approach may not always provide the desired disulfide pairing. Here, we compare the single‐step approach with a systematic evaluation of the sequential approach. We employed the acetamidomethyl group to protect each pair of cysteines involved in disulfide bridges, i.e. Cys¹⁸² to Cys¹⁹⁶, Cys¹⁹⁰ to Cys²¹⁰ and Cys²¹² to Cys^221. This reduced the number of possible disulfide patterns from 15 to three in the first folding step. We compared the efficiencies of folding for each protected pair using RP‐HPLC, mapped the disulfide connectivity of the predominant product and then formed the final disulfide from the partially folded intermediate via I₂ oxidation. Only the peptide having the Cys¹⁸²–Cys¹⁹⁶ pair blocked with acetamidomethyl forms the desired disulfide isomer (Cys¹⁹⁰–Cys²¹⁰/Cys²¹²–Cys²²¹) as a single homogeneous product. By optimizing both approaches, as well as other steps in the synthesis, we can now rapidly provide large‐scale syntheses of NDF_eα/β and other novel EGF‐like peptides.     

Ac10c: a medium‐ring,cycloaliphatic Cα,α‐disubstituted glycine. Incorporation into model peptides and preferred conformation

Abstract: Two complete series of N‐protected oligopeptide esters to the pentamer level from 1‐amino‐cyclodecane‐1‐carboxylic acid (Ac₁₀c), an α‐amino acid conformationally constrained through a medium‐ring C^α_i ? C^α_i cyclization, and either the l ‐Ala or Aib residue, along with the N‐protected Ac₁₀c monomer and homo‐dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT‐IR absorption and ¹H NMR techniques. Furthermore, the molecular structures of two derivatives (Z‐Ac₁₀c‐OH and Fmoc‐Ac₁₀c‐OH) and two peptides (the dipeptide ester Z‐Ac₁₀c‐l ‐Phe‐OMe and the tripeptide ester Z‐Aib‐Ac₁₀c‐Aib‐OtBu) were determined in the crystal state using X‐ray diffraction. The experimental results support the view that β‐bends and 3₁₀‐helices are preferentially adopted by peptides rich in Ac₁₀c, the third largest cycloaliphatic C^α,α‐disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1‐amino‐cycloalkane‐1‐carboxylic acid (Ac_nc, with n = 3–12) series, which represents the prerequisite for our recent proposal of the ‘Ac_nc scan’ concept.     

Zinc‐promoted simple synthesis of oligomer‐freeNα‐Fmoc‐amino acids using Fmoc‐Cl as an acylating agent under neutral conditions

Abstract: A range of N^α‐Fmoc‐protected amino acids, including those that contain t‐butyl moiety, have been synthesized by employing Fmoc‐Cl utilizing the activated, commercial zinc dust‐promoted synthesis of carbamates under neutral conditions.A general procedure is described that circumvents the oligomerization side reaction normally noticed in Schotten–Baumann conditions. It is a simple, convenient and clean method. Thus, Fmoc‐amino acids are obtained in high yield (85–92%) and purity as checked by thin‐layer chromatography, high‐performance liquid chromatography and other physical methods.     

Simple and stereospecific homologation of urethane‐protected α‐amino acids to their higher homologs using HBTU1

Abstract: A simple, efficient and stereospecific approach for the homologation of urethane‐protected α‐amino acids to β‐amino acids by the Arndt–Eistert method employing Fmoc‐/Boc‐α‐amino acid and 2‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyl‐uronium hexafluorophosphate mixture for the acylation of diazomethane synthesizing the key intermediates Fmoc‐/Boc‐α‐aminodiazomethanes as crystalline solids is described.     

A convenient incorporation of conformationally constrained 5,5‐dimethylproline into the ribonuclease A 89–124 sequence by condensation of synthetic peptide fragments

Abstract: The presence of l ‐5,5‐dimethylproline (dmP) within an amino acid sequence results in the formation of an X‐dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C‐terminal 36‐residue peptide, corresponding to the 89–124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro⁹³. The peptide was assembled by condensation of protected 5‐ and 31‐residue peptide fragments, which were synthesized by solid‐phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc‐Ser(^tBu)‐Ser(^tBu)‐Lys(Boc)‐Tyr(^tBu)‐dmP‐OH pentapeptide (residues 89–93) with efficient acylation of the sterically hindered dmP residue. In a comparative study, the reagent O‐(7‐azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate was found to be superior to bromo‐tris‐pyrrolidino‐phosphonium hexafluorophosphate and tetramethylfluoroformamidinium hexafluorophosphate for the synthesis of the ‐Tyr(^tBu)‐dmP‐ peptide bond in solution as well as on a resin.     

An efficient and highly selective deprotection of N‐Fmoc‐α‐amino acid and lipophilic N‐Fmoc‐dipeptide methyl esters with aluminium trichloride and N,N‐dimethylaniline

Abstract: A novel procedure for the deprotection of the carboxyl group of amino acid methyl esters is presented. The process is carried out by the reagent system aluminium trichloride/N,N‐dimethylaniline that can successfully be applied to unblock the carboxyl moiety either of N‐Fmoc‐protected amino acid methyl esters and N‐Fmoc‐protected short dipeptide methyl esters. The chiralities of the optically pure amino acid or peptide precursors are maintained totally unchanged.     

Evaluation of new base‐labile2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc)‐amino acids for solid‐phase peptide synthesis

Abstract: The 2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base‐labile protecting group for solid‐phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with N^α‐Nsc‐protected amino acids for the synthesis of some melanotropins important to our research, namely, γ‐melanocyte‐stimulating hormone (γ‐MSH), its [Nle³]‐analogue, and a cyclic α‐MSH/β‐MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in > 98% purity. The γ‐MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc‐chemistry but was accompanied by side products. Finally, the Nle³‐γ‐MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc‐chemistry might not be the best methodology for certain sequences.     

