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The use of 1H–31P GHMBC and covariance NMR to unambiguously determine phosphate ester linkages in complex polysaccharide mixtures

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Abstract

Poly- and oligo-saccharides are commonly employed as antigens in many vaccines. These antigens contain phosphoester structural elements that are crucial to the antigenicity, and hence the effectiveness of the vaccine. Nuclear Magnetic Resonance (NMR) is a powerful tool for the site-specific identification of phosphoesters in saccharides. We describe here two advances in the characterization of phosphoesters in saccharides: (1) the use of 1H–31P GHMBC to determine the site-specific identity of phosphoester moieties in heterogeneous mixtures and (2) the use of Unsymmetrical/Generalized Indirect Covariance (U/GIC) to calculate a carbon-phosphorus 2D spectrum. The sensitivity of the 1H–31P GHMBC is far greater than the “standard” 1H–31P GHSQC and allows long-range 3–5JHP couplings to be readily detected. This is the first example to be reported of using U/GIC to calculate a carbon-phosphorus spectrum. The U/GIC processing affords, in many cases, a fivefold to tenfold or greater increase in signal-to-noise ratios in the calculated spectrum. When coupled with the high sensitivity of 1H–31P HMBC, U/GIC processing allows the complete and unambiguous assignments of phosphoester moieties present in heterogeneous samples at levels of ~5% (or less) of the total sample, expanding the breadth of samples that NMR can be used to analyze. This new analytical technique is generally applicable to any NMR-observable phosphoester.

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Abbreviations

αRha:

l-α-Rhamnose

βGlc:

β-d-Glucose

β-ManNAc:

β-2-Acetamido-mannose

β-GlcNAc:

β-2-Acetamido-glucose

α-GlcNAc:

α-2-Acetamido-glucose

NMR:

Nuclear magnetic resonance spectroscopy

GHMBC:

Gradient heteronuclear multiple bond correlation

GHMQC:

Gradient heteronuclear multiple quantum correlation

GHSQC:

Gradient heteronuclear single quantum correlation

PC:

Phosphocholine

U/GIC:

Unsymmetrical/generalized indirect covariance

MS:

Mass spectrometry

References

  • Abeygunawardana C, Williams TC, Sumner JS, Hennessey JP (2000) Development and validation of an NMR-based identity assay for bacterial polysaccharides. Anal Biochem 279:226–240

    Article  Google Scholar 

  • Aubin Y, Jones C, Freedberg DI (2010) Using NMR Spectroscopy to obtain the higher order structure of biopharmaceutical products. Biopharm Int Supplement, 28–34

  • Bax A, Summers MF (1986) Proton and carbon-13 assignments from sensitivity enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J Am Chem Soc 108:2093–2094

    Article  Google Scholar 

  • Berger S (2010) 2D carbon-heteroatom correlation. In: Morris GA, Emsley JW (eds) Multidimensional NMR methods for the solution state. Wiley, New York, pp 365–372

    Google Scholar 

  • Blinov KA, Larin N, Williams AJ, Zell M, Martin GE (2006a) Long-range carbon–carbon connectivity via unsymmetrical indirect covariance processing of HSQC and HMBC NMR data. Magn Reson Chem 44:107–109

    Article  Google Scholar 

  • Blinov KA, Larin N, Williams AJ, Mills K, Martin GE (2006b) Unsymmetrical covariance processing of COSY or TOCSY and HSQC NMR data to obtain the equivalent of HSQC-COSY and HSQC-TOCSY Spectra. J Heterocycl Chem 43:163–166

    Article  Google Scholar 

  • Catoire LJ (2004) Phosphorus-31 transverse relaxation rate measurements by NMR spectroscopy: insight into conformational exchange along the nucleic acid backbone. J Biomol NMR 28:179–184

    Article  Google Scholar 

  • Chary KVR, Rastogi VK, Govil G (1993) An Efficient 2D NMR technique helco for heteronuclear [P-31-H-1] long-range correlation. J Magn Reson Ser B 102:81–83

    Article  Google Scholar 

  • Chen YB, Zhang FL, Snyder D, Gan ZH, Bruschweiler-Li L, Bruschweiler R (2007) Quantitative covariance NMR by regularization. J Biomol NMR 38:73–77

    Article  Google Scholar 

  • Claridge TDW, Perez-Victoria I (2003) Enhanced C-13 resolution in semi-selective HMBC: a band-selective, constant-time HMBC for complex organic structure elucidation by NMR. Org Biomol Chem 1:3632–3634

    Article  Google Scholar 

  • Davies DB, Eaton RJ, Baranovsky SF, Veselkov AN (2000) NMR investigation of the complexation of daunomycin with deoxytetranucleotides of different base sequence in aqueous solution. J Biomol Struct Dyn 17:887–901

    Google Scholar 

  • Draing C, Pfitzenmaier M, Zummo S, Mancuso G, Geyer A, Hartung T, von Aulock S (2006) Comparison of lipoteichoic acid from different serotypes of Streptococcus pneumoniae. J Biol Chem 281:33849–33859

    Article  Google Scholar 

  • Furrer J (2010) A robust, sensitive, and versatile HMBC experiment for rapid structure elucidation by NMR: IMPACT-HMBC. Chem Comm 46:3396–3398

    Article  Google Scholar 

  • ICH Harmonised Tripartite Guideline. Specifications: test procedures and acceptance criteria for biotechnological/biological products Q6B

  • Jennings HJ, Rosell KG, Carlo DJ (1980) Structural determination of the capsular polysaccharide of Streptococcus pneumoniae type-19 (F-19). Can J Chem 58:1069–1074

    Article  Google Scholar 

  • John CM, Liu MF, Jarvis GA (2009a) Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction. J Lipid Res 50:424–438

    Article  Google Scholar 

  • John CM, Liu MF, Jarvis GA (2009b) Natural phosphoryl and acyl variants of lipid a from Neisseria meningitidis strain 89I differentially induce tumor necrosis factor-alpha in human monocytes. J Biol Chem 284:21515–21525

    Article  Google Scholar 

  • Jones C (1985) Identification of the tetrasaccharide repeating-unit of the Streptococcus pneumoniae type-23 polysaccharide by high-field proton NMR-spectroscopy. Carbohydr Res 139:75–83

    Google Scholar 

  • Kamerling JP (2000) Pneumococcal polysaccharides: a chemical view. In: Tomasz A (ed) Streptococcus pneumoniae molecular biology and mechanisms of diseases. Mary Ann Liebert, Larchmont, pp 81–114

    Google Scholar 

  • Keniry MA (1996) Pulsed field gradient pulse sequence for heteronuclear [P-31-H-1] long range correlation. Magn Reson Chem 34:33–35

    Article  Google Scholar 

  • Kozminski W, Bednarek E, Bocian W, Sitkowski J, Kozerski L (2003) The new HMQC-based technique for the quantitative determination of heteronuclear coupling constants. Application for the measurement of (3)J(H′(i,) Pi+1) in DNA oligomers. J Magn Reson 160:120–125

    Article  ADS  Google Scholar 

  • Lindon JC, Baker DJ, Farrant RD, Williams JM (1986) H-1, C-13 and P-31 Nmr-spectra and molecular-conformation of myoinositol 1,4,5-trisphosphate. Biochem J 233:275–277

    Google Scholar 

  • Luy B, Marino JP (2001) J. Am Chem Soc 123:11306–11307

    Article  Google Scholar 

  • Majumdar A, Shah MH, Bitok JK, Hassis-LeBeau ME, Meyers CLF (2009) Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using H-1-P-31-P-31 correlation NMR spectroscopy. Mol Biosyst 5:935–944

    Article  Google Scholar 

  • Majumdar A, Sun Y, Shah M, Meyers CLF (2010) Versatile H-1-P-31-P-31 COSY 2D NMR techniques for the characterization of polyphosphorylated small molecules. J Org Chem 75:3214–3223

    Article  Google Scholar 

  • Malon M, Koshino H (2007) H(C)P and H(P)C triple-resonance experiments at natural abundance employing long-range couplings. Magn Reson Chem 45:770–776

    Article  Google Scholar 

  • Marek R, Lyčka R, Lyčka A (2002) 15N NMR spectroscopy in structural analysis. Curr Org Chem 6:35–66

    Article  Google Scholar 

  • Marek R, Lyčka A, Kolehmainen E, Sieväen E, Toušek J (2007) 15N NMR spectroscopy in structural analysis: an update (2001–2005). Curr Org Chem 11:1154–1205

    Article  Google Scholar 

  • Martin GE (2002a) Qualitative and quantitative exploitation of heteronuclear coupling constants. In: Webb GA (ed) Annual report on NMR spectroscopy, vol 46. Academic Press, New York, pp 37–101

    Google Scholar 

  • Martin GE (2002b) Cryogenic NMR probes. In: Grant DM, Harris RK (eds) Encyclopedia of nuclear magnetic resonance, vol 9 supplement. Wiley, New York, pp 33–35

    Google Scholar 

  • Martin GE (2005) Small-volume and high-sensitivity NMR probes. In: Webb GA (ed) Annual report on NMR spectroscopy, vol 56. Elsevier, Amsterdam, pp 2–96

    Google Scholar 

  • Martin GE, Hadden CE (2000) Techniques for long-range 1H–15N heteronuclear shift correlation at natural abundance and applications to structural problems. J Nat Prod 63:543–585

    Article  Google Scholar 

  • Martin GE, Williams AJ (2005) Long-range 1H–15N heteronuclear shift correlations. In: Webb GA (ed) Annual report on NMR spectroscopy, vol 55. Elsevier, Amsterdam, pp 2–119

    Google Scholar 

  • Martin GE, Williams AJ (2010) Utilizing long-range 1H–15N 2D NMR spectroscopy for chemical structure elucidation and confirmation: encyclopedia of NMR spectroscopy, Wiley, New York (in press)

  • Martin GE, Hilton BD, Blinov KA, Williams AJ (2007a) 13C–15N correlation via unsymmetrical indirect covariance NMR: application to vinblastine. J Nat Prod 70:1966–1970

    Article  Google Scholar 

  • Martin GE, Irish PA, Hilton BD, Blinov KA, Williams AJ (2007b) Utilizing unsymmetrical indirect covariance processing to define 15 N–13C connectivity networks. Magn Reson Chem 45:624–627

    Article  Google Scholar 

  • Martin GE, Hilton BD, Blinov KA (2011a) HSQC-ADEQUATE: a new paradigm for establishing molecular skeletons. Magn Reson Chem 49:248–252

    Article  Google Scholar 

  • Martin GE, Hilton BD, Willcott MR III, Blinov KA (2011b) HSQC-ADEQUATE: a further Investigation of data requirements. Magn Reson Chem 49:350–358

    Article  Google Scholar 

  • Pujar NS, Huang NF, Daniels CL, Dieter L, Gayton MG, Lee AL (2004) Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides. Biopolymer 75:71–84

    Article  Google Scholar 

  • Richards JC, Perry MB (1988) Structure of the specific capsular polysaccharide of Streptococcus pneumoniaetype-23F (American Type-23). Biochem Cell Biol 66:758–771

    Article  Google Scholar 

  • Roy A, Roy N (1984) Structure of the capsular polysaccharide from Streptococcus pneumoniae type-23. Carbohydr Res 126:271–277

    Article  Google Scholar 

  • Sakamoto Y, Kondo K, Onozato M, Aoyama T (2006) Conformational analysis of 2-(diphenylphosphanyl)-N, N-dimethyl-1-benz amide and 2-(diphenylphosphanyl)-phenyl-pyrrolidin-1-YL-methanone by NMR spectroscopy. Polycycl Aromat Compd 26:59–68

    Article  Google Scholar 

  • Schoefberger W, Schlagnitweit J, Muller N (2011) Ann. Rep NMR Spectrosc 72:1–60

    Article  Google Scholar 

  • Sims LD, Soltero LR, Martin GE (1989) Carbon-phosphorous heteronuclear shift correlation via multiple quantum coherence. Magn Reson Chem 27:599–602

    Article  Google Scholar 

  • Snyder DA, Brüschweiler R (2009) Generalized indirect covariance NMR Formalism for the establishment of multidimensional spin correlations. J Chem Phys A 113:12898–12902

    Article  Google Scholar 

  • Snyder DA, Brüschweiler R (2010) Multi-dimensional correlation spectroscopy by covariance NMR. In: Morris GA, Emsley JW (eds) Multidimesional NMR methods in the solution state. Wiley, New York, pp 97–105

    Google Scholar 

  • Veselkov AN, Eaton RJ, Pakhomov VI, Djimant LN, Davies DB (2001) Structural and thermodynamic analysis of daunomycin binding with desoxyhexanucleotides with different base sequences by NMR spectroscopy. J Struct Chem 42:193–206

    Article  Google Scholar 

  • Watson JD, Crick FHC (1953) A structure for deoxyribose nucleic acid. Nature 171:737–738

    Article  ADS  Google Scholar 

  • Williamson MP, Griffin CE (1968) Three- and four-bond 31P–1H coupling constants and geminal proton nonequivalence in ethyl esters of phosphorus acids. J Phys Chem 72:4043–4047

    Article  Google Scholar 

  • Willker W, Leibfritz D (1995) Determination of heteronuclear long-range H, X coupling-constants from gradient-selected Hmbc spectra. Magn Reson Chem 33:632–638

    Article  Google Scholar 

  • Xu QW, Abeygunawardana C, Ng AS, Sturgess AW, Harmon BJ, Hennessey JP (2005) Characterization and quantification of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharide preparations. Anal Biochem 336:262–272

    Article  Google Scholar 

  • Zartler ER, Porambo RJ, Anderson CL, Chen LH, Yu JG, Nahm MH (2009) Structure of the capsular polysaccharide of pneumococcal serotype 11A reveals a novel acetylglycerol that is the structural basis for 11A subtypes. J Biol Chem 284:7318–7329

    Article  Google Scholar 

Download references

Acknowledgments

We would like to thank Kirill Blinov of ACD, Moscow Department and Carlos Cobas of Mestrenova for implementing covariance processing in their software. ERZ would like to thank Dave Detlefsen for the initial idea to use HMBC instead of HSQC and John Limtiaco for discussions of the HMBC sequence and pointing out the use of constant-time HMBC to eliminate twisting in the indirect dimension.

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Authors and Affiliations

  1. Vaccine Analytical Development, Merck Research Labs, Merck & Co., West Point, PA, 19486, USA

    Edward R. Zartler

  2. Structure Elucidation Group, Merck Research Labs, Merck & Co., Summit, NJ, 07901, USA

    Gary E. Martin

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  1. Edward R. Zartler
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  2. Gary E. Martin
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Correspondence to Edward R. Zartler.

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Zartler, E.R., Martin, G.E. The use of 1H–31P GHMBC and covariance NMR to unambiguously determine phosphate ester linkages in complex polysaccharide mixtures. J Biomol NMR 51, 357–367 (2011). https://doi.org/10.1007/s10858-011-9563-8

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