Beta-turns in bridged proline-containing cyclic peptide models

The synthesis, CD, ir spectroscopic, and conformational studies of a series of bridged cyclic peptides of the general formula, cyclo[NH-(CH₂)_n-CO-Gly-Pro-Y-Gly] (2a–d, Y = Gly or Ser(OBu^t), n = 4 or 2) is reported. As indicated by difference nuclear Overhauser enhancement and Fourier transform ir experiments, the tetrapeptide sequence of cyclo[NH-(CH₂)₄-CO-Gly-Pro-Gly-Gly] (2a) and cyclo[NH-(CH₂)₂-CO-Gly-Pro-Gly-Gly] (2b) adopts a 1 ← 4 hydrogenbonded type II β-turn conformation in solution, while cyclo[NH-(CH₂)₄-CO-Gly-Pro-Ser(OBu^t) -Gly] (2c) features a type I β-turn, fixed by 1 ← 4 and O^γ … NH intramolecular H bonds. In aqueous solution 2a and 2c show class B and class C CD spectra, respectively. This is the first case reported of a typical class C CD pattern in aqueous solution for a conformationally mobile system having a type I β-turn. Based on the comparison of the band intensities of the bridged models with those of linear and cyclic model systems reported earlier, a set of subspectra with reduced band intensities is suggested for use in the CD analysis of the conformation of polypeptides in solution.     

Nmr investigation of CYCLO7,10[C7,X9,C10,Nle12] analogues of the α-factor from saccharomyces cerevisiae

The cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²], cyclo^7,10[Cys⁷,D -Ala⁹,Cys¹⁰,Nle¹²], and cyclo^7,10[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] analogues of the α-factor mating pheromone (WHWLQLKPGQPMY) of the yeast Saccharomyces cerevisiae were studied in DMSO/water (80 : 20) and aqueous solution by nmr spectroscopy. In addition, the cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²] α-factor was examined in DMSO/water. Nuclear Overhauser effect (NOE) and NH dδ/dT data indicate that the cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²] α-factor adopts a type II β-turn in DMSO/water and that the cyclo^7,10[Cys⁷,D -Ala⁹,Cys¹⁰,Nle¹²] - and cyclo^7,10-[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] α-factor analogues adopt type II and type I/III β-turns, respectively, in both DMSO/water and aqueous solutions. In aqueous solution, residues 8 and 9 of the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²] α-factor appear to adopt at least two distinct conformations, one of these being identified as a type I/III β-turn. In contrast, the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²] α-factor appears to adopt predominately a type II β-turn in DMSO/water. Quantitative NOE measurements of the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²]-, cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²]-, and cyclo^7,10[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] α-factors in DMSO/water were used to derive three-dimensional structures of the cyclo^7,10[Cys⁷,Pro⁸,X⁹Cys¹⁰] portion of these analogues. © 1994 John Wiley & Sons, Inc.     

Studies on proline-containing tetrapeptide models of beta-turns

The synthesis of a series of protected tetrapeptides of the general formula Cbz-Gly-X²-Y³-Gly-OR (R = stearyl or methyl, X and/or Y = proline) is described. Detailed CD studies have been performed to evaluate the contribution of proline-containing β-turns to the CD spectra of proteins. The CD spectra of all the models are dominated by the chiral contribution of the proline residue. In polar, proton-donating solvents, a poly-proline II-like spectrum was observed in almost all cases. The tetrapeptide model Cbz-Gly-Gly-Pro-Gly-OStearyl, in acetonitrile shows a type C spectrum that has not been previously reported for linear peptides. The ir and nmr data on this model support the assumption of that of a type III β-turn, exhibiting a type C spectrum, participate in the conformational equilibrium. The most interesting finding of the CD studies is the observation of a type D spectrum (according to the classification of Woody [Woody, R. W. (1974) in Peptides, Polypeptides and Proteins, Blout, E. R. Bovey, F. A. Lotan, N. & Goodman, M. (Eds.), Wiley, New York]) for models Cbz-Gly-Pro-Asp(OBu^t)-Gly-OStearyl and Cbz-Gly-Pro-Ser(OBu^t)-Gly-OStearyl in cyclohexane. The results of the CD measurements ae discussed in correlation with ir and nmr data and with recent literature.     