Solvolysis and aminolysis on peptidyl‐Kaiser oxime resin assisted by Ca2+and Eu3+: a mild procedure to prepare α‐methyl and ‐ethyl esters of protected peptides

Abstract: Ca²⁺ and Eu³⁺ were able to assist solvolysis on peptidyl‐Kaiser oxime resins generating α‐methyl and ‐ethyl esters of protected peptides. The methanolysis assistance was at least twice as effective as that of acetic acid, the common catalyst used in aminolysis of the ester oxime linkage. No molar excess of Ca²⁺ or Eu³⁺ was needed to enhance this reaction efficiency. Ca²⁺ also assisted aminolysis on peptidyl‐Kaiser oxime resins. Solvolysis and aminolysis rates depended on the nature of theC‐terminal residue attached to the resin and on the alcohol used. Both reactions were selective to the ester oxime linkage since no significant amount of secondary products, resulting from rearrangements or simultaneous transesterification of the β‐benzyl or cyclohexyl esters, was detected in the reaction media. The α‐methyl and ‐ethyl esters of Ac‐Ala‐Gly‐X [where, X = Gly, Ala, Phe or Lys (2‐Cl‐Z)] and of Ac‐Ile‐Ser (Bzl)‐Asp(OZ) (where, Z = Bzl or cHex) were essentially the only products formed in the solvolyses performed. Ac‐Ile‐Ser(Bzl)‐Asp(OcHex)Arg(HCl)‐OMe and Ac‐Ile‐Ser(Bzl)‐Asp(OcHex)Arg (HCl)‐OEt were the major products formed in the aminolysis reactions. In the presence of the metal ions, the resin‐cleavage yields were > 50%. In their absence, they were < 15%.     

Synthesis and immunological studies of α‐conotoxin chimera containing an immunodominant epitope from the 268–284 region of HSV gD protein

Abstract: We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as ‘guest’ sequence in the ‘host’ structure of α‐conotoxin GI, a 13‐residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276–284 region of HSV gD‐1 selected for these studies is highly hydrophilic and adopts a β‐turn. The α‐conotoxin GI also contains a β‐turn in the 8–12 region, stabilized by two disulfide bridges at positions 2–7 and 3–13. Thus, the tetramer sequence of α‐conotoxin, ⁸Arg‐His‐Tyr‐Ser¹² has been replaced by Asp‐Pro‐Val‐Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3–13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys²‐Cys⁷ three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 m HCl or Tl(tfa)₃ in trifluoroacetic acid (TFE)] were compared. The high‐performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimeric peptide and native α‐conotoxin GI showed similar circular dichroism spectra in phosphate‐buffered saline (PBS) and in a PBS‐TFE 1 : 1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M‐ and IgG‐type antibody responses showed that the bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV‐α‐[Tyr¹]‐conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV‐α‐[Tyr¹]‐conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG‐specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.     

177Lu‐labeled monomeric,dimeric and multimeric RGD peptides for the therapy of tumors expressing α(ν)β(3) integrins

The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target‐specific molecular recognition. Peptides based on the cyclic Arg‐Gly‐Asp (RGD) sequence have been reported as high‐affinity agents for the α(ν)β(3) integrin. The aim of this research was to prepare a multimeric system of ¹⁷⁷Lu‐labeled gold nanoparticles conjugated to c(RGDfK)C (cyclo(Arg‐Gly‐Asp‐Phe‐Lys)Cys) and to compare the radiation‐absorbed dose with that of ¹⁷⁷Lu‐labeled monomeric and dimeric RGD peptides to α(ν)β(3) integrin‐positive U87MG tumors in mice. DOTA‐GGC (1,4,7,10‐tetraazacyclododecane‐N‐N′,N″,N?‐tetraacetic acid‐Gly‐Gly‐Cys) and c(RGDfK)C peptides were synthesized and conjugated to AuNPs by a spontaneous reaction of the thiol groups. Transmission electron microscopy, ultraviolet–visible, X‐ray photoelectron spectroscopy, Raman and far‐infrared spectroscopy techniques demonstrated that AuNPs were functionalized with the peptides. For the ¹⁷⁷Lu‐AuNP‐c(RGDfK)C to be obtained, the ¹⁷⁷Lu‐DOTA‐GGC radiopeptide was first prepared and added to a solution of AuNPs followed by c(RGDfK)C (25 µl, 5 µ m ) at 18 °C for 15 min. ¹⁷⁷Lu‐DOTA‐GGC, ¹⁷⁷Lu‐DOTA‐cRGDfK and ¹⁷⁷Lu‐DOTA‐E‐c(RGDfK)₂ were prepared by adding ¹⁷⁷LuCl₃ (370 MBq) to 5 µl (1 mg/ml) of the DOTA derivative diluted with 50 µl of 1 m acetate buffer pH 5. The mixture was incubated at 90 °C in a block heater for 30 min. Radiochemical purity was determined by ultrafiltration and HPLC analyses. Biokinetic studies were accomplished in athymic mice with U87MG‐induced tumors. The radiochemical purity for all ¹⁷⁷Lu‐RGD derivatives was 96 ± 2%. ¹⁷⁷Lu‐absorbed doses per injected activity delivered to U87MG tumors were 0.357 ± 0.052 Gy/MBq (multimer), 0.252 ± 0.027 Gy/MBq (dimer) and 0.102 ± 0.018 Gy/MBq (monomer). ¹⁷⁷Lu‐labeled dimeric and multimeric RGD peptides demonstrated properties suitable for targeted radionuclide therapy of tumors expressing α(ν)β(3) integrins. Copyright © 2012 John Wiley & Sons, Ltd.