Investigation of c ions formed by N‐terminally charged peptides upon collision‐induced dissociation

Peptide fragments such as b and y sequence ions generated upon low‐energy collision‐induced dissociation have been routinely used for tandem mass spectrometry (MS/MS)‐based peptide/protein identification. The underlying formation mechanisms have been studied extensively and described within the literature. As a result, the ‘mobile proton model’ and ‘pathways in competition model’ have been built to interpret a majority of peptide fragmentation behavior. However, unusual peptide fragments which involve unfamiliar fragmentation pathways or various rearrangement reactions occasionally appear in MS/MS spectra, resulting in confused MS/MS interpretations. In this work, a series of unfamiliar c ions are detected in MS/MS spectra of the model peptides having an N‐terminal Arg or deuterohemin group upon low‐energy collision‐induced dissociation process. Both the protonated Arg and deuterohemin group play an important role in retention of a positive charge at the N‐terminus that is remote from the cleavage sites. According to previous reports and our studies involving amino acid substitutions and hydrogen–deuterium exchange, we propose a McLafferty‐type rearrangement via charge‐remote fragmentation as the potential mechanism to explain the formation of c ions from precursor peptide ions or unconventional b ions. Density functional theory calculations are also employed in order to elucidate the proposed fragmentation mechanisms. Copyright © 2016 John Wiley & Sons, Ltd.     

Novel N‐(2,2‐Dimethyl‐2H‐azirin‐3‐yl)‐L‐prolinates as Aib‐Pro Synthons

The syntheses of phenacyl N‐(2,2‐dimethyl‐2H‐azirin‐3‐yl)‐L ‐prolinate and allyl N‐(2,2‐dimethyl‐2H‐azirin‐3‐yl)‐L ‐prolinate are reported. Reactions of these 2H‐azirin‐3‐amine derivatives with Z‐protected amino acids have shown them to be suitable synthons for the Aib‐Pro unit in peptide synthesis. After incorporation into the peptide by means of the ‘azirine/oxazolone method’, the C‐termini of the resulting peptides were deprotected selectively with Zn in AcOH or by a mild Pd⁰‐promoted procedure, respectively.     

Syntheses and CD‐Spectroscopic Investigations of Longer‐Chain β‐Peptides: Preparation by Solid‐Phase Couplings of Single Amino Acids,Dipeptides, and Tripeptides

The synthesis and CD‐spectroscopic analysis of eleven water‐soluble β‐peptides composed of all‐β³ or alternating β²‐ and β³‐amino acids is described. Different approaches for the efficient syntheses of longer‐chain β‐peptides (>9 residues) were investigated. They were synthesized on solid phase with Fmoc‐protected amino acids or Fmoc‐protected di‐ or tripeptide fragments (assembled using solution‐phase synthesis). The use of preformed fragments significantly increased the purity of the crude peptides and facilitated purification. Especially, the use of Fmoc‐protected β²/β³‐dipeptides for the synthesis of a ‘mixed' β²/β³‐nonapeptide proved to be remarkably effective, yielding the crude peptide in 95% purity and without detectable epimerization of the β²‐amino acid residues. This is a significant improvement over previously reported procedures for the solid‐phase synthesis of β‐peptides, and foreshadows that the field of β‐peptide research will now switch from synthesis to the design and study of complex functional ‘β‐proteins'.     

3,5‐Dihydro‐2H‐thieno[2,3‐c]pyrrole 1,1‐Dioxide – A New Simple Pyrrole Unit. Preliminary Communication

2,3‐Dihydrothiophene 1,1‐dioxide (‘2‐sulfolene’) reacted with tosylmethyl isocyanide (TsMIC) in the presence of a base to give the hitherto unknown 3,5‐dihydro‐2H‐thieno[2,3‐c]pyrrole 1,1‐dioxide (‘β′‐sulfolenopyrrole’) from the expected cyclocondensation. A serendipitous formation of this β′‐sulfolenopyrrole was found earlier, when we investigated synthetic routes to a 3,5‐dihydro‐1H‐thieno[3,4‐c]pyrrole 2,2‐dioxide (a ‘β″‐sulfolenopyrrole’) from TsMIC and 2,5‐dihydrothiophene 1,1‐dioxide (‘3‐sulfolene’). Here, we present the synthesis and characterization of β′‐sulfolenopyrrole. The X‐ray crystal‐structure analyses of β′‐sulfolenopyrrole and the isomeric β″‐sulfolenopyrrole are also reported here. This β′‐sulfolenopyrrole is a new type of a functionalized pyrrole, which is likely to be of interest for pharmaceutical purposes.     

