Elsevier

Analytical Biochemistry

Volume 580, 1 September 2019, Pages 14-20
Analytical Biochemistry

Solid-state vibrational circular dichroism studies of L- and D-serine

https://doi.org/10.1016/j.ab.2019.05.016Get rights and content

Highlights

  • Vibrational circular dichroism (VCD) spectroscopy determines absolute configuration of chiral biomolecules.

  • d-serine is a key endogenous substance in the human body.

  • d- and l-serine can be spectroscopically discriminated using solid-state VCD in the mid-infrared region.

  • The VCD features of d-serine is characteristic, compared to d-alanine.

Abstract

d-Serine is considered a key endogenous substance involved in several enzymatic reactions in the human body. In this study, solid-state vibrational circular dichroism (VCD) measurements of enantiomeric serine were performed for spectroscopic distinction between d- and l-serine. The mirror-image VCD signals of the isomers in the KBr pellets were observed in the mid-infrared range of 1800–1250 cm−1. The calculated infrared (IR) and VCD spectra for the optimized serine structures were in good agreement with the corresponding observed spectra. In addition, the chemical shift values estimated from the shielding constants of the optimized structure of serine corresponded with the observed values in 13C and 15N solid-state nuclear magnetic resonance spectra, supporting the VCD assignments. Our results suggest the need for further study of VCD to develop a sensitive and high-resolution spectroscopic technique for the detection of d-amino acids.

Introduction

Many d-amino acids, which are previously thought to rarely exist in living organisms, have lately been discovered in higher organisms and play a decisive role in life functions [1]. d-serine is considered a key endogenous substance involved in several enzymatic reactions in mammalian tissues. d-Amino acid oxidase (DAAO), a well-known flavin adenine dinucleotide (FAD)-dependent enzyme, is a stereospecific oxidative deamination enzyme for the D-isomers of polar amino acids to obtain α-keto acids, etc. [[2], [3], [4]]. Thus, DAAO regulates the d-serine concentration in mammalian brain [5]. d-Serine plays an important role in neurotransmission by glutamate as a co-agonist of the ion-channel-type N-methyl-d-aspartic acid (NMDA) receptor [[6], [7], [8], [9]]. High concentrations of d-serine are present in the central nervous system of mammals, including humans, and are related to the activation of NMDA receptors involved in learning and memory functions. The level of d-serine, which is associated with various diseases involving dysfunctional NMDA receptors, e.g. amyotrophic lateral sclerosis (ALS) and Alzheimer's disease [10,11]. Since accompanying increases were observed in the level of expression and activity of DAAO, DAAO inhibitors are useful for increasing the d-serine levels in human serum and cerebrospinal fluid of schizophrenic patients [12]. d-Serine also regulates synaptic plasticity by binding to the δ2 glutamate receptor [13]. The detection methods for d-serine are technically important for understanding various biological reactions. Biochemical detection methods using antibodies capable of specifically interacting with d-serine have been reported [14]. Furthermore, separation and detection of free d-amino acid mixtures, containing d-serine, by advanced two-dimensional HPLC in combination with tandem mass spectrometry, have been reported [15]. Using circular dichroism (CD) spectra obtained in the vacuum ultraviolet (VUV)/UV region with circularly polarized light, of d-alanine in amorphous solid state can be distinguished from a mixture of amino-acids [16].

Vibrational circular dichroism (VCD) using different absorptions of left- and right-polarized infrared light is a powerful tool to investigate chiral molecules, such as d- and l-amino acids [17,18]. For instance, the VCD spectra of serine and alanine have been reported by Nafie et al. in the C–H stretching region of 3200–2000 cm−1 in solid state and aqueous solution [19]. Solid-state VCD experiments of l-isoleucine, d-isoleucine, l-allo-isoleucine, and d-allo-isoleucine show unique patterns based on the interplay between the two chiral centers [20]. The mechanism of enhanced VCD signals for packed alanine crystals is revealed using quantum calculations [21]. VCD has also been used to investigate the structure of antimicrobial peptide and protein [22,23]. β-sheet structures in fibril peptides like amyloid beta and α-synuclein peptides in a solution have been well studied by VCD spectroscopy [[24], [25], [26], [27]]. Sampling technique is an important part of CD measurements in the solid state; i.e., single crystals, KBr pellets, films and Nujol Mulls [19,28]. The KBr pellet has a feature that the background signals are almost negligible on VCD spectra in the mid-infrared region. In combination with first principles calculations, like density functional theory (DFT), VCD is a fine approach to investigate the three-dimensional structures of amino acids [29]. VCD based on the infrared light, which can be penetrated human skin, is less sensitive but may play a role in the recognition of d-amino acids in the human body.

