Enantiospecific tritium labeling of 28‐homocastasterone Elbert, Tomáš; Patil, Mahadeo R.; Marek, Aleš
Journal of labelled compounds & radiopharmaceuticals,
March 2017, 2017-03-00, 20170301, Letnik:
60, Številka:
3
Journal Article
Recenzirano
A regiospecific and enantiospecific synthesis of tritium‐labeled 28‐homocastasterone is reported. Appropriate chlorocarbonate, efficiently synthesized from the starting 28‐homocastasterone in an ...overall yield of 46%, undergoes catalytic tritium dechlorination by the T2/Pd0/Et3N system, providing 28‐3β‐3Hhomocastasterone, in a good yield, radiochemical purity (>97%), and with a high specific activity (5.8 Ci/mmol).
The 6‐step synthetic sequence starting from the brassinosteroid to be labeled provides the desired tritium stereospecifically and regiospecifically labeled brassinosteroids in satisfactory yields and with radiochemical purities >97% as required for biological experiments. This paper describes the synthesis of 28‐3β‐3Hhomocastasterone with a high SA of 5.8 Ci/mmol as a proof‐of‐concept of this new methodology.
Electron transfer dissociation of peptide ions with the diazirine-containing residue photomethionine (M*) results in side-chain dissociations by loss of C
3
H
7
N
2
radicals in addition to standard ...backbone cleavages. The side-chain dissociations are particularly prominent upon activation of long-lived, charge-reduced, cation radicals (GM*GGR + 2H)
+●
. Investigation of these cation radicals by near-UV photodissociation and collisional activation revealed different fragmentation products and mechanisms resulting from these ion activation modes. The dissociations observed for photomethionine were dramatically different from those previously reported for the lower homologue photoleucine; here, a difference by a single methylene group in the side chain had a large effect on the chemistries of the cation radicals upon ETD and further activation. ETD intermediates and products were probed by tandem 355-nm UV photodissociation-collision induced dissociation and found to contain chromophores that resulted from electron attachment to the diazirine ring. The nature of the newly formed chromophores and ion energetics and kinetics were investigated by electron structure calculations combining ab initio and density functional theory methods and Rice-Ramsperger-Kassel-Marcus (RRKM) theory. The dramatic difference between the dissociations of L* and M* containing peptide cation radicals is explained by electronic effects that play a role in stabilizing critical reaction intermediates and steer the dissociations into kinetically favored reaction channels. In addition, a new alternating UVPD-ETD-UVPD MS
4
experiment is introduced and utilized for ion structure elucidation.
Graphical Abstract
ᅟ
We report non‐chiral amino acid residues cis‐ and trans‐1,4‐diaminocyclohexane‐1‐carboxylic acid (cyclo‐ornithine, cO) that exhibit unprecedented stereospecific control of backbone dissociations of ...singly charged peptide cations and hydrogen‐rich cation radicals produced by electron‐transfer dissociation. Upon collision‐induced dissociation (CID) in the slow heating regime, peptide cations containing trans‐cO residues undergo facile backbone cleavages of amide bonds C‐terminal to trans‐cO. By contrast, peptides with cis‐cO residues undergo dissociations at several amide bonds along the peptide ion backbone. Diastereoisomeric cO‐containing peptides thus provide remarkably distinct tandem mass spectra. The stereospecific effect in CID of the trans‐cO residue is explained by syn‐facially directed proton transfer from the 4‐ammonium group at cO to the C‐terminal amide followed by neighboring group participation in the cleavage of the CO―NH bond, analogous to the aspartic acid and ornithine effects. Backbone dissociations of diastereoisomeric cO‐containing peptide ions generate distinct bn+‐type fragment ions that were characterized by CID‐MS3 spectra. Stereospecific control is also reported for electron‐transfer dissociation of cis‐ and trans‐cO containing doubly charged peptide ions. The stereospecific effect upon electron transfer is related to the different conformations of doubly charged peptide ions that affect the electron attachment sites and ensuing N―Cα bond dissociations.
Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. ...Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions.