Divergent Solid‐Phase Synthesis of Natural Product‐Inspired Bipartite Cyclodepsipeptides: Total Synthesis of Seragamide A

Macrocyclic natural products (NPs) and analogues thereof often show high affinity, selectivity, and metabolic stability, and methods for the synthesis of NP‐like macrocycle collections are of major current interest. We report an efficient solid‐phase/cyclorelease method for the synthesis of a collection of macrocyclic depsipeptides with bipartite peptide/polyketide structure inspired by the very potent F‐actin stabilizing depsipeptides of the jasplakinolide/geodiamolide class. The method includes the assembly of an acyclic precursor chain on a polymeric carrier, terminated by olefins that constitute complementary fragments of the polyketide section and cyclization by means of a relay‐ring‐closing metathesis (RRCM). The method was validated in the first total synthesis of the actin‐stabilizing cyclodepsipeptide seragamide A and the synthesis of a collection of structurally diverse bipartite depsipeptides.     

Heterospirocyclic N‐(2H‐Azirin‐3‐yl)‐L‐prolinates: New Dipeptide Synthons

The synthesis of methyl N‐(1‐aza‐6‐oxaspiro[2.5]oct‐1‐en‐2‐yl)‐L ‐prolinate ( 1e ) has been performed by consecutive treatment of methyl N‐[(tetrahydro‐2H‐pyran‐4‐yl)thiocarbonyl]‐L ‐prolinate ( 5 ) with COCl₂, 1,4‐diazabicyclo[2.2.2]octane (DABCO), and NaN₃ (Scheme 1). As the first example of a novel class of dipeptide synthons, 1e has been shown to undergo the expected reactions with carboxylic acids and thioacids (Scheme 2). The successful preparation of the nonapeptide 16 , which is an analogue of the C‐terminal nonapeptide of the antibiotic Trichovirin I 1B, proved that 1e can be used in peptide synthesis as a dipeptide building block (Scheme 3). The structure of 7 has been established by X‐ray crystal‐structure analysis (Figs. 1 and 2).     

The N‐Terminal Nonapeptide of Cephaibols A and C: A Naturally Occurring Example of Mismatched Helical Screw‐Sense Control

The N‐terminal nonapeptide domain of the fungal nonribosomal peptide antibiotics cephaibol A and cephaibol C (AcPheAib₄LeuIvaGly‐ Aib) is reported to adopt a right‐handed helical conformation in the crystalline state. However, this conformation is at odds with the left‐handed helicity observed in solution in related synthetic oligomers capped with Ac‐L ‐PheAib₄ fragments. We report the synthesis of four diastereoisomers of the cephaibol N‐terminal nonapeptide, and show by NMR and CD spectroscopy that the peptide containing the chiral amino acids Phe and Leu in the naturally occurring relative configuration exists in solution as an interconverting mixture of helical screw‐sense conformers. In contrast, the nonapeptide containing the unnatural relative configuration at Phe and Leu adopts a single, stable helical screw‐sense, which is left handed when the N‐terminal Phe residue is L and right‐handed when the N‐terminal Phe residue is D .     

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.     

Synthesis of (S)‐3‐heteroaryl‐2‐hydroxy‐l‐propyl benzoates by ‘ring switching’ methodology

(S)‐5‐Benzoyloxymethyl‐3‐[(E)‐(dimethylamino)methylidene]tetrahydrofuran‐2‐one (6), prepared in 5 steps from L‐glutamic acid (1), was used as precursor in a one step ‘ring switching’ synthesis of (S)‐2‐hydroxy‐3‐heteroaryl‐l‐propyl benzoates 13‐18, 23, 24. In the reaction of 6 with 2‐aminopyridine (21) and 2‐amino‐4,6‐dimethylpyrimidine (22) the corresponding dimethylamine substitution products (25, 26) were obtained.     

