Kinetics of hydrogen-deuterium exchange of tryptophan and tryptophan peptides in deutero-trifluoroacetic acid using proton magnetic resonance spectroscopy

Norton, Raymond S.; Bradbury, J. H.

doi:10.1007/bf01731556

Cited by 12 publications

(1 citation statement)

References 81 publications

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“…The indole ring proton signals were assigned according to (see below) published data using '3C and 'H-NMR spectra of tryptophan in trifluoroacetic acid and D20 solutions (8,25,28). For our work, acetone-d6 was the best solvent.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of 4,5,6,7 and 2,4,5,6,7 Deuterium-labeled Indole-3-Acetic Acid for Use in Mass Spectrometric Assays

Magnus

Bandurski²,

Schulze³

1980

Plant Physiol.

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Syntheses are described for tetra and pentadeutero indole-3-acetic acid (IAA) labeled in positions 4, 5, 6, 7 or 2, 4, 5, 6, 7 of the indole moiety. Polydeuterated IAA is proposed as an internal standard for gas chromatographic-mass spectrometric analysis of IAA by selected ion monitoring. Nanogram amounts of IAA may be assayed by monitoring the base peak of IAA at m/z = 130 (134 for d4-IAA) and the molecular ion of the methyl ester of IAA at 189 (193 for d4-IAA). Deuterium in positions 4,5,6,and 7 and, to only a slightly lesser extent, that in position 2 of IAA is retained during alkali treatment, thus permitting use of these compounds as internal standards for assay of IAA released by alkaline hydrolysis of ester and amide conjugates. The use of polydeutero internal standards separates the standards from the "isotope cluster" caused by the normal abundance of heavy isotopes and also permits use of reduced mass resolution, thus leading to a 10-fold increase in sensitivity.Tetradeutero IAA was used as an internal standard for determining free plus ester IAA in alkaline hydrolysates of Zea mays, and showed exact agreement between estimates based on the molecular ion of the methyl ester and those based upon base peak. Application of the method to measuring free IAA in the upper and lower halves of geotropically stimulated Zea shoots showed 61 ± 4% of the free IAA to be on the lower side.Indolylic compounds, other than tryptophan, occur in plant (cf. 3, 4) and in animal tissue (cf. 23) in ylM amounts. Low tissue concentrations of the indoles and their lability (16,22,24) make assay by GC-SIM-MS4, with deuterated IAA as an internal standard, an attractive procedure. A number of procedures have been described using side chain d2-indoles as standards for assay of IAA and 5-hydroxy-IAA in plants and animals (1, 6, 10, 26). However, plants contain most of their IAA as ester or amide conjugates, conveniently assayed as free IAA, following hydrolysis of the esters by 1 N NaOH and the amide conjugates in 7 N NaOH (4). Alkaline hydrolysis precludes use of d2-IAA with deuterium in the side chain, since these deuterium are lost during base treatment. Deuterium in positions 4, 5, 6, and 7 is not exchanged with hydrogen during alkaline hydrolysis. Even deuterium in the 2 position is only slightly exchanged. ' To whom reprint requests should be directed.4Abbreviations: GC-SIM-MS, gas chromatography-selected ion monitoring-mass spectrometry; mp, melting point; NMR, nuclear magnetic resonance. synthesis for 4,5,6,7-d4-IAA and 2,4,5,6,7-d5-IAA and use of these compounds as internal standards.In addition to alkali stability, polydeuterated compounds provide additional advantages as internal standards for GC-SIM-MS. The ions generated by d4-and d5-IAA during MS are remote from the ions of IAA and, thus, the background caused by the normal abundances of heavy ions at I and 2 mass units above the IAA is eliminated. Further, providing sample purity is adequate, mass resolution may be broadened to ±+ amu (fat peak monitoring) y...

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Section: Resultsmentioning

confidence: 99%

Synthesis of 4,5,6,7 and 2,4,5,6,7 Deuterium-labeled Indole-3-Acetic Acid for Use in Mass Spectrometric Assays

Magnus

Bandurski²,

Schulze³

1980

Plant Physiol.

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Co‐oligopeptides containing two aromatic residues spaced by glycyl residues. X. Proton magnetic resonance study of co‐oligopeptides of tryptophan and glycine

Skrabal¹,

Rizzo²,

Baici³

et al. 1979

Biopolymers

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SynopsisThe 'H-nmr spectra of co-oligopeptides of tryptophan and glycine with structure H-GlyTrp-(Gly),-Trp-Gly-OH (n = 0-2) and those of several di-and tripeptides have been recorded a t 360 MHz with CDBOD solutions containing 0.1N NaOD. The assignment of resonance signals was generally possible by comparing the spectra of structurally related peptides with each other. In order to solve the remaining ambiguities in the assignment, H-(cuL,BS)(cu,@-dz)Trp-OH, H-Trp-((~L,BS)(cu,P-ds)Trp-0H, and H -T r p -( 6 1 , r 2 ,~,~~,~2 -d~) T r p -O H have been prepared and their spectra compared with those of the undeuterated compounds. The distribution of rotamers around the X I and (in two cases) x 2 torsion angles of the side chains has been obtained from the vicinal coupling constants ' J J H~J H~ and from the long-range coupling constants 4 J~a~a~. These data and an analysis of the chemical shifts of the Gly-CC( protons suggest that the orientation of the aromatic side chain is influenced by the following order of decreasing interaction with the functional groups a t N-and C-side: -NH2 > -NHCO-> -CONH-> -COO-. This rule does not hold for the second Trp residue of diand tripeptides containing the -Trp -7'rp-sequence, which has tentatively been attributed to steric effects.

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Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

Greathouse

Vostrikov

McClellan

et al. 2008

Journal of Peptide Science

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Lactoferricin (LfB) is a 25-residue innate immunity peptide released by pepsin from the N-terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR-NH2) retains antimicrobial activity and is thought to constitute the "antimicrobial active-site" (Tomita, Acta Paediatr Jpn. 1994; 36: 585-91). Here we report on N-acylation of 1-Me-Trp5-LfB6, Cn-RRWQ[1-Me-W]R-NH2, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp5) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn-RRWQ[1-Me-W]R-NH2 (deuterated 1-Me-Trp5 underlined), were monitored by solid state 31P NMR and 2H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn-RRWQ[&1-Me-W]R-NH2 peptides added at a 1:25 peptide to lipid ratio. 2H-NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The 2H NMR quadrupolar splittings suggest that the 1-Me-Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. 31P-NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0-RRWQ[1-Me-W]R-NH2, resulted in a 3-4 fold increase in antimicrobial activity against E. coli. N-acylation with a C12 fatty acid enhanced activity almost 90 fold.

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Kinetics of hydrogen-deuterium exchange of tryptophan and tryptophan peptides in deutero-trifluoroacetic acid using proton magnetic resonance spectroscopy

Cited by 12 publications

References 81 publications

Synthesis of 4,5,6,7 and 2,4,5,6,7 Deuterium-labeled Indole-3-Acetic Acid for Use in Mass Spectrometric Assays

Synthesis of 4,5,6,7 and 2,4,5,6,7 Deuterium-labeled Indole-3-Acetic Acid for Use in Mass Spectrometric Assays

Co‐oligopeptides containing two aromatic residues spaced by glycyl residues. X. Proton magnetic resonance study of co‐oligopeptides of tryptophan and glycine

Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

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