# Solid-state NMR observation of chitin in whole cells by indirect 15N detection with NC, NCC, CNC and CNCC polarization transfers

Loic Delcourte, Mélanie Berbon, Marion Rodriguez, Laurence Delhaes, Birgit Habenstein, Antoine Loquet

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## To cite this version:

Loic Delcourte, Mélanie Berbon, Marion Rodriguez, Laurence Delhaes, Birgit Habenstein, et al. Solid-state NMR observation of chitin in whole cells by indirect 15N detection with NC, NCC, CNC and CNCC polarization transfers. Solid State Nuclear Magnetic Resonance, 2025, 137, pp.102002. 10.1016/j.ssnmr.2025.102002. hal-05383459

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# Solid-state NMR observation of chitin in whole cells by indirect <sup>15</sup>N detection with NC, NCC, CNC and CNCC polarization transfers

Loic Delcourte<sup>a</sup>, Mélanie Berbon<sup>a</sup>, Marion Rodriguez<sup>b</sup>, Laurence Delhaes<sup>b,c</sup>, Birgit Habenstein<sup>a</sup>, Antoine Loquet<sup>a,∗</sup>

## ARTICLE INFO

Handling Editor: Prof D Bryce

## ABSTRACT

Chitin is the most important nitrogen-containing polysaccharide found on Earth. This polysaccharide is a polymer of an N-acetylglucosamine and it is a crucial structural component of fungal cell walls and crustaceans. Magic-angle spinning solid-state NMR is emerging as a powerful analytical approach to study polysaccharides in the context of intact cell walls and whole cells. The presence of an acetamido group in chitin is attractive for <sup>15</sup>N solid-state NMR. Here we investigate the use of various multi-step polarization transfer experiments incorporating indirect <sup>15</sup>N detection at moderate spinning frequency, adapted from pulse sequences commonly employed for residue resonance assignment in biosolid proteins. The <sup>13</sup>C,<sup>15</sup>N chitin spin topology slightly differs from amino acids, and we discussed the use of frequency-selective <sup>15</sup>N-<sup>13</sup>C cross-polarization transfers followed by broadband or frequency-selective homonuclear <sup>13</sup>C-<sup>13</sup>C transfers to detect chitin resonances. Demonstrated here for chitin found in the cell wall of the fungus *Aspergillus fumigatus*, the use of indirect <sup>15</sup>N detection through multi-step polarization transfers could be advantageous to investigate more complex nitrogen-containing polysaccharides found in whole cells and peptidoglycan samples.

## 1. Introduction

Most cells from algal, fungal, yeast and bacterial species possess a so-called cell wall, which is a crucial surface component to protect the cell to the external environment. These cell walls have numerous functions and roles depending the considered species, e.g. a protective layer providing mechanical integrity, a selective barrier, or a structural shell to resist internal turgor pressure [1-3]. In addition, cell wall components can also play key roles in the process of pathogenesis, by masking pathogen-associated molecular patterns prior to the infection or by directly interact with receptors of immune cells to trigger devastating effects [4].

In fungi, the mechanisms of cell growth, cell division and morphotype changes critically rely on the plasticity and rigidity of the cell wall [5]. These macroscopic parameters are directly associated to the molecular composition and structural organization of polysaccharides in the cell wall, that should maintain enough structural rigidity to offer a

solid protective shell, while allowing enough plasticity to enable morphological transitions such as the spore germination. Fungal cell walls are located outside the plasma membrane and they constitute the first component seen by host cells and neighboring cells. They are mostly composed of polysaccharides, namely glucans and chitin [6]; additionally, proteins, lipids and water are also present. Because most polysaccharides found in fungi are unique, they constitute attractive targets for the development of antifungal drugs [7]. However, there are currently only few antifungal molecules available and efficient against human fungal pathogens, and the current rise of antifungal resistance is critical and the increase of drug-resistant strains is a major threat [8]. There is therefore an important interest to develop tools to investigate the molecular organization of polysaccharides in fungal cell walls and understand the molecular mechanisms of antifungal drug resistance.