Synthesis and Transformation of Methyl 2‐(6‐Hydroxy‐2‐phenylpyrimidin‐4‐yl)acetate: Simple Preparation of Pyrimidines with Heterocyclic Substituents

A simple and efficient synthesis of four new substituted pyrimidines, compounds 9a – d , from the title compound 3 is described. Conversion of 3 to methyl (E)‐3‐(dimethylamino)‐2‐(6‐methoxy‐2‐phenylpyrimidin‐4‐yl)prop‐2‐enoate ( 4 ), followed by condensation with various dinucleophiles according to the ‘enaminone methodology’, afforded the target compounds 9 in medium‐to‐good yields.     

The Aminopropenal‐ and Aminopentadienal Rearrangement

Scope and mechanism of the aminopropenal rearrangement are reviewed: Various 3‐acyloxy‐3‐dialkylaminopropenals ( 2 , formed by addition of acids to ‘push‐pull’‐acetylenes 1 ) rearranged quantitatively to give 3‐acyloxyacrylic amides ( 3 , Schemes 1 and 22). Since these activated enol esters reacted very selectively with amino groups of polyfunctional amino acids, ‘push‐pull’‐acetylenes are versatile peptide reagents. Similarly, 5‐X‐5‐dialkylaminopentadienals ( 38 , formed by addition of acids to ‘push‐pull’‐enynes 37 ) could be rearranged (aminopentadienal rearrangement). In this case, the rearrangement 38 → 40 → 42 (Schemes 16 and 22) normally stopped at the level of the quite stable 2‐dialkylamino‐pyrylium salts 40 . Ring opening 40 → 42 of these intermediates was quite tricky, but could be realized in several cases.     

Solid‐phase Total Synthesis of (−)‐Apratoxin A and Its Analogues and Their Biological Evaluation

Two approaches for the solid‐phase total synthesis of apratoxin A and its derivatives were accomplished. In synthetic route A, the peptide was prepared by the sequential coupling of the corresponding amino acids on trityl chloride SynPhase Lanterns. After cleavage from the polymer‐support, macrolactamization of 10 , followed by thiazoline formation, provided apratoxin A. This approach, however, resulted in low yield because the chemoselectivity was not sufficient for the formation of the thiazoline ring though its analogue 33 was obtained. However, in synthetic route B, a cyclization precursor was prepared by solid‐phase peptide synthesis by using amino acids 13 – 15 and 18 . The final macrolactamization was performed in solution to provide apratoxin A in high overall yield. This method was then successfully applied to the synthesis of apratoxin analogues. The cytotoxic activity of the synthetic derivatives was then evaluated. The epimer 34 was as potent as apratoxin A, and O‐methyl tyrosine can be replaced by 7‐azidoheptyl tyrosine without loss of activity. The 1,3‐dipolar cycloaddition of 38 with phenylacetylene was performed in the presence of a copper catalyst without affecting the thiazoline ring.     

Synthesis of Large‐Pore ECNU‐19 Material (12 × 8‐R) via Interlayer‐Expansion of HUS‐2 Lamellar Silicate

A large‐pore ECNU‐19 material with unique pore system consisting of 12‐ring (12R) pore channels intersected by 8R channels was post‐synthesized via interlayer‐expansion of HUS‐2 lamellar silicate with silylating agent of 1,3‐dimethyltetramethoxydisiloxane (DMTMDS). In consideration of the fact that the HUS‐2 precursor possessed a special structure with a malposition of the neighboring layers as well as silicon vacancies on layer surface, a “detemplating disassembly – intercalation reassembly – silylation” strategy was proposed to realize a successful interlayer‐expansion and structural amending. An acid treatment was firstly performed to remove a part of the structure‐directing agent molecules, which favored the following intercalation by bulk organic species. The intercalation not only rearranged the relative position of up‐down layers but also provided enough interlayer space for the insertion of dimeric silane molecules. Two –OH groups attached to one silicon atom of the silane molecule reacted with two close silanols on the up‐surface layer, while the other two –OH groups condensed with two silanols on the down‐surface layer, which then connected the two layers via ‐Si‐O‐Si‐ pillars and constructed new 12R pores along a axis and 8R pores along c axis, respectively.     

