Facile synthesis of polymer–peptide conjugates via direct amino acid coupling chemistry

Polymer–peptide conjugates (also known as biohybrids) are attracting considerable attention as injectable materials owing to the self‐assembling behavior of the peptide and the ability to control the material properties using the polymer component. To this end, a simple method for preparing poly(ethylene oxide)‐oligophenylalanine polymer–peptide conjugates (mPEO_m‐F_n‐OEt) using isobutylchloroformate as the activating reagent has been identified and developed. The synthetic approach reported employs an industrially viable route to produce conjugates with high yield and purity. Moreover, the approach allows judicious selection of the precursor building blocks to produce libraries of polymer–peptide conjugates with complete control over the molecular composition. Control over the molecular make‐up of the conjugates allows fine control of the physicochemical properties, which will be exploited in future studies into the prominent self‐assembling behavior of such materials. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 4853–4859     

Single‐Crystal to Single‐Crystal Linker Substitution,Linker Place Exchange,and Transmetalation Reactions in Interpenetrated Pillared–Bilayer Zinc(II) Metal–Organic Frameworks

A twofold interpenetrated pillared–bilayer framework, {[Zn₃( L )₂( L₂ )(DMF)] ? (18DMF)(6H₂O)}_n ( 1 ), has been synthesized from the ligands tris(4′‐carboxybiphenyl)amine ( H₃L ) and 1,2‐bis(4‐pyridyl)ethylene ( L₂ ). The structure contains [Zn₃(COO)₆] secondary building units (SBUs), in which three Zn^II ions are almost linear with carboxylate bridging. This framework undergoes reversible pillar linker substitution reactions at the terminal Zn^II centers with three different dipyridyl linkers of different lengths to afford three daughter frameworks, 2 – 4 . Frameworks 2 – 4 are interconvertible through reversible linker substitution reactions. Also, competitive linker‐exchange experiments show preferential incorporation of linker L₃ in the parent framework 1 . The larger linker L₅ does not undergo such substitution reactions and framework 5 , which contains this linker, can be synthesized solvothermally as a twofold interpenetrated structure. Interestingly, when framework 5 is dipped in a solution of L₃ in DMF, linker substitution takes place as before, but linker L₅ now moves and diagonally binds two Zn^II centers to afford 6 as a nonpenetrated single framework. This linker place exchange reaction is unprecedented. All of these reactions take place in a single‐crystal to single‐crystal (SC‐SC) manner, and have been observed directly through X‐ray crystallography. In addition, each 3D framework undergoes complete copper(II) transmetalation.     

Improved Methodology for the Synthesis of a Cathepsin B Cleavable Dipeptide Linker,Widely Used in Antibody-Drug Conjugate Research

Antibody-drug conjugates (ADCs) represent an emerging class of biopharmaceutical agents that deliver highly potent anticancer agents (payloads) selectively to tumors or components associated with the tumor microenvironment. The linker, responsible for the connection between the antibody and payload, is a crucial component of ADCs. In certain examples the linker is composed of a cleavable short peptide which imparts an additional aspect of selectivity. Especially prevalent is the cathepsin B cleavable Mc-Val-Cit-PABOH linker utilized in many pre-clinical ADC candidates, as well as the FDA approved ADC ADCETRIS^® (brentuximab vedotin). An alternative route for the synthesis of the cathepsin B cleavable Mc-Val-Cit-PABOH linker is reported herein that involved six steps from l-Citrulline and proceeded with a 50% overall yield. In this modified route, the spacer (a para-aminobenzyl alcohol moiety) was incorporated via HATU coupling followed by dipeptide formation. Importantly, this route avoided undesirable epimerization and proceeded with improved overall yield. Utilizing this methodology, a drug-linker construct incorporating a potent small-molecule inhibitor of tubulin polymerization (referred to as KGP05), was synthesized as a representative example.     

205Tl NMR studies: Ion pairing of Tl+ and (CH3)2Tl+ with NO 3 − and ClO 4 − in various solvents

The concentration dependence of the²⁰⁵Tl chemical shifts of Tl⁺ and of (CH₃)₂Tl⁺ ions was determined in several solvents with NO ₃ ^– and ClO ₄ ^– counterions. In general, increased ion pairing caused a low-frequency shift of the²⁰⁵Tl resonance, with the exceptions of (CH₃)₂TlNO₃ inn-butylamine and TlNO₃ in N,N-dimethylformamide (DMF) and in hexamethylphosphorotriamide (HMPA). In HMPA,²⁰⁵Tl linewidths of both Tl⁺ and (CH₃)₂Tl⁺ increased dramatically with dilution below 0.1M. Analysis of the data allowed ion-pair formation constants and²⁰⁵Tl chemical shifts for the ion-paired cation and for the free (solvated) cation to be estimated for some of the solvents.     

