Synthesis of Stereoarray Isotope Labeled (SAIL) Lysine via the “Head-to-Tail” Conversion of SAIL Glutamic Acid

Terauchi, Tsutomu; Kamikawai, Tomoe; Vinogradov, M. G.; Starodubtseva, E. V.; Takeda, Mitsuhiro; Kainosho, Masatsune

doi:10.1021/ol1026766

Cited by 10 publications

(16 citation statements)

References 28 publications

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“…2.2, this strategy based solely on the amino acid sequence information worked perfectly [7]. In addition to the unambiguous assignment, this method provided even more crucial information about the state of the scissile bond in the SSI-subtilisin complex, through the 13 C chemical shift of Met 73 and the 13 C- 15 N spin coupling values between Met 73 and Val 74 . We finally proved that the Michaelis complex with the intact, undistorted scissile bond is the only stable form of the SSI-subtilisin complex in solution [8,9].…”

Section: Stereo-specific Deuteration Of Prochiral Methylenementioning

confidence: 91%

“…Since the scissile peptide bond of SSI is formed between Met 73 and Val 74 , its state in the proteinase complex would be precisely manifested by the 13 C-NMR signal for the carbonyl carbon of Met 73 , if we could observe a single 13 13 C and 15 N, which are known to be about 15 Hz. As illustrated in Fig.…”

Section: Stereo-specific Deuteration Of Prochiral Methylenementioning

confidence: 99%

“…2.1.2 Selective 13 C, 15 N Double-Labeling Method for the Sequential Assignment of Backbone Amide NMR Signals in Large Proteins…”

Section: Stereo-specific Deuteration Of Prochiral Methylenementioning

confidence: 99%

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Isotope-Aided Methods for Biological NMR Spectroscopy: Past, Present, and Future

Kainosho

Miyanoiri

Takeda³

2017

Experimental Approaches of NMR Spectroscopy

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This chapter starts by providing a historical background of our research endeavors over the past half-century to develop various isotope-aided methods in biological NMR spectroscopy, since innovations bloom only on the rich ground cultivated by previous investigators. We then focused on the stereo-array isotope-labeling (SAIL) method, one of our recent accomplishments, which culminates the isotope-aided NMR technologies for structural studies of proteins from various aspects: accurate structural determinations of large proteins, elaboration for automated structural determination, highly efficient and versatile residue-selective methyl labeling with newly developed auxotrophic E. coli strains, large-amplitude slow-breathing motion (LASBM) as revealed by the aromatic ring flipping of the residues in ligand-binding interfaces, and applications of the deuterium-induced 13 C-NMR isotope shift to investigate the hydrogen exchange phenomena of side-chain polar groups. Meanwhile, the expected role of NMR spectroscopy has been rapidly shifting from structure determinations to dynamics studies of biologically interesting targets, such as membrane proteins and larger protein complexes. The dynamic aspects of protein-protein and protein-ligand interactions are closely related to their biological functions and can be efficiently studied by using proteins residue selectively labeled with amino acids bearing optimized labeling

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Section: Stereo-specific Deuteration Of Prochiral Methylenementioning

confidence: 91%

Section: Stereo-specific Deuteration Of Prochiral Methylenementioning

confidence: 99%

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Isotope-Aided Methods for Biological NMR Spectroscopy: Past, Present, and Future

Kainosho

Miyanoiri

Takeda³

2017

Experimental Approaches of NMR Spectroscopy

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“…In the case of Lys residues, the NMR signals of the ζ -amino 15 N and εor δ-carbon 13 C signals would be plausible candidates for probing the deuterium substitution effects. There have several reports on the isotope shifts of the δand ε-13 C for the Lys residues induced by the deuteration of ζ -amino groups (Ladner et al, 1975;Led and Petersen, 1979;Hansen, 1983;Dziembowska et al, 2004;Tomlinson et al, 2009;Platzer et al, 2014). However, it seems no comprehensive studies have applied the deuterium-induced isotope shifts on 13 C ε signals to characterize the ionization states of Lys residues.…”