Herein, VCD measurements of d- and l-serine in KBr pellets were performed using the mid-infrared region of 1800–1250 cm−1, which can be attributed to Cdouble bondO stretching, and –OH and –NH bending. Furthermore, VCD signals were calculated based on optimized structures confirmed by solid-state NMR analysis.

Section snippets

Amino acids

White crystals of l- and d-serine (purity ≥ 99.0%) were purchased from FUJIFILM Wako Pure Chemical Corporation (191–00401 and 191–08821). l-/d-alanine and l-/d-threonine were purchased from FUJIFILM Wako Pure Chemical Corporation (012–01041/015-01031 and 206–01321/206-07661), respectively.

Solid-state VCD experiments

Amino acid samples for VCD experiments were prepared by mixing an amino acid and KBr at a ratio of 1:100 and placing the solid mixture in a transparent 10-mm-diameter pellet. VCD and infrared (IR) signals were

VCD/IR experiments

The experimental VCD and IR spectra of l-serine and d-serine are shown in Fig. 1(a) and (b). The wavenumber of functional groups of amino acids have been related with molecular structure in the IR spectra [34]. The obtained VCD signals (peak numbers 1–8) of l- and d-serine were almost exact mirror images, corresponding to the absolute configurations (S and R, respectively) at the chiral α-carbon of the amino acids. Large couplet peaks around 1620 cm−1 in both spectra were assigned to the

Conclusions

Herein we have studied the spectroscopic chiral discrimination for l- and d-serine in the solid state using VCD combined with DFT approaches. We found well-resolved VCD signals of d-serine in the mid-infrared region, which is sufficiently distinguishable from l-serine, L/d-alanine, and L/d-threonine. Solid-state NMR analysis supported the optimized structure of associated serine to make accurate VCD signal assignments.

Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Culture, Sports, Science and Technology of Japan (JSPS KAKENHI) to I. K. (16H00828 and 18H02387), to H. S. (16H00840 and 17H03044), and Japan Science Technology Agency (JST) MIRAI grants to H. S. and I. K. (JPMJMI18GC). The computations were performed at the Research Center for Computational Science, Okazaki, Japan.

References (42)

  • L. Pollegioni et al.

    Physiological functions of D-amino acid oxidases: from yeast to humans

    Cell. Mol. Life Sci.

    (2007)
  • S. Sacchi et al.

    Structure-function relationships in human D-amino acid oxidase

    Amino Acids

    (2012)
  • X. Ding et al.

    Localization of D-serine and serine racemae in neurons and neuroglias in mouse brain

    Neurol. Sci.

    (2011)
  • J. De Miranda et al.

    Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl- D-aspartate (NMDA) receptor coagonist D-serine

    Proc. Natl. Acad. Sci. U.S.A.

    (2002)
  • Y. Tanii et al.

    Stereoselective antagonism by enantiomers of alanine and serine induced hyperactivity, stereotypy and ataxia in the rat

    J. Pharmacol. Exp. Ther.

    (1994)
  • H. Wolosker

    NMDA receptor regulation by D-serine: new findings and perspectives

    Mol. Neurobiol.

    (2007)
  • P. Paoletti et al.

    NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease

    Nat. Rev. Neurosci.

    (2013)
  • J. Sasabe et al.

    D-serine is a key determinant of glutamate toxicity in amyotrophic lateral sclerosis

    EMBO J.

    (2007)
  • C. Madeira et al.

    D-serine levels in Alzheimer's disease: implications for novel biomarker development

    Transl. Psychiatry

    (2015)
  • W. Kakegawa et al.

    D-serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor

    Nat. Neurosci.

    (2011)
  • L.A. Nafie et al.

    Vibrational circular dichroism in amino acids and peptides. 8. A chirality rule for methine C*a-H stretching modes

    J. Am. Chem. Soc.

    (1983)
  • Cited by (12)

    View all citing articles on Scopus
    View full text