Luminescence and magnetic properties of mixed-ligand europium trifluoroacetates

The luminescence and magnetic properties of mixed-ligand europium trifluoroacetates with nitrogen- and phosphorus-containing neutral ligands [Eu(TFA)₃ · 3H₂O]₂ and Eu(TFA)₃ · 2D · nH₂O were studied (here TFA is the trifluoroacetic acid anion, and D is Phen (1,10-phenanthroline), n = 1; DMF (dimethylformamide), n = 1; or TPPO (triphenylphosphine oxide), n = 3). The molar magnetic susceptibility increased in the series of complex compounds Eu(TFA)₃ · 2Phen · H₂O < [Eu(TFA)₃ · 3H₂O]₂ < Eu(TFA)₃ · 2DMF · H₂O < Eu(TFA)₃ · 2TPPO · 3H₂O. Correlations were found between the luminescent and magnetic characteristics of the complex compounds.     

Peptide‐poly(tert‐butyl methacrylate) conjugate into composite micelles in organic solvents versus peptide‐poly(methacrylic acid) conjugate into spherical and worm‐like micelles in water: Synthesis and self‐assembly

Peptide–polymer conjugates are versatile class of biomaterials composed of a peptide block covalently linked with a synthetic polymer block. This report demonstrates the synthesis of peptide‐poly(tert‐butyl methacrylate) (Peptide‐P^tBMA) conjugates of varying molecular weights via a “grafting from” atom transfer radical polymerization (ATRP) technique using as‐synthesized peptide‐based initiator in toluene. Peptide‐P^tBMA conjugate is soluble in many organic solvents and undergoes self‐assembly into micro/nanospheres in DMF/THF as observed from both FESEM and DLS results. The conjugate micro/nanospheres are nothing but the composite micelles formed by the secondary aggregation of primary micelles generated initially in these organic solvents. The hydrolysis of tert‐butyl groups of Peptide‐P^tBMA conjugate leads to the formation of peptide‐poly(methacrylic acid) (Peptide‐PMA) conjugate. The circular dichroism (CD) analysis exhibits the presence of β‐sheet conformation of peptide moiety in synthesized conjugates. The formed Peptide‐PMA conjugate is soluble in water and owing to its amphiphilic character, the conjugate molecules self‐assemble into spherical micelles as well as worm‐like micelles upon increasing the concentration of conjugate in water. However, the sodium salt of Peptide‐PMA conjugates (Peptide‐PMAS) self‐assembles into only spherical swollen micelles in water at higher (pH ～10). The critical aggregation concentrations (CACs) of both Peptide‐PMA and Peptide‐PMAS micelles are measured by fluorescence spectroscopy. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 3019–3031     

Synthetic dsDNA‐Binding Peptides Using Natural Compounds as Model

We have developed a series of short DNA‐binding peptides containing newly synthesized, unnatural as well as natural amino acid building blocks. By a combinatorial‐library approach, oligopeptides were developed with moderate dsDNA‐binding affinities. Two strategies were used to further enhance the binding affinity of the lead peptides: Ac‐Arg‐Ual‐Sar‐Chi‐Chi‐Chi‐Arg‐NH₂ and Ac‐Arg‐Cbg‐Cha‐Chi‐Chi‐Tal‐Arg‐NH₂. Site‐selective amino acid substitutions increased the binding affinities up to 2 × 10^?5 M . Further enhancement of the binding affinities could be achieved by coupling of an acridine intercalating unit, using linker arms of different length and flexibility. With the introduction of a new lysine‐based acridine unit, different types of oligopeptide–acridine conjugates were designed using known dsDNA‐binding ligands as model compounds. The binding capacities of these new oligopeptide–acridine conjugates have been investigated by a fluorescent intercalator (ethidium bromide) displacement (FID) assay. With the synthesis of the dipeptide–acridine conjugates, binding affinities in the low micromolar range were obtained (6.4 × 10^?6 M ), which is similar to the binding strength of the well‐known DNA binder Hoechst 33258.     

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.     