Section: Characterization Of the Ionization State Of Thementioning

confidence: 99%

Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method

et al. 2021

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Abstract. Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated using NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the Δ+PHS/V66K variant of staphylococcal nuclease (SNase), which contains 21 Lys residues, including the engineered Lys-66 with an unusually low pKa of ∼ 5.6. All of the NMR signals for the 21 Lys residues were sequentially and stereospecifically assigned using the stereo-array isotope-labeled Lys (SAIL-Lys), [U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys. The complete set of assigned 1H, 13C, and 15N NMR signals for the Lys side-chain moieties affords useful structural information. For example, the set includes the characteristic chemical shifts for the 13Cδ, 13Cε, and 15Nζ signals for Lys-66, which has the deprotonated ζ-amino group, and the large upfield shifts for the 1H and 13C signals for the Lys-9, Lys-28, Lys-84, Lys-110, and Lys-133 side chains, which are indicative of nearby aromatic rings. The 13Cε and 15Nζ chemical shifts of the SNase variant selectively labeled with either [ε-13C;ε,ε-D2]-Lys or SAIL-Lys, dissolved in H2O and D2O, showed that the deuterium-induced shifts for Lys-66 were substantially different from those of the other 20 Lys residues. Namely, the deuterium-induced shifts of the 13Cε and 15Nζ signals depend on the ionization states of the ζ-amino group, i.e., −0.32 ppm for Δδ13Cε [NζD3+-NζH3+] vs. −0.21 ppm for Δδ13Cε [NζD2-NζH2] and −1.1 ppm for Δδ15Nζ[NζD3+-NζH3+] vs. −1.8 ppm for Δδ15Nζ[NζD2-NζH2]. Since the 1D 13C NMR spectrum of a protein selectively labeled with [ε-13C;ε,ε-D2]-Lys shows narrow (> 2 Hz) and well-dispersed 13C signals, the deuterium-induced shift difference of 0.11 ppm for the protonated and deprotonated ζ-amino groups, which corresponds to 16.5 Hz at a field strength of 14 T (150 MHz for 13C), could be accurately measured. Although the isotope shift difference itself may not be absolutely decisive to distinguish the ionization state of the ζ-amino group, the 13Cδ, 13Cε, and 15Nζ signals for a Lys residue with a deprotonated ζ-amino group are likely to exhibit distinctive chemical shifts as compared to the normal residues with protonated ζ-amino groups. Therefore, the isotope shifts would provide a useful auxiliary index for identifying Lys residues with deprotonated ζ-amino groups at physiological pH levels.

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“…The condensation of aldehyde 110 with N -acetyl phosphonato glycine ethyl ester ( 20 , Scheme 9) in the presence of DBU afforded product 111 . Asymmetric hydrogenation with (+)-1,2-bis[(2S,5S)-2,5-diethylphospholano] benzene-(cyclooctadiene)-rhodium(I)-trifluoro-methanesulfonate [(S,S)-Et-DuPhos-Rh] and deprotection of the amino function by refluxing in HCl, followed by the hydrazine treatment afforded (2 S ,3 R ,4 R ,5 S ,6 R )-[3,4,5,6- 2 H 4 ;1,2,3,4,5,6- 13 C 6 ;2,6- 15 N 2 ]-lysine [89]. …”

Section: Synthesis Of 22 Amino Acidsmentioning

confidence: 99%

Access to Any Site Directed Stable Isotope (2H, 13C, 15N, 17O and 18O) in Genetically Encoded Amino Acids

Dawadi

Lugtenburg

2013

Molecules

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Proteins and peptides play a preeminent role in the processes of living cells. The only way to study structure-function relationships of a protein at the atomic level without any perturbation is by using non-invasive isotope sensitive techniques with site-directed stable isotope incorporation at a predetermined amino acid residue in the protein chain. The method can be extended to study the protein chain tagged with stable isotope enriched amino acid residues at any position or combinations of positions in the system. In order to access these studies synthetic methods to prepare any possible isotopologue and isotopomer of the 22 genetically encoded amino acids have to be available. In this paper the synthetic schemes and the stable isotope enriched building blocks that are available via commercially available stable isotope enriched starting materials are described.

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Synthesis of Stereoarray Isotope Labeled (SAIL) Lysine via the “Head-to-Tail” Conversion of SAIL Glutamic Acid

Cited by 10 publications

References 28 publications

Isotope-Aided Methods for Biological NMR Spectroscopy: Past, Present, and Future

Isotope-Aided Methods for Biological NMR Spectroscopy: Past, Present, and Future

Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method

Access to Any Site Directed Stable Isotope (2H, 13C, 15N, 17O and 18O) in Genetically Encoded Amino Acids

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