Computer Modeling of the Solution Conformation of Cyclic Enkephalins

Summary The probable conformations of two cyclic enkephalin analogs, DNS-cyclo[d-Dab-Gly-Trp-Leu] (I) and DNS-cyclo[d-Dab-Gly-Trp-d-Leu] (II) (DNS=dansyl), were determined by combining the results of NOE, vicinal coupling constant and fluorescence energy transfer measurements with theoretical calculations. The common feature of the conformations for both peptides is the presence of a β-turn at residues 2 and 3.     

Structural and ICAM-1-Docking Properties of a Cyclic Peptide from the I-domain of LFA-1: An inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion

Abstract

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)- Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7-Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 α-subunit. We found that cyclic peptide 1 can bind to the D1- domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1- domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable βII'-turn at Asp4-Gly5-Glu6-Ala7 and a βI-turn at Pen1-Ile2-Thr3-Asp4; a less stable βV-turn is found at the C-terminal region. The β-turn at Asp4-Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1- domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

New Phosphitylating Reagents Containing P-F Bond and Their Application in the Synthesis of P-Fluoro Nucleosidyl Phosphates,Thiophosphates and Selenophosphates

Abstract

An efficient and general synthesis of phosphorofluoridates RO-P(X)(OH)F (X=O) and their analogues (X=S, Se) based on two new phosphitylating reagents: 2-cyanoethyl-N,N-diisopropylfluorophosphoroamidite F-P(NPrⁱ ₂)OCH₂CH₂CN and tertbutyl-N,N-diisopropylfluorophosphoroamidite F-P(NPrⁱ ₂)OBu^t is described.     

A 13C spin-lattice relaxation study of dipeptides containing glycine and proline: mobility of the cyclic proline side chain.

Molecular dynamics of the cyclic dipeptides cyclo(Gly-L -Pro), cyclo-(L-Pro-L -Pro), and cyclo(L-Pro-D-Pro) and the linear dipeptides L-Pro-Gly and cis and trans Gly-L -Pro were studied in neutral aqueous solution by ¹³C nuclear magnetic resonance. Spinlattice relaxation times (T₁) were determined for each individual carbon atom. The correlation times, τ, were derived from a semiquantitative analysis of the T₁ data. The correlation times of the proline ring carbons, β, γ, and δ suggest that the cyclic dipeptides have more restriction of conformational freedom in the proline ring than the linear dipeptides. This effect is most pronounced on the γ carbon.     

Crystal structures of two cyclic pseudopentapeptides containing ψ[CH2S] and ψ[CH2SO] backbone surrogates

The solid state conformations of cyclo[Gly–Proψ[CH₂S]Gly–D –Phe–Pro] and cyclo[Gly–Proψ[CH₂–(S)–SO]Gly–D –Phe–Pro] have been characterized by X-ray diffraction analysis. Crystals of the sulfide trihydrate are orthorhombic, P2₁2₁2₁, with a = 10.156(3) Å, b = 11.704(3) Å, c = 21.913(4) Å, and Z = 4. Crystals of the sulfoxide are monoclinic, P2₁, with a = 10.662(1) Å, b = 8.552(3) Å, c = 12.947(2) Å, β = 94.28(2), and Z = 2. Unlike their all-amide parent, which adopts an all-trans backbone conformation and a type II β-turn encompassing Gly-Pro-Gly-D -Phe, both of these peptides contain a cis Gly¹-Pro² bond and form a novel turn structure, i.e., a type II′ β-turn consisting of Gly–D –Phe–Pro–Gly. The turn structure in each of these peptides is stabilized by an intramolecular H bond between the carbonyl oxygen of Gly¹ and the amide proton of D -Phe⁴. In the cyclic sulfoxide, the sulfinyl group is not involved in H bonding despite its strong potential as a hydrogen-bond acceptor. The crystal structure made it possible to establish the absolute configuration of the sulfinyl group in this peptide. The two crystal structures also helped identify a type II′ β-turn in the DMSO-d₆ solution conformers of these peptides. © 1993 John Wiley & Sons, Inc.     

