A Major Step in Opening the Black Box of High-Molecular-Weight

Oct 30, 2017 - Dissolved organic nitrogen (DON) comprises the largest pool of fixed N in the surface ocean, yet its composition has remained poorly ...
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A Major Step in Opening the Black Box of High-Molecular-Weight Dissolved Organic Nitrogen by Isotopic Labeling of Synechococcus and Multibond Two-Dimensional NMR Xiaoyan Cao,† Margaret R. Mulholland,‡ John R. Helms,§,∥ Peter W. Bernhardt,‡ Pu Duan,† Jingdong Mao,*,§ and Klaus Schmidt-Rohr*,† †

Department of Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02453, United States Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, 4600 Elkhorn Avenue, Norfolk, Virginia 23529, United States § Department of Chemistry and Biochemistry, Old Dominion University, 4541 Hampton Boulevard, Norfolk, Virginia 23529, United States ∥ Biology and Chemistry Department, Morningside College, 1501 Morningside Avenue, Sioux City, Iowa 51106, United States ‡

S Supporting Information *

ABSTRACT: Dissolved organic nitrogen (DON) comprises the largest pool of fixed N in the surface ocean, yet its composition has remained poorly constrained. Knowledge of the chemical composition of this nitrogen pool is crucial for understanding its biogeochemical function and reactivity in the environment. Previous work has suggested that high-molecularweight (high-MW) DON exists only in two closely related forms, the secondary amides of peptides and of N-acetylated hexose sugars. Here, we demonstrate that the chemical structures of high-MW DON may be much more diverse than previously thought. We couple isotopic labeling of cyanobacterially derived dissolved organic matter with advanced twodimensional NMR spectroscopy to open the “black box” of uncharacterized high-MW DON. Using multibond NMR correlations, we have identified novel N-methyl-containing amines and amides, primary amides, and novel N-acetylated sugars, which together account for nearly 50% of cyanobacterially derived highMW DON. This study reveals unprecedented compositional details of the previously uncharacterized DON pool and outlines the means to further advance our understanding of this biogeochemically and globally important reservoir of organic nitrogen.

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reactivity of its components, and its often low concentration in the oceans. The few available structural studies of DON have mostly focused on the fraction that is isolated by ultrafiltration, referred to as high-molecular-weight DON (high-MW DON, usually >1 kDa) or ultrafiltered DON, and representing ∼30% of the DON pool.5−7,9,10 15N nuclear magnetic resonance (NMR) studies have found that amides account for >90% of nitrogen-containing moieties in high-MW DON.5,6,10 15N NMR spectra of dissolved organic matter (DOM) samples isolated on XAD-8 and XAD-4 resins also showed heterocyclic in addition to the dominant amide nitrogen.11 On the basis of 15 N NMR coupled with mild hydrolysis of high-MW DON, Aluwihare et al.10 concluded that nearly half of the high-MW DON in the surface ocean was composed of N-acetyl amino polysaccharides. The remaining high-MW DON was distrib-

issolved organic nitrogen (DON) is the largest reservoir (∼58−77%) of fixed dissolved N in most aquatic systems, with the exception of the nitrate-rich deep ocean and the highnutrient low-chlorophyll Southern Ocean, and has been implicated in a number of important upper-ocean processes.1 For instance, DON serves as a key intermediate in the microbial loop, being generated by and fueling primary as well as heterotrophic microbial production in surface waters.2−4 As the molecules that are associated with DON are also part of the dissolved organic carbon (DOC) pool, they have major implications for the global carbon cycle and consequently for sequestration of CO2 from the atmosphere. DON is predominantly of biotic origin5 and has a wide compositional range including biologically labile compounds such as amino acids and primary amines,1,2,6 and long-lived, biologically inert materials such as degradation-resistant residues of bacterial cell walls.7−9 Despite observations that many components of the DON pool are biologically reactive, much of the DON pool is uncharacterized due to the complexity of its composition, the variable biogeochemical © XXXX American Chemical Society

Received: June 16, 2017 Accepted: October 16, 2017

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DOI: 10.1021/acs.analchem.7b02335 Anal. Chem. XXXX, XXX, XXX−XXX