Many analytical approaches to study cell wall polysaccharides used routinely in the field of fungal biology rely on the derivation of cell wall components by chemical extraction, thermic treatment and/or

This article is part of a special issue entitled: SSNMR of Biomaterials published in Solid State Nuclear Magnetic Resonance.

E-mail address: antoine.loquet@cnrs.fr (A. Loquet).

<footnote><sup>a</sup> Univ Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, Pessac, France</footnote>

<footnote><sup>b</sup> CNR des Aspergilloses Chroniques, Mycology-Parasitology Department, CHU Bordeaux, 33000, Bordeaux, France</footnote>

<footnote><sup>c</sup> Centre de Recherche Cardio-Thoracique de Bordeaux, Inserm, UMR 1045, Univ Bordeaux, 33000, Bordeaux, France</footnote>

<footnote>* Corresponding author.</footnote>
enzymatic essays to selectively isolate and solubilize polysaccharides. Such approaches have been widely used in the last three decades [6,9], however they are limited to investigate the native organization of the cell wall in the context of whole cells. For chitin, chemical extraction procedures involving acids and alkali are commonly employed [10,11], followed by analytical techniques such as mass spectrometry, infrared and solution NMR spectroscopies or X-ray diffraction analysis [12].

Chitin is the second most abundant biopolymer found on Earth after the cellulose, and the most abundant nitrogen-containing natural resource. It is a crucial constitutive component of fungal cell wall, as well as the shell of crab, insects or lobsters [13]. Chitin is insoluble in water and in most organic acids used in analytical chemistry and biochemistry. Moreover, most solvents used in the industry for chitin extraction (e.g. dichloroacetic acid, trichloroacetate, hexa-fluoroisopropyl alcohol, lithium thiocyanate) are toxic, corrosive and might lead to unwanted chitin hydrolysis. As a consequence, chitin analysis procedures often rely on its deacetylation into chitosan, already present in lower amount in native cell walls, which is a more soluble form used massively in the cosmetic and food industries [14].

In the past decade, solid-state NMR has emerged as a powerful technique to investigate polysaccharides embedded in the cell envelope, using isolated cell walls or intact cells [15–25] as well as using peptidoglycan samples [26–28]. The technique possesses the key advantage to be applicable to whole cells without chemical extraction and therefore deliver relevant structural and dynamics information of polysaccharides in a near-native environment. The group of Mei Hong have pioneered the use of multi-steps ¹³C-detected polarization transfer using magic-angle spinning NMR (MAS NMR) to propose a methodology to investigate the organization of cell envelope, as demonstrated for plant cell wall samples [29]. In the past five years, Wang and coworkers provided a precise description of the polysaccharide organization in the cell wall of several fungal species using multidimensional MAS NMR [30]. In particular, the structural polymorphism of chitin present in fungal cell walls has been characterized in details [31] using ¹³C-detected experiments, such as two dimensional ¹³C–¹³C correlation spectra using the CORD pulse sequence [32] and 2D¹³C–¹³C INADEQUATE. Extracted chitin samples from other sources have also been investigated by ¹D¹³C MAS NMR [33–36]. These ¹³C-detected MAS NMR techniques are efficient to investigate the structural polymorphism of chitin through ¹³C chemical shifts and line width measurements, however the spectral crowding observed for whole cell samples in the ¹³C dimension can be detrimental to distinguish chitin to non-chitinous molecular species found in the cell wall.

In the present study, we take advantage of the presence of an acetamido group in this polymer to examine the use of indirect ¹⁵N detection to enhance the specific detection of chitinous species by MAS NMR. We performed our study on intact whole cells of *Aspergillus fumigatus*, a pathogenic fungus causing aspergillosis such as invasive pulmonary aspergillosis, for which we recently described the cell wall evolution through its germination process [22]. We employed several multi-step polarization transfer techniques (pulse sequences in Supplementary Fig. 1), involving an indirect ¹⁵N dimension, that are routinely used for the resonance assignment of biosolid protein samples. By comparing the spin topology of chitin to amino acids found in protein samples, we discussed the advantages of these techniques for indirect ¹⁵N chitin detection via through-space polarization transfers. Demonstrated here on whole cells containing a rather simple ¹⁵N-containing polysaccharide, these techniques could be advantageous to investigate more complex cell envelopes containing several chemically distinct ¹⁵N-containing biopolymers or peptidoglycans.

# 2. Materials and methods

## 2.1. Sample preparation

Spores of *Aspergillus fumigatus* (ATCC-204305) were harvested from a

solid Sabouraud medium by scraping at the surface of the medium immersed in aqueous Tween 0.5 %. Conidia were separated from hyphae by vortexing and filtration through Miracloth® filter. Then, the conidial suspension was inoculated into 100 mL of M9 minimal medium containing U-¹³C₆-glucose (20 g/L) and ¹⁵N-labeled ammonium chloride (10 g/L) and incubated for 72 h at 30 °C at 150 rpm. The resulting mycelium was washed with deionized water twice. The centrifuged pellet was used to pack a 4 mm NMR rotor. Pure chitin was purchased from MP Biomedicals.

## 2.2. Solid-state NMR spectroscopy

All experiments on *A. fumigatus* cells were recorded at 600 MHz ¹H frequency (Bruker spectrometer) at 11 kHz MAS using a 4 mm HCN Bruker probe. A cross-polarization contact times of 1 ms (for ¹H-¹³C and ¹H-¹⁵N) and high-power (90 kHz) SPINAL-64 [37] proton decoupling were used. The temperature-dependent position of the water proton resonance was used to set the sample temperature at 5 °C [38]. ¹D¹³C and ¹⁵N cross-polarization (CP) spectra (Fig. 1) were recorded with 512 scans. ¹D (¹H-¹⁵N)- [13]C SPECIFIC-CP experiments (Fig. 2) were recorded with 512 scans, with a¹³C carrier frequency set at 55.7 and 177 ppm during the heteronuclear CP step for the two conditions described in the manuscript. A continuous wave decoupling of 83 kHz was used during the SPECIFIC-CP step, the contact was set to 5.5 ms. ¹D (¹H-¹⁵N-¹³C)- [13]C SPECIFIC-CP PDSD experiment (Fig. 3) were recorded with 512 scans, using a 50 ms mixing time, using the same conditions as described for the SPECIFIC-CP experiments. ¹D (¹H-¹⁵N-¹³C)- [13]C SPECIFIC-CP DREAM experiments (Fig. 4) were recorded with 512 scans, using an “up” tangential amplitude modulation of 3 ms with 1000 discrete steps, centered at 5.5 kHz with a ±10 % modulation and an estimated homonuclear dipolar coupling of 3640 kHz. During the DREAM sweep, a ¹H continuous wave (CW) decoupling with a field strength of 83 kHz was applied. ¹D (¹H-¹³C-¹⁵N)-¹³C experiments (Fig. 5) were recorded with 512 scans, with a¹³C carrier frequency shifted to 55.7 and 177 ppm during the heteronuclear CP step for the two conditions described in the manuscript. The contact time for the two heteronuclear CP was set to 5.5 ms. ¹D (¹H-¹³C-¹⁵N-¹³C)-¹³C DREAM experiment (Fig. 6) were recorded with 2048 scans, with a¹³C carrier frequency shifted to 95.5 ppm during the DREAM step, with the same conditions used for the ¹D (¹H-¹⁵N-¹³C)-¹³C SPECIFIC-CP DREAM. ¹D¹³C CP spectrum of commercial chitin was recorded at 500 MHz using 11 kHz MAS frequency with 256 scans.