β-Alanine containing cyclic peptides with predetermined turned structure. V

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution structural characterization by nmr spectroscopy, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(Pro-Phe-β-Ala-Phe-Phe-β-Ala). The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylenechloride solution using N, N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol/ethyl acetate. The molecule adopts in the solid state a conformation characterized by cis β-Ala⁶-Pro¹ peptide bond. The α-amino acid residues are at the corner positions of turned structures. The Pro¹-Phe² segment is incorporated in a pseudo type I β-turn, while Phe⁴-Phe⁵ is in a typical type I β-turn. Assignment of all ¹H and ¹³C resonances was achieved by homo- and heteronuclear two-dimensional techniques in dimethylsulfoxide (DMSO) solutions. The conformational analysis was based on inter-proton distances derived from rotating frame nuclear Overhauser effect spectroscopy spectra and homonuclear coupling constants. Restrained molecular dynamic simulation in vacuo was also performed to built refined molecular models. The molecule is present in DMSO solution as two slowly interconverting conformers, characterized by a cis-tran isomerism around the β-Ala⁶-Pro¹ peptide bond. This work confirms our expectations on the low propensity of β-alanyl residues to be positioned at the corners of turned structure. © 1994 John Wiley & Sons, Inc.     

Studies on cyclic peptides. IV. Conformation of cyclo(Sar-Sar-Gly)2, cyclo(Sar)6 and cyclo(Sar-Gly-Gly)2 and their conformational change induced by alkali thiocyanates

Conformation of cyclo (Sar-Sar-Gly)₂, cyclo(Sar)₆, and cyclo(Sar-Gly-Gly)₂ was investigated by nmr spectroscopy. cyclo(Sar-Sar-Gly)₂, were shown to assume various conformations in dimethysulfoxide. It was attributed to the distribution of cis as well as trans Gly-Sar or Sar-Sar amide links along the peptide backbone. In particular, cyclo(Sar-Sar-Gly)₂ took five or six different conformations: one or three C₂-symmetric conformations and four or three asymmetric conformations, respectively. Three of nine NH resonance signals were ascribed to the internally hydrogen-bonded glycine residues. cyclo(Sar-Sar-Gly)₂ and cyclo(Sar)₆ showed a spectral change on the addition of alkali thiocyanates, indicating a conformational change induced by a complex formation with the alkali cations. The complex nmr spectrum due to a hybridization of different conformations changed with the salt addition into a simple nmr spectrum, suggesting a preponderence of a new, single conformation. On the basis of the spectral change, the strength for the cations binding the cyclic peptides was found to be in the order of K⁺ > Na⁺ > Rb⁺ > Cs⁺ for cyclo(Sar-Gly-Gly)₂ and K⁺ > Rb⁺ > Cs⁺ for cyclo(Sar)₆. On the other hand, cyclo(Sar-Gly-Gly)₂ in dimethylsulfoxide assumed a single C₂ conformation having two glycyl peptide protons shielded from solvent and the other two exposed to solvent. This conformation did not change with the salt addition. Finally, the conformations of several cyclic peptides containing the sarcosine residue such as cyclo(Sar)₆ cyclo(Sar-Sar-Gly)₂ cyclo(Pro-Sar-Gly)₂, and cyclo (Sar-Gly-Gly)₂ were compared. It appeared that proline and glycine residues reduced the conformational multiplicity of the cyclic peptide backbone, and the ability to bind alkali metal cations decreased in the above order.     