A New Base-Labile Anchoring Group for Polymer-Supported Peptide Synthesis

A new base-labile anchoring group, derived from 9-(hydroxymethyl)fluorene-4-carboxylic acid (HO₂CFmoH or HOFmCO₂H; 7), for polymer-supported peptide synthesis os described. The synthesis of 7 starting from 2,2′-biphenyldicarboxylic acid ( 1 ) proceeds in an overall yield of 53%. The group HO₂CFmo exhibits properties similar to the well known Fmoc protecting group: It is stable to acidic conditions and cleavable by 15% piperidine in DMF. In combination with acid labile N^α-protecting groups (e.g. Boc, Ddz, Bpoc, Nps etc.), it renders more flexibility to the stepwise synthesis using polymer supports. The versatility of the new anchoring group in solid- and liquid-phase peptide synthesis is demonstrated for the synthesis of a model peptide.     

Iron(III) Chloride Catalyzed Oxidative Coupling Reaction of 1,2‐Diarylethylene Derivatives

A nontoxic FeCl₃ catalyzed intramolecular oxidative coupling reaction was developed for mild synthesis of a series of phenanthrenes with different substituents. The method involves cross dehydrogenative coupling of a variety of 1,2‐diarylethylene derivatives with di‐tert‐butylperoxide (DTBP) as a sole oxidant at room temperature in CH₂Cl₂/TFA (9:1 V/V) to yield phenanthrenes in good to excellent yields.     

Identification of the Structural Determinants for Anticancer Activity of a Ruthenium Arene Peptide Conjugate

Organometallic Ru(arene)–peptide bioconjugates with potent in vitro anticancer activity are rare. We have prepared a conjugate of a Ru(arene) complex with the neuropeptide [Leu⁵]‐enkephalin. [Chlorido(η⁶‐p‐cymene)(5‐oxo‐κO‐2‐{(4‐[(N‐tyrosinyl‐glycinyl‐glycinyl‐phenylalanyl‐leucinyl‐NH₂)propanamido]‐1H‐1,2,3‐triazol‐1‐yl)methyl}‐4H‐pyronato‐κO)ruthenium(II)] ( 8 ) shows antiproliferative activity in human ovarian carcinoma cells with an IC₅₀ value as low as 13 μM , whereas the peptide or the Ru moiety alone are hardly cytotoxic. The conjugation strategy for linking the Ru(cym) (cym=η⁶‐p‐cymene) moiety to the peptide involved N‐terminal modification of an alkyne‐[Leu⁵]‐enkephalin with a 2‐(azidomethyl)‐5‐hydroxy‐4H‐pyran‐4‐one linker, using Cu^I‐catalyzed alkyne–azide cycloaddition (CuAAC), and subsequent metallation with the Ru(cym) moiety. The ruthenium‐bioconjugate was characterized by high resolution top‐down electrospray ionization mass spectrometry (ESI‐MS) with regard to peptide sequence, linker modification and metallation site. Notably, complete sequence coverage was obtained and the Ru(cym) moiety was confirmed to be coordinated to the pyronato linker. The ruthenium‐bioconjugate was analyzed with respect to cytotoxicity‐determining constituents, and through the bioconjugate models [{2‐(azidomethyl)‐5‐oxo‐κO‐4H‐pyronato‐κO}chloride (η⁶‐p‐cymene)ruthenium(II)] ( 5 ) and [chlorido(η⁶‐p‐cymene){5‐oxo‐κO‐2‐([(4‐(phenoxymethyl)‐1H‐1,2,3‐triazol‐1‐yl]methyl)‐4H‐pyronato‐κO}ruthenium(II)] ( 6 ) the Ru(cym) fragment with a triazole‐carrying pyronato ligand was identified as the minimal unit required to achieve in vitro anticancer activity.     

Synthesis of C-11 linked active ester derivatives of vitamin D3 and their conjugations to 42-residue helix-loop-helix peptides

Derivatives of vitamin D₃ carrying an 8-carbon linker at C-11 terminating in an active ester were synthesized from commercial vitamin D₃ using a disassembly-reassembly strategy. Vitamin D₃ was cleaved at the C6-C7 double bond and the ‘upper’ fragment was converted, via a series of reactions, to derivatives substituted at C-11 with an 8-carbon linker terminating in an ethyl ester. Reassembly with modified ‘lower’ fragments using Horner-Wittig olefination followed by linker ester hydrolysis and re-esterification with p-nitrophenol gave C-11 substituted p-nitrophenyl esters. These vitamin D derivatives were conjugated to 42-amino acid helix-loop-helix peptides by reaction of their p-nitrophenyl esters with lysyl side-chain amino groups on the peptides. The vitamin D—peptide conjugates, being potential specific binder candidates for vitamin D-binding protein, were characterized by mass spectroscopy and CD measurements.     

Synthesis and evaluation of 2-(2-fluoro-4-hydroxymethyl-5-methoxy-phenoxy)acetic acid as a linker in solid-phase synthesis monitored by gel-phase (19)F NMR spectroscopy

Gel-phase (19)F NMR spectroscopy is a useful monitoring technique for solid-phase organic chemistry due to the high information content it delivers and swift acquisition times, using standard NMR spectrometers. This paper describes the synthesis of the novel linker 2-(2-fluoro-4-hydroxymethyl-5-methoxy-phenoxy)acetic acid in 29% yield over seven steps, using nucleophilic aromatic substitutions on 2,4,5-trifluorobenzonitrile as key steps. Following standard solid-phase synthesis a peptide could be cleaved from the linker using 20% TFA in CH(2)Cl(2) in 30 minutes, in contrast to a previously described monoalkoxy linker that requires 90% TFA in water at elevated temperature. A resin-bound peptide could be successfully glycosylated using only two equivalents of a thioglycoside donor, activated with N-iodosuccinimide and trifluoromethanesulfonic acid, and subsequent cleavage and deprotection gave the target glycopeptide. Direct glycosylation of the linker itself followed by mild acidic cleavage gave a fully protected hemiacetal for further chemical manipulation.     

Synthesis and Biological Evaluation of RGD Peptidomimetic–Paclitaxel Conjugates Bearing Lysosomally Cleavable Linkers

Two small‐molecule–drug conjugates (SMDCs, 6 and 7 ) featuring lysosomally cleavable linkers (namely the Val–Ala and Phe–Lys peptide sequences) were synthesized by conjugation of the α_vβ₃‐integrin ligand cyclo[DKP–RGD]‐CH₂NH₂ ( 2 ) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP–RGD]–PTX conjugate with a nonpeptide “uncleavable” linker ( 8 ) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified α_Vβ₃‐integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8 , which possesses a nonpeptide “uncleavable” linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF‐CEM (α_Vβ₃?) and its subclone CCRF‐CEM α_Vβ₃ (α_Vβ₃+). Fairly effective integrin targeting was displayed by the cyclo[DKP–RGD]–Val–Ala–PTX conjugate ( 6 ), which was found to differentially inhibit proliferation in antigen‐positive CCRF‐CEM α_Vβ₃ versus antigen‐negative isogenic CCRF‐CEM cells. The total lack of activity displayed by the “uncleavable” cyclo[DKP–RGD]–PTX conjugate ( 8 ) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.     

Synthesis of a secretoglobin 3A2 type C (98–139) fragment by Dawson’s native chemical ligation

A secretoglobin 3A2 type C (98–139) peptide was synthesized by native chemical ligation between ¹¹⁵Ile and ¹¹⁶Cys residues using Dawson’s linker. The peptide-N-acyl-benzimidazolinone-glycine amide, a C-terminal thioesters precursor, was provided from 3-amino-4-(methylamino)benzoic acid. In addition, an N-terminal cysteine fragment, the (116–139) peptide, was prepared by ordinary Fmoc-solid phase peptide synthesis. Native chemical ligation of the (98–115) fragment with the Dawson’s linker and the (116–139) peptide smoothly proceeded to give SCGB3A2 type C (98–139) peptide.     

Synthesis,radiolabelling and biodistribution of HYNIC-Tyr<Superscript>3</Superscript> octreotide: a somatostatin receptor positive tumour imaging agent

In vivo imaging of tumours using radiolabelled somatostatin (SST) analogues has become an accepted clinical tool in oncology. HYNIC-Tyr³ octreotide and Tyr³ octreotide were synthesized by FMOC solid-phase peptide synthesis using a semi-automated synthesizer. These were analyzed and purified by RP-HPLC, mass spectroscopy, IR spectroscopy, ¹H NMR and ¹³C NMR. The prochelator 6-BOC-HYNIC was also synthesised and characterised indigenously. HYNIC-Tyr³ octreotide was labelled with ^99mTc using Tricine and EDDA as coligand by SnCl₂ method. Labelling with ^99mTc was performed at 100 °C for 15 min and radiochemical analysis by ITLC and HPLC methods. The radiochemical purity of the complex was over 98% and log p value was found to be −1.27 ± 0.12. The stability of radiolabelled peptide complex was checked at 37 °C up to 24 h. Blood clearance and protein-binding study was also performed. In vivo biodistribution studies in rat showed uptake of ^99mTc-HYNIC-TOC in kidney than any other organs. The blood clearance was faster with rapid excretion through kidneys and relatively low uptake in liver.     