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After filtering through a prerinsed 0.22 μm cartridge filter, the filtrate (i.e., the operationally defined “dissolved” fraction) was passed through a tangential flow ultrafiltration system with a poly(ether sulfone) >1 kDa cartridge (Separation Engineering Inc.) to isolate the high-MW DOM fraction. Elemental analysis (C, H and N) of this sample was performed by Huffman Laboratories (Golden, Colorado).25 Particulate samples were collected on GF/F filters to determine total enrichment of cells using a Europa 20/20 Isotope Ratio Mass Spectrometer equipped with an automated nitrogen and carbon analyzer. The final enrichment of the cultured Synechococcus cells was >44.0% 15N and >74.4% 13C. More experimental details on Synechococcus cultures and isolation of high-MW DOM by ultrafiltration are given in Supporting Information (SI). NMR Spectroscopy. The NMR experiments were performed using a Bruker DSX400 spectrometer at 400 MHz for 1H, 100 MHz for 13C, and 40 MHz for 15N. A Bruker 4 mm triple-resonance magic-angle spinning (MAS) probe head was used. 13C chemical shifts were referenced to tetramethylsilane, using the COO resonance of glycine at 176.46 ppm as a secondary reference. 15N chemical shifts were indirectly referenced to liquid ammonia by setting the signal of Nacetyl-D-valine to 122 ppm. 15 N cross-polarization magic angle spinning (CP/MAS) spectra were acquired with or without 280-μs of gated decoupling for dipolar dephasing, with a CP time of 1 ms at an MAS frequency of 7 kHz. The Hartmann−Hahn condition for cross-polarization was set carefully on the nonprotonated N of 15N-tBOC-L-proline. The 90° pulse length on the 15N channel was 10.7 μs and the recycle delay was 2 s. Two-pulse phasemodulation (TPPM) decoupling was used during detection. Quantitative 13C direct polarization magic angle spinning (DP/ MAS) spectra were acquired at 13.5 kHz MAS. A Hahn echo requiring two rotation periods (tr) was used before detection to avoid baseline distortions, and TPPM decoupling was applied during detection. The recycle delay was 40 s. Carbons near 15N were selected by 13C{15N} rotational-echo double-resonance (REDOR).26,27 Two recoupling periods of 4 tr and 12 tr, corresponding to total REDOR times of 0.57 and 1.71 ms, respectively, were used. 13C spectra recorded with (S) and without (S0) 180° pulses on the 15N channel contain signals mostly of C not bonded to N and the full reference signal S0, respectively. The difference spectrum ΔS = S0 − S derives selectively from C bonded to N, with 25% excess in NCO peak area relative to the integrated NCHn intensity in Figure 2b. With 10 ms spin exchange, these primary amide nitrogen compounds showed cross peaks to OCH, but not to anomeric carbons (Figure S6e), unlike secondary amide N. Such connectivity, CH(−O)−CH(−O)− C(O)−NH2, is corroborated by the cross peak from 173ppm NCO to 71-ppm CH−OH in 15N−13C filtered 2D 13 C−13C exchange spectra (Figures 4 and S6f). Such a distinct cross peak to OCH will not arise from N-acetyl groups since their amide carbon is not bonded to an OCH and is at a threebond distance to multiple OCH with various chemical shifts. Possible origins of primary amides are discussed in SI. N-Acetylated Sugars. 2D 15N−13C NMR and 13C{15N} filtered 13C−13C exchange NMR spectra identified three types of NCH structures with distinct carbon chemical shifts (50.5, 56, and 60 ppm) (Figures 3 and 4). Among those, the 56-ppm NCH and 50.5-ppm NCH carbons were bonded to amide nitrogen, and therefore in HC−N−CO linkages and potentially present in N-acetylated sugars. Previous identifications and quantifications of N-acetylated sugars were mostly based on diagnostic masses using direct temperature-resolved (in-source pyrolysis) mass spectrometry,49 quantification of specific amino sugars using high-performance anion-exchange chromatography following HCl hydrolysis,50 quantification of acetic acid after mild hydrolysis,10,37 or integration of acetyl CH3 NMR signals at ∼23 ppm for 13C or ∼2 ppm for 1H.10,37 The advanced 2D NMR techniques employed here enable more accurate and specific assignments, and reveal new types of N-acetylated sugars different from previously identified abundant N-acetyl-glucosamine and N-acetyl-galactosamine in high-MW DOM.51 This could partly explain why the NMRderived estimates of N-acetylated amino polysaccharides are much higher than those from molecular-level amino sugar analyses focused on N-acetyl-glucosamine, N-acetyl-galactosamine, and muramic acid (40−50% vs 2−7% of nitrogen in surface high-MW DON).10,51 N-acetylated di- or trideoxyglucose was, for the first time, identified in the cyanobacterially derived high-MW DON. The DQ/exchange/SQ spectra (Figure S7b) exhibited clear cross peaks showing that the O−C−HC−N carbon pair (sum frequency of 73 + 56 ppm, indicated by a green arrow) was in close proximity to CH3. The strong cross peak between H3C− (HC−O) (DQ frequency of 73 + 19 ppm) and HC−N at 56 ppm in DQ/exchange/SQ spectra (Figure S7b and c) with a 3 ms spin exchange time further confirms the H3C−(HC−O)− (HC−N) three-carbon fragment. The 56 to 19 ppm cross peak even at a short spin exchange time (10 ms) in the 2D 13C−13C exchange spectrum (Figure S8a) also shows that HC-N and CH3 are in close proximity. The DQ/SQ spectra also establish that the 56-ppm NCH with a DQ frequency of 103.5 + 56 ppm (indicated by a green arrow, Figure S7a) is bonded to an anomeric O−(CH)−O carbon. This might suggest the following 4-carbon structure (with N-acetyl side group): H3C−(HC−O)−CH(−HN−(CO)−CH3)−O−(CH)−O. However, since a regular sugar ring has six or five carbons, one must consider whether there are two or more OCH carbons in the center of the structure; 2,4-diacetamido-2,4,6-trideoxy-Dglucose with and without ether linkage at C3, see Figure S7d, could explain the observed chemical shifts.52 But other structures with C6 in a methyl group, N-acetylated C4, and