Potent activity of a PK/PBAN analog with an (E)-alkene,trans-Pro mimic identifies the Pro orientation and core conformation during interaction with HevPBANR-C receptor

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a multifunctional role in an array of important physiological processes in insects, including regulation of sex pheromone biosynthesis in moths. A cyclic PK/PBAN analog (cyclo[NTSFTPRL]) retains significant activity on the pheromonotropic HevPBANR receptor from the tobacco budworm Heliothis virescens expressed in CHO-K1 cells. Previous studies indicate that this rigid, cyclic analog adopts a type I β-turn with a transPro over residues TPRL within the core PK/PBAN region. An analog containing an (E)-alkene, trans-Pro mimetic motif was synthesized, and upon evaluation on the HevPBANR receptor found to have an EC₅₀ value that is not statistically different from a parent C-terminal PK/PBAN hexapeptide sequence. The results, in aggregate, provide strong evidence for the orientation of Pro and the core conformation of PK/PBAN neuropeptides during interaction with the expressed PBAN receptor. The work further identifies a novel scaffold with which to design mimetic PBAN analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting PK/PBAN-regulated pheromone signaling systems.     

Omega amino acids in peptide design: incorporation into helices

Incorporation of easily available achiral ω-amino acid residues into an oligopeptide results in substitution of amide bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptidomimetic. The central Gly-Gly segment of the helical octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-Ome(1) has been replaced by δ-amino-valeric acid (δ-Ava) residue in the newly designed peptide Boc-Leu-Aib-Val-δ-Ava-Leu-Aib-Val-OMe(2). ¹H-nmr results clearly suggest that in the apolar solvent CDCl₃, the δ-Ava residue is accommodated into a folded helical conformation, stabilized by successive hydrogen bonds involving the NH groups of Val(3), δ-Ava(4), and Leu(5). The δ-Ava residue must adopt a gauche-gauche-trans-gauche-gauche conformation along the central polymethylene unit of the aliphatic segment, a feature seen in an energy-minimized model conformation based on nmr parameters. The absence of hydrogen bonding functionalities, however, limits the elongation of the helix. In fact, in CDCl₃, the folded conformation consists of an N-terminal helix spanning residues 1–4, followed by a Type II β-turn at residues 5 and 6, whereas in strongly solvating media like (CD₃)₂SO, the unfolding of the N-terminal helix results in β-turn conformations at Leu(1)-Aib(2). The Type II β-turn at the Leu(5)-Aib(6) segment remains intact even in (CD₃)₂SO. CD comparisons of peptides 1 and 2 reveal a “nonhelical” spectrum for 2 in 2,2,2-trifluoroethanol. © 1996 John Wiley & Sons, Inc.     

An efficient strategy for the large-scale synthesis of head-to-tail cyclic peptides

Summary An efficient and generally applicable method for the synthesis of head-to-tail cyclic peptides with HBTU (2-(1H-benzotriazol-1-yl)-1,1,3, 3-tetramethyluronium hexafluorophosphate) has been developed. The utility of this approach was exemplified with the multigram preparation of a potent endothelin receptor-selective (ET_A) antagonist BQ-123 (cyclo[-d-Trp-d-Asp-Pro-d-Val-Leu-]). This methodology can be readily applied to the small-and largescale synthesis of other head-to-tail cyclic peptides.     

Influence of glucose-templated proline mimetics on the β-turn conformation of the peptide fragment Ac-Leu-d-Phe-Pro-Val-NMe2 found in Gramicidin S