Analytical Applications of 2,6‐Diacetylpyridine‐bis‐4‐phenyl‐3‐thiosemicarbazone (2,6‐DAPBPTSC): Determination of Cd(II) in Foods and Water Samples

To the determination of trace amount of Cd(II) present in food and water samples, a selective and extractive spectrophotometric method were developed with 2,6‐diacetylpyridine‐bis‐4‐phenyl‐3‐thiosemicarbazone as a complexing agent. The yellowish orange colored metal complex, Cd(II)‐2,6‐DAPBPTSC with 1:1 (M:L) composition was extracted in to cyclohexanol at pH 9.5 and was shows maximum absorbance at λ_max 390 nm. This method obeys Beer's law in the range of 1.12‐11.25 ppm with 0.972 correlation coefficient of Cd(II)‐2,6‐DAPBPTSC complex, which is indicates linearity between the two variables. The molar absorptivity and sandell's sensitivity were found to be 6.088 × 10⁴ L mol^?1 cm^?1 and 0.0018 μg cm^?2, respectively. The instability constant calculated from Asmus' method (1.447 × 10^?4)at room temperature. The precision and accuracy of the method were checked by relative standard deviation (n = 5), 0.929 and its detection limit, 0.0060 μg mL^?1. The interfering effects of various cations and anions were also studied. The proposed method was successfully applied to the determination of Cd(II) in foods and water samples, and was evaluated its performance in terms of Student ‘t’ test and Variance ‘f’ test, which indicates the significance of present method. The inter comparison of the experimental values, using atomic absorption spectrometer (AAS), was also repoted.     

Synthesis and Cytotoxic Evaluation of Novel 1,2,3‐Triazole‐4‐Linked (2E,6E)‐2‐Benzylidene‐6‐(4‐nitrobenzylidene)cyclohexanones

This work describes the synthesis of novel 1,2,3‐triazole‐4‐linked (2E,6E)‐2‐benzylidene‐6‐(4‐nitrobenzylidene)cyclo‐hexanones starting from cyclohexanone. 1‐(Cyclohex‐1‐en‐1‐yl)piperidine, the enamine from cyclohexanone and piperidine, reacted with 4‐nitrobenzaldehyde to obtain 2‐(4‐nitrobenzylidene)cyclohexanone. Condensation of the latter compound with (prop‐2‐yn‐1‐yloxy)benzaldehyde derivatives under acidic conditions gave (4‐nitrobenzylidene)‐[(prop‐2‐yn‐1‐yloxy)‐benzylidene]cyclohexanones. Finally, ‘click reaction’ of these derivatives and various organic azides led to the title compounds. All compounds were examined by MTT assay for cytotoxic activity in one human breast cancer cell line, MDA‐MB‐231.     

Tetrakis(bicyclo[2.2.2]oct‐2‐ene)‐Fused Calix[4]pyrrole

Tetrakis(bicyclo[2.2.2]oct‐2‐ene)‐fused calix[4]pyrrole, 5 , was obtained starting from (E)‐1,2‐bis(phenylsulfonyl)ethylene. This new calixpyrrole derivative is the prospective precursor of tetrabenzocalix[4]pyrrole, a potential ion‐pair receptor and an attractive species as a possible deep‐walled ‘molecular container’.     

Construction of a High‐Mannose‐Type Glycan Library by a Renewed Top‐Down Chemo‐Enzymatic Approach

A comprehensive method for the construction of a high‐mannose‐type glycan library by systematic chemo‐enzymatic trimming of a single Man9‐based precursor was developed. It consists of the chemical synthesis of a non‐natural tridecasaccharide precursor, the orthogonal demasking of the non‐reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high‐mannose‐type glycans in their mono‐ or non‐glucosylated forms. It employed glucose, isopropylidene, and N‐acetylglucosamine groups for blocking the A‐, B‐, and C‐arms, respectively. After systematic trimming of the precursor, thirty‐seven high‐mannose‐type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N‐acetylglucosaminyltransferase‐I toward M7–M3 glycans, clarifying the substrate specificity in the context of high‐mannose‐type glycans.     