Figure 6. Identification of N-methyl amine structures. Strip plots from (a) the 15N−13C spectrum without spin exchange; (b) same with 10 ms of spin exchange; (c) the 13C−13C with 13C{15N} REDOR difference spectrum with 30 ms spin exchange; and (d) the DQ/SQ 13 C−13C correlation spectrum. (e) Proposed N-methyl amine structural units, as well as chemical shifts from prediction (in parentheses) and experiment (in bold).

spectra measured in this study. The 2D 15N−13C NMR spectrum (Figure 3a and 6a) showed that this NCH3 carbon is bonded to amine nitrogen, proving a CH−NH−CH3 linkage. The next neighbors are OCH2 groups revealed by the doubly intense 66-ppm peak in the 15N−13C spectrum with 10 ms spin exchange (Figures 3b and 6b). The OCH2−CH−NH linkage was corroborated by the 66-ppm cross peaks in 15N−13C filtered 13C−13C (Figure 6c) and DQ/SQ (Figure 6d) spectra. The structure deduced from these connectivities is shown in Figure 6e. The absence of any other significant carbon cross peaks after 10 ms of spin exchange in Figure 6b confirmed that the network of directly bonded carbons is limited to the NCH carbon and two OCH2 carbons shown, which are separated from other carbons by heteroatoms. The predicted 13C chemical shifts (in parentheses), computed using the ACD/ NMR predictor, agree closely with our experimental chemical shifts (in bold). In particular, the NCH and OCH2 carbons in the deduced structure have characteristically higher chemical shifts than sugar-ring NCH carbons (bonded to acetyl groups) and OCH2 carbons, respectively, just as observed experimentally. Primary Amides. The 15N−13C correlation spectrum showed a significant amide signal component near 112 ppm 15 N, 173 ppm 13C (Figure 3a, see inset, marked in orange), but this 112-ppm 15N did not exhibit clear cross peaks to other carbons. In other words, the amide N in this group was bonded only to a CO carbon. This indicates that the moiety is a primary amide, −C(O)−NH 2 . This assignment was confirmed by the upfield 15N chemical shift of 112 ppm, which is typical of primary amides (e.g., in asparagine and glutamine side groups).24 The signal at 112 ppm (Figure S6a− F

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Figure 7. Previous and new views of the chemical composition of high-MW DON. The pie chart on the left shows the composition of high-MW DON from surface seawater, according to Aluwihare et al.,10 while that on the right displays the more diverse high-MW DON composition identified by our novel approach coupling isotopic labeling and multidimensional NMR.

Synechococcus-derived high-MW DON based on the fact that in the 2D spectrum (Figure 3a) the volume of primary amide signals accounted for ∼20% of the volume of amides, and that amide N accounted for 92% of all high-MW DON (Figure 1a). These three newly identified forms of DON accounted for ∼40% of all N in Synechococcus-derived high-MW DON. Mild acid hydrolysis of these molecular structures would not release the amino acids or acetic acid that were measured in previous studies.10 These compounds are quantitatively important, particularly because they are components of the uncharacterized DON fraction (30−40% of surface ocean high-MW DON, ∼75% of deep ocean high-MW DON). Novel N-acetyl structures contributed at least 11% of Synechococcus-derived high-MW DON, according to the relative volume integrals of NCO associated with 56-ppm NCH in the contour plot (Figure 3a) and the fraction of 56-ppm NCH that is linked to ether OC at C3. Thus, the previously uncharacterized forms of DON account for 40% (excluding N-acetyl) to 51% (including novel N-acetyl structures) of the total nitrogen present in the Synechococcus-derived high-MW DOM, significantly altering our view of the reactivity of nitrogen in DOM.