The synthesis of tetrapeptide-based β-turn mimetics containing spirocyclic glucose-templated 3-hydroxyproline hybrids Glc3′(S)-5′(R)(CH₂OH)HypH and Glc3′(S)-5′(S)(CH₂OH)HypH as proline mimetics is presented. NMR-based conformational analysis of Ac-Leu-d-Phe-[Glc3′(S)-5′(R)(CH₂OH)HypH]-Val-NMe₂ and Ac-Leu-d-Phe-[Glc3′(S)-5′(S)(CH₂OH)HypH]-Val-NMe₂ demonstrates the presence of β-turn conformations. Different turn structures were observed by changing the stereochemistry at 5′-position of Glc3′(S)-5′(R)(CH₂OH)HypH. The major prolyl amide cis isomer of glucose-protected tetrapeptide Ac-Leu-d-Phe-[Glc(MOM)₄3′(S)-5′(R)(CH₂OMOM)HypH]-Val-NMe₂11 and glucose unprotected Ac-Leu-d-Phe-[Glc3′(S)-5′(R)(CH₂OH)HypH]-Val-NMe₂13 forms a type VI β-turn conformation. In contrast, the major prolyl amide trans rotamer of tetrapeptide Ac-Leu-d-Phe-[Glc(MOM)₄3′(S)-5′(S)(CH₂OMOM)HypH]-Val-NMe₂12 conserves a similar β-turn conformation as the Gramicidin S-based peptide fragment Ac-Leu-d-Phe-Pro-Val-NMe₂16.     

Conformations of cyclic peptide/calcium complexes in solution

Synthetic cyclic octapeptides of general structure cyclo[Glu(γOBzl)-Sar-Gly-(N-R)Gly]₂ (R = n-hexyl and cyclohexyl) transport calcium ions selectively across organic phases and phospholipid membranes. We have now used proton nmr spectroscopy (360 MHz) to study the solution conformation(s) of their calcium complexes. When Ca(ClO₄)₂ was added to solutions of these peptides in CDCl₃, nmr spectra of the resulting calcium complexes were characteristic of a single C₂-symmetric conformer. From a Karplus-Bystrov analysis of vicinal coupling constants in both the peptide backbone and Glu side chain (treated as an ABCC′MX spin system), in conjuction with model-building studies, a structure was proposed in which the calcium ion is bound in an octahedral-type complex by the four (coplanar) carbonyl groups of the (all-trans) Glu-Sar and Gly-(N-R)Gly peptide bonds. Occurrence of preferred rotamers about Glu side chain C^α–C^β bonds indicated that restricted rotation in peptide side chains arises upon calcium binding.     

A nonhelical,multiple β-turn conformation in a glycine-rich heptapeptide fragment of trichogin A IV containing a single central α-aminoisobutyric acid residue

The conformational properties of the protected seven-residue C-terminal fragment the lipopeptaibol antibiotic Trichogin A IV (Boc-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe) has been examined in CDCl₃ and (CD₃)₂SO by ¹H-nmr. Evidence for a multiple β-turn conformation [type I′ at Gly(1)-Gly(2), type II at Leu(3)-Aib(4), and a type I′ at Aib(4)-Gly(5)] suggests that Leu(3) has preferred an extended or semiextended conformation over a helical conformation in CDCl₃. This structure is thus in contrast to earlier observations of seven-residue peptides containing a single central Aib preferring helical conformations in both solution and crystalline slates. A structural transition to a frayed right-handed helix is absented in (CD₃)₂SO. These results suggest that nonhelical conformations may be important in Gly-rich peptides containing Aib. Further, the presence of amino acids with contradictory influences on backbone conformational freedom can lead to well-defined conformational transitions even in small peptides. © 1995 John Wiley & Sons, Inc.     

Isodichroic point and the β–random coil transition of poly(S-carboxymethyl-L-cysteine) and poly(S-carboxyethyl-L-cysteine) in the absence of added salt

The β-coil transition of poly(S-carboxymethyl-L -cysteine) (poly[Cys(CH₂CO₂H)]) and poly(S-carboxyethyl-L -cysteine) (poly[Cys((CH₂)₂CO₂H)]) was followed by CD, potentiometric titration, and viscosity in the absence of added salt. These different properties give consistent results for poly[Cys((CH₂)₂CO₂H)]. The CD spectra of poly[Cys(CH₂CO₂H)] change considerably with the degree of neutralization α even for a low-molecular-weight sample incapable of forming the β-structure. Because of the superposition of this additional effect, the dependence of CD on α is inconsistent with titration data for the case of poly[Cys(CH₂CO₂H)], particularly when the nπ transition is used to follow the β-coil transition. The change of CD inherent to the β-coil transition is characterized by an isodichroic point: 215 nm for poly[Cys((CH₂)₂CO₂H)] and 218 nm for poly[Cys(CH₂CO₂H)]. A criterion supporting the stacking of the pleated sheet is suggested based on the isodichroic point.     