Graphical Abstract
ᅟ
Gas-phase conformations and electron transfer dissociations of pentapeptide ions containing the photo-Leu residue (L*) were studied. Exhaustive conformational search including molecular dynamics ...force-field, semi-empirical, ab initio, and density functional theory calculations established that the photo-Leu residue did not alter the gas-phase conformations of (GL*GGK + 2H)
2+
and (GL*GGK-NH
2
+ H)
+
ions, which showed the same conformer energy ranking as the unmodified Leu-containing ions. This finding is significant in that it simplifies conformational analysis of photo-labeled peptide ions. Electron transfer dissociation mass spectra of (GL*GGK + 2H)
2+
, (GL*GGK-NH
2
+ 2H)
2+
,(GL*GGKK + 2H)
2+
, (GL*GLK + 2H)
2+
, and (GL*LGK + 2H)
2+
showed 16 %–21 % fragment ions originating by radical rearrangements and cleavages in the diazirine ring. These side-chain dissociations resulted in eliminations of N
2
H
3
, N
2
H
4
, N
2
H
5
, and NH
4
O neutral fragments and were particularly abundant in long-lived charge-reduced cation-radicals. Deuterium labeling established that the neutral hydrazine molecules mainly contained two exchangeable and two nonexchangeable hydrogen atoms from the peptide and underwent further H/D exchange in an ion–molecule complex. Electron structure calculations on the charge-reduced ions indicated that the unpaired electron was delocalized between the diazirine and amide π* electronic systems in the low electronic states of the cation-radicals. The diazirine moiety in GL*GGK-NH
2
was calculated to have an intrinsic electron affinity of 1.5 eV, which was further increased by the Coulomb effect of the peptide positive charge. Mechanisms are proposed for the unusual elimination of hydrazine from the photo-labeled peptide ions.
Figure
ᅟ
A convenient method for the synthesis of tritium-labeled brassinosteroids with very high specific activity is reported. A
3
H-labeled 24-epicastasterone was isolated in high yield (40 mCi), ...radiochemical purity (>97%) and a specific activity up to 99 Ci mmol
−1
. The labeling strategy was designed to employ a radiolabeling step at the late stage of the synthetic sequence.
A convenient method for the synthesis of tritium-labeled brassinosteroids with very high specific activity is reported.
3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of 3H-1 ...is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity, and we demonstrate in vivo brain penetration. In vitro characterization of 3H-1 binding shows high specificity to the high-affinity GHB binding sites.
A detailed study of regiospecific silylation, oxidation, and reduction of sterols is reported. The different reactivity of 2α and 3α hydroxy groups is demonstrated on a ...2α,3α‐dihydroxy‐5α‐pregnane‐6,20‐dione as a model substrate. A regiospecific silylation of the 2α‐alcohol occurred when silyl chloride was used under mild reaction conditions. The selective oxidation of one of the 2α,3α‐diols by dimethyldioxirane or the Dess–Martin periodinane led to a mixture of α‐hydroxyketones with high prevalence of 3,6,20‐trione over 2,6,20‐trione in the ratio 10:1. A regiospecific reduction of a model substrate by lithium tri‐tert‐butoxyaluminium hydride gives the C6 alcohol.
Just one of them! A practical and simple strategy for selective transformations of one of the hydroxy and/or ketone groups on sterol substrates is reported.
A new and efficient procedure for the preparation of brassinosteroids labeled with hydrogen isotopes was developed. A four-step reaction sequence started with the selective oxidation of a 2,3-diol ...group to an α-hydroxy ketone, which was converted stereospecifically into a chlorocarbonate by reaction with triphosgene. A subsequent Pd-catalyzed reductive dechlorination with deuterium gas yielded deuterium-labeled brassinosteroid 2,3-carbonates. The reaction sequence was completed by base-catalyzed hydrolysis of the cyclic carbonate.
Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we ...evaluate the activity of 3′,3′-c-di(2′F,2′dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2′ position of 3′,3′-c-di(2′F,2′dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.
PDF