High‐performance liquid chromatography tandem mass spectrometry method for the determination of 2CC‐NBOMe and 25I‐NBOMe in human serum

2CC‐NBOMe {4‐chloro‐2,5‐dimethoxyphenethyl‐N‐[(2‐methoxyphenyl) methyl] ethanamine} and 25I‐NBOMe {2‐(4‐iodo‐2,5‐dimethoxyphenyl)‐N‐[(2‐methoxyphenyl) methyl] ethanamine} are of a class of N‐benzyl phenethylamine derivatives whose synthesis was first reported in the scientific literature in 2011. Recent reports from ‘personal drug experience websites’ and in the popular press indicate these drugs are the latest in a series of designer ‘Bath Salt’ drugs of abuse. The presented high‐performance liquid chromatography triple quadrupole mass spectrometry (HPLC/MS/MS) method was developed for the detection and quantification of 2CC‐NBOMe and 25I‐NBOMe in serum of intoxicated emergency department patients. The assay applies 2‐?(2,?5‐?dimethoxyphenyl)‐?N‐?(2‐?methoxybenzyl) ethanamine (25H‐NBOMe) as the internal standard. Samples were extracted using solid‐phase extraction columns. The chromatographic separation was performed on a Luna 3 µ C₈(2) 100 Å, 100 × 2.0 mm, column. Detection was accomplished by multiple‐reaction monitoring via an electrospray ionization source operating in the positive ionization mode. The calibration curves were linear over the investigated concentration range, 30–2000 pg/mL, with a lower limit of detection of 10 pg/mL for both 2CC‐NBOMe and 25I‐NBOMe. The method proved suitable for serum clinical toxicology testing. Two severely intoxicated emergency department patients were determined to have serum concentrations of 250 and 2780 pg/mL of 25I‐NBOMe using the presented method. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis and Cytotoxicity Studies of New (Dimethylamino)‐Functionalised and 7‐Azaindole‐Substituted ‘Titanocene’ Anticancer Agents (7‐Azaindole=1H‐Pyrrolo[2,3‐b]pyridine)

From the carbolithiation of 1‐(cyclopenta‐2,4‐dien‐1‐ylidene)‐N,N‐dimethylmethanamine (=6‐(dimethylamino)fulvene; 3 ) and different lithiated azaindoles 2 (1‐methyl‐7‐azaindol‐2‐yl, 1‐[(diethylamino)methyl]‐7‐azaindol‐2‐yl, and 1‐(methoxymethyl)‐7‐azaindol‐2‐yl), the corresponding lithium cyclopentadienide intermediates 4a – 4c were formed (7‐azaindole=1H‐pyrrolo[2,3‐b]pyridine). The latter underwent a transmetallation reaction with TiCl₄ resulting in the (dimethylamino)‐functionalised ‘titanocenes’ 5a – 5c . When the ‘titanocenes’ 5a – 5c were tested against LLC‐PK cells, the IC₅₀ values obtained were of 8.8, 12, and 87 μM , respectively. The most cytotoxic ‘titanocene’, 5a , with an IC₅₀ value of 8.8 μM is nearly as cytotoxic as cis‐platin, which showed an IC₅₀ value of 3.3 μM when tested on the epithelial pig kidney LLC‐PK cell line, and ca. 200 times better than ‘titanocene dichloride’ itself.     

Synthesis of Macrocyclic Lactones via Ring Transformation of 4‐(ω‐Hydroxyalkyl)‐1,3‐oxazol‐5(4H)‐ones

The synthesis of α‐benzamido‐α‐benzyl lactones 23 of various ring size was achieved either via ‘direct amide cyclization’ by treatment of 2‐benzamido‐2‐benzyl‐ω‐hydroxy‐N,N‐dimethylalkanamides 21 in toluene at 90 – 110° with HCl gas or by ‘ring transformation’ of 4‐benzyl‐4‐(ω‐hydroxyalkyl)‐2‐phenyl‐1,3‐oxazol‐5(4H)‐ones under the same conditions. The precursors were obtained by C‐alkylations of 4‐benzyl‐2‐phenyl‐1,3‐oxazol‐5(4H)‐one ( 15 ) with THP‐ or TBDMS‐protected ω‐hydroxyalkyl iodides. Ring opening of the THP‐protected oxazolones by treatment with Me₂NH followed by deprotection of the OH group gave the diamides 21 , whereas deprotection of the TBDMS series of oxazolones 25 with TBAF followed by treatment with HCl gas led to the corresponding lactones 23 in a one‐pot reaction.