C3 linked to an ether oxygen, are also possible; for instance, the coexistence of N-acetyl-glucosamine (or β-N-acetyl-galactosamine) and of 4-acetamido-4,6-dideoxy-D-glucose (Figure S7d) would also explain the data. Although the assignment of 56ppm NCH to C2 of β-N-acetyl-galactosamine (which differs only in C4 configuration from N-acetyl-glucosamine) cannot be completely ruled out, the agreement of experimental NCH chemical shifts with N-acetylated glucose is better. 53 Furthermore, N-acetyl-galactosamine with an NCH resonance at 50.5 ppm was documented by the multibond NMR methods (see SI; labeled in magenta in Figures 3, 4, and S7a). Synopsis. The complex DON reservoir has frequently been treated as a “black box” containing a heterogeneous mixture of mostly uncharacterized organic compounds. For instance, ≤14% of marine DON is identified at the molecular level,54 and this small characterizable DON pool includes several compound classes such as urea, amino acids, nuclei acids, amino sugars, purines, pyrimidines, pteridines, creatine, as well as methylamines in mono-, di-, and trimethyl forms of ammonia.1 The dominant, molecularly uncharacterized DON component is accessible only to advanced analytical techniques. A study monitoring the effects of mild acid hydrolysis on 1H- and 15N NMR spectra concluded that high-MW DON consists of two chemically distinct pools of amide (N-acetyl amino polysaccharides vs unhydrolyzed amide) plus uncharacterized amine.10 Combining isotopic labeling and innovative multibond 2D NMR, we have “opened this black box”, demonstrating that the high-MW DON of a ubiquitous phytoplankton group, which exhibits 15N and 13C NMR signatures typical of marine DOM, contained a much more diverse array of N-containing structures than previously recognized (Figure 7). The relative amounts of the newly discovered nitrogen forms identified in this study (Figure 7) were estimated based on NMR peak integrals. The N-methyl amines accounted for ∼8% of Synechococcus-derived high-MW DON, determined from the well-resolved amine peak in the 15N spectrum (Figure 1a). NMethyl amides linked to sugar rings contributed twice as much N, or approximately 15%, of the Synechococcus-derived highMW DON, according to the 1.9:1 ratio of the 13C NMR peak areas of 27-ppm NCH3 to 31-ppm NCH3 (Figure 2(b,c)). The abundance of primary amides was estimated to be ∼18% of

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CONCLUSIONS This work demonstrates that coupling isotope-labeling of cyanobacterially derived DOM and multidimensional NMR opens a new analytical window for solving the complex structure of organic nitrogen in natural waters. Growing Synechococcus cultures on medium enriched with H13CO3− and 15NO3− has produced 13C- and 15N-enriched DOM with 13 C and 15N NMR signatures closely matching those of natural DOM. Advanced multibond NMR methods have enabled us to interrogate, in unprecedented detail, structures of 13C- and 15Nenriched DOM and revealed that the chemical structures of organic nitrogen in DOM are more diverse than previously recognized. Notably, several of the newly discovered moieties are not hydrolyzable and therefore invisible to wet chemical analyses. Our study outlines new avenues for further structural studies aimed at a better understanding of biogeochemically and globally important reservoirs of organic nitrogen. G

DOI: 10.1021/acs.analchem.7b02335 Anal. Chem. XXXX, XXX, XXX−XXX

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b02335. More details on Synechococcus cultures and isolation of high-MW DOM by ultrafiltration, possible origins of primary amides, N-acetyl-galactosamine, distinctive sugars without N, a conceptual graph explaining the multibond NMR approach, 13C spectra obtained as partial projections from 2D 15N−13C spectra, identification of carbon−carbon pairs and nearby carbons, carbons bonded to tertiary nitrogen, structural information from 13C(O)−13CH(−OH) spin pairs, primary amide structures, proximity of C-containing moieties, and 15N NMR spectra of extracellular DON and natural water DON (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xiaoyan Cao: 0000-0001-7571-6482 Klaus Schmidt-Rohr: 0000-0002-3188-4828 Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.analchem.7b02335 Anal. Chem. XXXX, XXX, XXX−XXX