Conformational interconversions in peptide β-turns: Discrimination between enantiomeric conformations by chiral perturbation

The crystal structure of the model tripeptide Boc-Aib-Gly-Leu-OMe ( 1 ) reveals two independent molecules in the asymmetric unit that adopt “enantiomeric” type I and type I′ β-turn conformations with the Aib and Gly residues occupying the corner (i + 1 and i + 2) positions. ¹³C cross polarization and magic angle sample spinning spectra in the solid state also support the coexistence of two conformational species. ¹³C-nmr in CDCl₃ establishes the presence of a single species or rapid exchange between conformations. 400 MHz ¹H-nmr provides evidence for conformational exchange involving a major and minor species, with β-turn conformations supported by the low solvent exposure of Leu(3) NH and the observation of N_iH ↔ N_i+1H nuclear Overhauser effects. CD bands in the region 190–230 nm are positive, supporting a major population of type I′ β-turns. The isomeric peptide, Boc-Gly-Leu-Aib-OMe ( 2 ), adopts an “open” type II′ β-turn conformation in crystals. Solid state and solution nmr support population of a single conformational species. Chiral perturbation introduced outside the folded region of peptides may provide a means of modulating screw sense in achiral sequences. © 1998 John Wiley & Sons, Inc. Biopoly 45: 191–202, 1998     

Type II β-turn conformation of pivaloyl-L-prolyl-α-aminoisobutyryl-N-methylamide: Theoretical,spectroscopic, and X-ray studies

Pivaloyl-L -Pro-Aib-N-methylamide has been shown to possess one intramolecular hydrogen bond in (CD₃)₂SO solution, by ¹H-nmr methods, suggesting the existence of β-turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II β-turn conformations are about 2 kcal mol^?1 more stable than Type III structures. A crystallographic study has established the Type II β-turn in the solid state. The molecule crystallizes in the space group P2₁ with a = 5.865 Å, b = 11.421 Å, c = 12.966 Å, β = 97.55°, and Z = 2. The structure has been refined to a final R value of 0.061. The Type II β-turn conformation is stabilized by an intramolecular 4 → 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are ?_Pro = ?57.8°, ψ_Pro = 139.3°, ?_Aib = 61.4°, and ψ_Aib = 25.1°. The Type II β-turn conformation for Pro-Aib in this peptide is compared with the Type III structures observed for the same segment in larger peptides.     

Melanoma targeting with α-melanocyte stimulating hormone analogs labeled with fac-[99mTc(CO)3]+: effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled α-melanocyte stimulating hormone (α-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized α-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of α-MSH (Ac-Nle-cyclo[Asp-His-dPhe-Arg-Trp-Lys]-NH₂]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of α-MSH analogs by comparing the pharmacokinetics profile of the ^99mTc-labeled cyclic peptide βAla-Nle-cyclo[Asp-His-d-Phe-Arg-Trp-Lys]-NH₂ with that of the linear analog βAla-Nle-Asp-His-dPhe-Arg-Trp-Lys-NH₂ in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of βAla, and the resulting pz–peptide conjugates were reacted with the fac-[^99mTc(CO)₃]⁺ moiety. The ^99mTc(CO)₃-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic ^99mTc(CO)₃-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 °C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 ± 0.83 and 11.31 ± 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear ^99mTc(CO)₃-pz–peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle⁴,dPhe⁷)-αMSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively).