Rapid and high-yield solution-phase synthesis of DOTA-Tyr-octreotide and DOTA-Tyr-octreotate using unprotected DOTA

An improved method for the solution-phase derivatization of Tyr³-Lys⁵(Dde)-octreotide (TOC(Dde)) and Tyr³-Lys⁵(Dde)-octreotate (TATE(Dde)) with the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-N′,N″,N?,N?-tetraacetic acid) has been developed. The fully protected parent peptides were assembled via solid-phase peptide synthesis (SPPS) using Fmoc-strategy. After cleavage from the solid support, disulfide bond formation was carried out using H₂O₂. Both TOC(Dde) and TATE(Dde) were successfully coupled with DOTA in the presence of NHS, EDCI and DIPEA in a water/DMF solvent system. Yields of the coupling reaction were >98% within only 2 h with no detectable formation of sideproducts. This method for the preparation of DOTATOC, DOTATATE and other DOTA-peptide conjugates is therefore a rapid and economic alternative to the currently used methods.     

Application of tris-allyl-DOTA in the preparation of DOTA-peptide conjugates

The synthesis of tris-allyl-DOTA starting from cyclen and its application in the preparation of DOTA-peptide conjugates is reported. Clinically important conjugates such as DOTA-Tyr³-octreotide (DOTA-TOC), DOTA-Tyr³-octreotate (DOTA-TATE) as well as a DOTA-RGD peptide were synthesized in high yields with Fmoc solid phase peptide synthesis. The final, extremely reliable de-allylation was achieved on solid phase by different methods identifying morpholine/Pd(0) as the most suitable one obtaining all DOTA peptide conjugates in high yields. All DOTA-peptides were purified by reversed phase HPLC and structural identity was proved using MALDI-TOF mass spectrometry.     

Synthesis of a Tyr-octreotate conjugated closo-carborane [HC2B10H10]: a potential compound for boron neutron capture therapy

A novel Tyr³-octreotate conjugated closo-carborane as a potential compound for boron neutron capture therapy was obtained via Fmoc solid phase peptide synthesis. The boron cluster [C₂B₁₀H₁₁] was introduced through the reaction of 6,9-bis(acetonitrile)decaborane and 5-hexynoic acid yielding a new closo-carborane conjugated carboxylic acid which was coupled subsequently with solid phase conjugated Tyr³-octreotate. The final boron-containing peptide was purified by preparative reverse phase HPLC and structural identity was proved applying MALDI-TOF mass spectrometry.     

Synthesis and electrochemical studies of disubstituted ferrocene/dipeptide conjugates with sulfur-containing side chains

A series of 1,1′-disubstituted ferrocenoyl peptides incorporating dipeptide sidearms has been synthesized and studied electrochemically. The target peptides include ferrocene as an electrochemical reporter, sulfur-containing amino acids (l-methionine, S-methyl-l-cysteine, S-trityl-l-cysteine, S-benzhydryl-l-cysteine) as metal binding agents, and amino acids with non-polar side chains (l-alanine, l-valine, l-phenylalanine) as spacers between reporter and metal binding groups. Ferrocene/dipeptide conjugates were prepared using solution phase peptide synthesis methods employing a BOC-protecting group strategy and HBTU- (O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) mediated peptide coupling. The electrochemical properties of these 1,1′-substituted ferrocenoyl peptides have been characterized using cyclic voltammetry. All exhibit fully reversible one electron oxidation steps; forward sweep half wave peaks (E_F), reverse sweep half wave peaks (E_R), peak separations (ΔE_P) and half wave potentials (E_1/2) are reported. Finally, towards the goal of utilizing ferrocenoyl peptides to detect heavy metals in solution, the response of these ferrocene/dipeptide conjugates to metal cations (zinc(II), mercury(II), cadmium(II), lead(II), silver(I)) has been examined. Monitoring changes in the potential of the Fe(II)/Fe(III) redox couple to follow peptide/metal interactions, we have probed the influence of the spacer unit between the redox reporter and the metal-binding amino acid, and shown that these systems respond to mercury(II) more strongly than to other heavy metal ions.     

Facile solid phase synthesis of N-cycloguanidinyl-formyl peptides

A novel strategy of solid phase synthesis of N-cycloguanidinyl-formyl peptides has been established and investigated which involved coupling orthogonal protected diaminoacid with resin bound peptide, α-amino group deprotection, guanidinylation of α-amino group by bis-Cbz-1H-pyrazole-1-carboxamidine followed by cleavage and cyclization in solution, and finally removing Cbz by palladium catalyzed hydrogenation. Through this method, cycloguanidine could be introduced to either N-terminus or sidechain of designated peptides. The reaction conditions were facile, straightforward, and totally adaptive to common solid phase peptide synthesis strategy.     

Pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction

In one of the peptide condensation methods termed thioester method, an amino protecting group is required in the lysine side chain. In this study, to investigate the efficiency of the pyruvoyl group as an amino protecting group, we synthesized N^α-fluorenylmethoxycarbonyl (Fmoc)-N^ε-pyruvoyl-lysine and introduced it into peptides and glycopeptides by the ordinary Fmoc-based solid phase peptide synthesis. The pyruvoyl peptide could be condensed with a peptide thioester by the thioester method, and this protecting group was easily removed by o-phenylenediamine treatment without significant side reactions.     

Investigation for the cyclization efficiency of linear tetrapeptides: Synthesis of tentoxin B and dihydrotentoxin

Investigation of the cyclization efficiency of N-methyl linear tetrapeptides using a molecular modeling study and chemical synthesis is described. The linear peptide with two N-methyl groups, MeAla-Leu-MePhe-Gly, forms γ-turn like conformation with the amine at N-terminus and the carbonyl at C-terminus in closer proximity to give the desired cyclic tetrapeptide, dihydrotentoxin. In addition, synthesis of tentoxin B by the combination of Fmoc solid-phase peptide synthesis and cyclization in solution phase has been reported. An unusual amino acid, an L-N-methyl-β-hydroxyphenylalanine derivative, which was assembled on solid support, was prepared from ethyl cinnamate. Cyclic tetrapeptide formation and cleavage of benzyl ether were optimized with DIPCI/HOBt/DIPEA and Et₃SiH/Pd(OH)₂, respectively.     

Fmoc-based solid-phase synthesis of adenylylated peptides using diester-type adenylylated amino acid derivatives

Phosphodiester-type adenylylated (AMPylated) Ser, Thr, and Tyr derivatives were developed for Fmoc solid phase peptide synthesis of AMPylated peptides. One-pot/sequential reaction consisting of condensation of an N-nonprotected adenosine derivative and Fmoc-Ser/Thr/Tyr-OAllyl using allyl-N,N-diisopropylchlorophosphoramidite and subsequent oxidation with m-chloroperbenzoic acid gave phosphotriester-type AMPylated Ser/Thr/Tyr derivatives. After Pd(0)-mediated deprotection of allyl groups, the resulting phosphodiester-type AMPylated Ser/Thr/Tyr derivatives were successfully incorporated into peptides by standard Fmoc solid phase peptide synthesis without significant side reactions including dehydroalanine formation.     

Metal labeling for accurate multiplexed peptide quantification via matrix-assisted laser desorption/ionization mass spectrometry

Two peptide quantification strategies, the isobaric tags for relative or absolute quantitation (iTRAQ) labeling methodology and a metal-chelate labeling approach, were compared using matrix-assisted laser desorption/ionization-TOF/TOF MS and MS/MS analysis. Amino- and cysteine-directed labeling using the rare earth metal chelator 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) were applied for relative quantification of single peptides and a six-protein mixture. For analyte ratios close to one, iTRAQ and amino-directed DOTA labeling delivered overall comparable results regarding accuracy and reproducibility. In contrast, the MS-based quantification via amino-directed lanthanide-DOTA tags was more accurate for analyte ratios ≥5 and offered an extended dynamic range of three orders of magnitude. Our results show that the amino-directed DOTA labeling is an alternative relative quantification tool offering advantages like flexible multiplexing possibilities and, in particular, large dynamic ranges, which should be useful in sophisticated, targeted issues, where the accurate determination of extremely different protein or peptide concentration becomes relevant.     

N,N,N‐Trimethyl‐5‐[(2,3,5,6‐tetrafluorophenoxy)carbonyl]pyridin‐2‐aminium trifluoromethanesulfonate a precursor for the synthesis of 2,3,5,6‐tetrafluorophenyl 6‐[18F]‐fluoronicotinate

The synthesis, recrystallization, and X‐ray deterimination of N,N,N‐trimethyl‐5‐[(2,3,5,6‐tetrafluorophenoxy)carbonyl]pyridin‐2‐aminium trifluoromethanesulfonate (PyTFP‐precursor), C₁₅H₁₃F₄N₂O₂⁺·CF₃SO₃⁻, is described. This triflate salt precursor is required for the synthesis of 2,3,5,6‐tetrafluorophenyl 6‐[¹⁸F]‐fluoronicotinate ([¹⁸F]FPyTFP), a prosthetic group used to radiolabel peptides for positron emission tomography (PET), as peptides are increasingly being used as PET‐imaging probes in nuclear medicine. Radiolabeling of peptides is typically done using a `prosthetic group', a small synthon to which the radioisotope is attached in the first step, followed by attachment to the peptide in the second step. During the synthesis of the PyTFP‐precursor, displacement of a Cl atom with trimethylamine gas and anion replacement with a triflate counter‐ion is critical, as incomplete replacement would hinder radioisotopic incorporation of nucleophilic fluorine‐18 and result in diminished radiochemical yields. The structural determination of the PyTFP‐precursor by X‐ray crystallography helped confirm the anion exchange of chloride with triflate.     

Fragmentation of protonated thioether conjugates of acrolein using low collision energies

The protonated mercapturic acid conjugate of acrolein, S-(3-oxopropyl)-N-acetyl-L-cysteine (I), undergoes facile retro-Michael loss of acrolein in the gas phase. To determine whether extensive loss of acrolein would impede structural characterization of acrolein-peptide adducts, fragmentation reactions of a series of conjugates, formed by 1,4-Michael addition of acrolein to peptides and cysteine derivatives, were investigated at collision cell potentials up to ?50 V using a triple quadrupole mass spectrometer. Differences in fragmentation dynamics suggest protonation at the sulfur of the N-acetylcysteine conjugate I facilitates retro-Michael elimination of acrolein with a low activation energy relative to other fragmentations. Analogous fragmentation was eliminated after borohydride reduction of the aldehyde to an alcohol. Retro-Michael fragmentation was not significant for acrolein conjugates of glutathione derivatives, suggesting that proton sequestration occurs in peptides with multiple amide linkages even when the peptide does not contain a basic amino group. An unexpected outcome of these experiments was the observation of a facile gas-phase cleavage of peptides on the N-terminal side of S-(3-oxopropyl)cysteine residues. Such fragmentation behavior may prove useful for locating cysteine residues in peptides.     

First total synthesis of versicotide D and analogs

The first total synthesis of cyclotetrapeptide versicotide D has been achieved in 21% overall yield using solid phase peptide synthesis and solution cyclization. In addition, in the search for candidates of antimalarial new drugs, one cyclic tetrapeptide analog which differs in the sequence, and four cyclic pentapeptide containing N-methyl amino acids, were prepared. The obtained compounds were evaluated against P. falciparum 3D7. Versicotide D showed low micromolar antiplasmodial activity.     

Native peptide folding dominates over stereoelectronic effects of prolyl hydroxylation in loop 5 of the macrocyclic peptide kalata B1

Kalata B1 (4) is a prototypical, 29-residue, Möbius cyclotide with a cis prolyl peptide bond in loop 5. Two analogs were synthesized in which Pro²⁴ was substituted by trans-4-hydroxy-l-proline (peptide 5) and cis-4-hydroxy-l-proline (peptide 6). Linear peptides were assembled by solid phase peptide synthesis using Fmoc/tBu chemistry. Head-to-tail cyclization was performed using HATU, side-chain protecting groups removed and the cyclic peptides 2 and 3 isolated by RP-HPLC. Oxidation led to the formation of peptides 5 and 6, each incorporating three disulfide bonds. Analysis of TOCSY and NOESY spectra of the purified peptides enabled assignment of the backbone amide and Hα resonances. These showed a striking correlation with those of native kalata B1, indicating that folding had produced the same disulfide bridge topology. While somewhat surprising that stereoelectronic effects introduced by the hydroxyl substituents in this key region of the peptide had little impact, this reflects the strong thermodynamic driving force toward formation of the cyclic cystine knot scaffold.     

Solid-Phase Total Synthesis of Cyclosporine Analogues

Syntheses of cyclosporine analogues are reported wherein the peptide couplings were achieved in solid phase. The Wang resin was used as the solid support, and the peptide couplings commenced with the residue 11 of the cyclosporine skeleton. The couplings proceeded in a stepwide manner up to the residue MeBmt¹, using symmetric anhydrides. The peptides were then cleaved off the resin, and the cyclization was achieved in solution using Castro's reagent. The solid-phase synthesis described herein offers a very efficient method for the rapid synthesis of structurally diverse cyclosporine analogues in small quantities. The biological activities of the synthetic cyclosporine analogues were evaluated in two in vitro assays, including the IL-2 reporter gene assay and the cyclophilin binding assay. The structure-activity relationship is discussed.     