Use of Dibutyl[14C]formamide as a Formylating Reagent in the Vilsmeier–Haack Reaction and Synthesis of a 14C‐Labeled Novel Phosphodiesterase‐4 (PDE‐4) Inhibitor

A simple, high‐yielding synthesis of dibutyl[¹⁴C]formamide ([¹⁴C]DBF; 1 ) from ¹⁴CO₂ was developed (Scheme 1): reaction of LiBEt₃H and ¹⁴CO₂ followed by aqueous workup gave H¹⁴CO₂H in high yield. Conversion of the [¹⁴C]formic acid to 1 was effected by a standard carbodiimide coupling procedure. The utility of 1 as an alternative to dimethyl[¹⁴C]formamide ([¹⁴C]DMF) in alkylation reactions and in the [¹⁴C]Vilsmeier–Haack reaction was demonstrated for several substrates (Table 2). A ¹⁴C‐labeled phosphodiesterase‐4 (PDE‐4) inhibitor, [¹⁴C]‐ 2 , was synthesized by application of this technology (Scheme 2).     

Huge dielectric response and molecular motions in paddle-wheel [Cu(II)2(adamantylcarboxylate)4(DMF)2]⋅(DMF)2

The temperature‐dependent dynamic properties of [Cu^II₂(ADCOO)₄(DMF)₂]?(DMF)₂ ( 1 ) and [Cu^II₂(ADCOO)₄(AcOEt)₂] ( 2 ) crystals were examined by X‐ray crystallography, ¹H NMR spectroscopy, and measurements of the dielectric constants and magnetic susceptibilities (ADCOO=adamantane carboxylate, DMF=N,N‐dimethylformamide, and AcOEt=ethyl acetate). In both crystals, four ADCOO groups bridged a binuclear Cu^II? Cu^II bond, forming a paddle‐wheel [Cu^II₂(ADCOO)₄] structure. The oxygen atoms of two DMF molecules in crystal 1 and two AcOEt molecules in crystal 2 were coordinated at axial positions of the [Cu^II₂(ADCOO)₄] moiety, forming [Cu^II₂(ADCOO)₄(DMF)₂] and [Cu^II₂(ADCOO)₄(AcOEt)₂], respectively. Two additional DMF molecules were included in the unit cell of crystal 1 , whereas AcOEt was not included in the unit cell of crystal 2 . The structural analyses of crystal 1 at 300 K showed three‐fold rotation of the adamantyl groups, whereas rotation of the adamantyl groups of crystal 2 at 300 K was not observed. Thermogravimetric measurements of crystal 1 indicated a gradual elimination of DMF upon increasing the temperature above 300 K. The dynamic behavior of the crystallized DMF yielded significant temperature‐dependent dielectric responses in crystal 1 , which showed a huge dielectric peak at 358 K in the heating process. In contrast, only small frequency‐dependent dielectric responses were observed in crystal 2 because of the freezing of the molecular rotation of the adamantyl groups. The magnetic behavior was dominated by the strong antiferromagnetic coupling between the two S=1/2 spins of the Cu^II? Cu^II site, with magnetic exchange energies (J) of ?265 K (crystal 1 ) and ?277 K (crystal 2 ).     

A New ‘Off–On’ System Based on Core-Substituted Naphthalene Diimide with Dimethylamine for Reversible Acid–Base Sensing

A new ‘Off–On’ system designed and synthesised by functionalisation of a naphthalene diimide (NDI) core with dimethylamine produces 4,9-bis(dimethylamino)-2,7-dioctylbenzo[lmn][3,8]-phenanthroline-1,3,6,8-(2H,7H)-tetraone, abbreviated as DDPT ( 1 ). DDPT 1 was synthesised using a simple strategy, namely aromatic nucleophilic substitution using Br₂-NDI with dimethylamine at 110 °C. DDPT was characterized by ¹H and ¹³C NMR spectroscopy, ESI mass spectrometry and elemental analysis. DDPT 1 was then used for optical studies through protonation of its dimethylamine core with trifluoroacetic acid (TFA), blue-shifting the absorption band from 600 nm to 545 nm in solution. Interestingly, the fluorescence of DDPT 1 is weak in solution with a quantum yield Φ=0.09, which is significantly enhanced to Φ=0.78 upon addition of TFA. The limit of detection (LOD) was determined to 2.77 nm . Furthermore, DDPT 1 can be used for naked eyed detection not only under UV light (365 nm) but also using visible light, as clear changes can be clearly seen upon addition of TFA. The binding constant of DDPT was calculated to 2.1×10⁻³ m ⁻¹. Importantly, DDPT 1 showed reversible switching by alternative addition of acid (TFA) and base (triethylamine) without loss of activity. Immobilised on paper, DDPT 1 can be used for strip-test sensing in which the colour changes from blue to reddish when expose to TFA vapours and reverse in the presence of triethylamine vapours.