Synthesis of cyclic peptides via O-N-acyl migration

We describe here a novel and convenient synthesis of head-to-tail cyclic peptide avoiding racemization. Linear depsipeptides including a serine residue as the key element for ester bond formation and acyl transfer were synthesized on 2-chlorotrityl chloride resin. After cleavage from the resin, intramolecular head-to-tail cyclization was performed in solution by C-terminal activation of urethane protected O-acyl serine residue. After removal of the N^α-serine protecting group, the final step consisted in O-N-acyl migration reaction on the ‘switch’ or ‘click’ element to restore native cyclic peptides.     

Facile Synthesis of Internal and C‐Terminal Peptide α‐Ketoamides with Fmoc‐Solid Phase Peptide Synthesis

We report a general and operationally simple method for the solid phase synthesis of α‐ketoamide peptides using standard Fmoc solid phase peptide synthesis. The method delivers deprotected peptide α‐ketoamides directly upon resin cleavage without any additional steps, and tolerates all side chain functional groups. A small collection of C‐terminal and internal α‐ketoamide peptides – including two reported protease inhibitors (HCV and SUB1) – were prepared in good yields. In addition, we demonstrate that our method serves as versatile platform for the convenient preparation of cyclic α‐ketoamide peptides, photocagged peptide α‐ketoamides, and fluorescently labeled peptides.     

Understanding lipid recognition by protein-mimicking cyclic peptides

This paper describes our investigation of the structural determinants of a designed cyclic peptide (cLac, cyclic peptide mimicking lactadherin) (Zheng, H.; Wang, F.; Wang, Q.; Gao, J. J. Am. Chem. Soc.2011, 133, 15280–15283) for phosphatidylserine (PS) recognition. A highly efficient strategy that takes advantage of the native chemical ligation (NCL) chemistry has been developed for the synthesis and labeling of cyclic peptides in general. Ala scanning of the cLac peptide revealed a sophisticated model for PS binding, in which the peptide scaffold assembles multiple polar residues to balance the desolvation and electrostatic interactions (salt bridge and hydrogen bonding) to achieve lipid selectivity. The results suggest that cLac effectively mimics the membrane binding mechanism of the parent protein lactadherin.     

Modelling of prebiotic synthesis and selection of peptides under isothermal conditions and thermal cycling mode

The model peptide synthesis from mixtures of amino acids was carried out under the thermal cycling and isothermal modes. The compositions of the obtained mixtures of products and the primary amino acid sequence of the synthesized peptides were determined by Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry in combination with high-performance liquid chromatography with the application of de novo sequencing of the synthesized products. The processes of abiogenous synthesis of peptides were shown to occur under relatively mild temperature conditions and give a substantially less number of peptides as compared with the possible statistical set. The evolution of the system takes place in the process of the synthesis in solid phase with the disappearance of a series of the most unstable peptides. The selection process with the formation of complementary peptides takes place in peptide synthesis under the thermal cyclic mode.     

Introduction of the Aib-Pro unit into peptides by means of the ‘azirine/oxazolone method’ on solid phase

A method for the direct introduction of Aib-Pro into peptides on solid phase was developed. The Aib-Pro unit was introduced by means of the ‘azirine/oxazolone method’ using allyl N-(2,2-dimethyl-2H-azirin-3-yl)-l-prolinate as the synthon. After the reaction of the resin-bound amino or peptide acid with allyl N-(2,2-dimethyl-2H-azirin-3-yl)-l-prolinate, the allyl protecting group of the resulting extended peptide could be removed by a mild Pd⁰-promoted procedure. Cleavage of the peptide from the resin was performed with UV light at 352 nm and yielded C-terminal protected peptides. The method found a successful application in the syntheses of different Aib-Pro containing peptaibol segments. Furthermore, a protected derivative of the peptide antibiotic Trichovirin I 1B was prepared by segment condensation.     

Syntheses and lipophilicity measurement of N/N-terminus-1,1-dihydroperfluoroalkylated α-amino acids and small peptides

(1,1-Dihydroperfluoroalkyl)phenyliodonium N,N-bis(trifluoromethylsulfonyl)imides (4, n = 0-2) were synthesized and used to transfer the corresponding 1,1-dihydroperfluoroalkyl groups to the α-amino group of (l)tyrosine. The obtained N^α-2,2,2-trifluoroethylated (l)tyrosine (6, n = 0) was further used as the N-terminus in the solid phase peptide synthesis of leucine enkephalin analogue. The lipophilicity of the N^α-1,1-dihydroperfluoroalkylated (l)tyrosines (6, n = 0-2) and N-terminus-2,2,2-trifluoroethylated leucine enkephalin analogue (7), as well as the corresponding parent compounds, was measured.