# 3. Results and discussion

The simplified chemical structure of chitin is presented in Fig. 1A. It consists of a polymeric chain of N-acetylglucosamine (β-1,4 glucosidic linkage of 2-acetoamino-d-glucose). The β-1,4-linked glucose core units contain six carbon positions (C₁ to C₆), with ¹³C chemical shifts ranging to C₁~104 ppm, C₂~55 ppm, C₃~73 ppm, C₄~84 ppm, C₅~76 ppm, and C₆~60 ppm. The acetamido group contains a nitrogen (¹⁵N 120–125 ppm), a carbonyl C'~174 ppm and a methyl CH₃~23 ppm. We produced a culture of *A. fumigatus* cells, fully labeled on ¹³C and ¹⁵N positions. The ¹D ¹H-¹³C cross-polarization (CP) spectrum recorded at 600 MHz ¹H frequency and 11 kHz MAS of ¹³C,¹⁵N-labeled *A. fumigatus* cells is presented in Fig. 1C. Chitin ¹³C resonances are observed with the two other main polysaccharides found in the fungal cell wall, namely α- and β-1,3-glucan. The ¹³C chemical shift assignment has already been reported here [21,22]. The ¹⁵N signal obtained using a ¹H-¹⁵N CP (Fig. 1D) exhibits a broad signal and mainly encode for chitinous species, with additional ¹⁵N signals from surface proteins [21,22,39].

It is interesting to note that amino acid residues found in proteins and the chitin subunit share several similarities at the level of their ¹³C,¹⁵N spin topologies (Fig. 1B). The nitrogen of chitin possesses several topological features close to backbone amides found in proteins. First, the nitrogen is chemically attached to two carbon positions. For both chitin
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** $^{13}$C and $^{15}$N spin topology in chitin and protein residues. (A) Chitin simplified chemical structure. (B) Spin topology of two consecutive residues in a protein and spin topology of chitin. (C) 1D$^{13}$C cross-polarization spectrum of *Aspergillus fumigatus* cells (black) and commercial chitin (magenta). (D) 1D$^{15}$N cross-polarization spectrum of *A. fumigatus* cells. Spectra recorded at 600 MHz at 11 kHz MAS frequency. Chitin signals are highlighted.</figcaption></figure>

and amino acids, one of this carbon position is a carbonyl, with typical C' chemical shifts at ~175 ppm. It opens the possibility to perform indirect $^{15}$N chitin detection through direct detection of its carbonyl resonance, in a chemical shift range for which only carbonyl is detected. The second carbon position directly attached to $^{15}$N is Cα in proteins and C₂ for chitin. Protein Cα chemical shifts usually range between ~53 and 60 ppm with few exceptions (Glycine Cα ~45 ppm and proline Cα ~64 ppm). Cα chemical shift values depend on the amino acid type, as well as the secondary structure [40]. Considering a chitin C₂ chemical shift at ~55 ppm, the spin topology (i.e. C'-N-C) is highly similar between amino acids and chitin, and it opens an avenue to use heteronuclear $^{15}$N-$^{13}$C polarization transfers to transfer $^{15}$N magnetization to C' or C₂ spins.

Next, we considered the nature of the carbon(s) attached to the carbons covalently bonded to nitrogen. In protein residues, the pathway of polarization transfers can mainly go in three directions, i.e. N-C'-Cα, N-Cα-C' and N-Cα-Cx, with Cx encoding for side chain carbons. This spin topology, common to all residues except Proline and Glycine (no Cx), has promoted multi-step NCC polarization transfer as one the main spectroscopic tools for protein assignment by MAS NMR [41–44]. Examples include intra-residue side chain assignment with N<sub>i</sub>-Cα<sub>i</sub>-Cβ<sub>i</sub> and N<sub>i</sub>-Cα<sub>i</sub>-Cx<sub>i</sub> experiments and inter-residue sequential assignment with the N<sub>i</sub>-C'-i-1-Cx<sub>i-1</sub> experiment (with i and i-1 being adjacent residues in the protein sequence). Considering the chitin $^{15}$N,$^{13}$C spin topology, three pathways for polarization transfer can also be considered: N-C'-CH<sub>3</sub>, N-C<sub>2</sub>-C<sub>1</sub> and N-C<sub>2</sub>-C<sub>3</sub>. Note that CH<sub>3</sub>, C<sub>1</sub> and C<sub>3</sub> frequencies can be selectively selected on the basis on their distinct chemical shifts.

We took advantage of the similarities between the $^{15}$N,$^{13}$C topologies of chitin and amino acids to investigate the use of several multi-step polarization experiments involving an indirect $^{15}$N detection. These pulse sequences include a direct $^{13}$C detection and are commonly used at slow to moderate MAS frequencies (10–25 kHz) to perform intra-residue and inter-residue resonance assignment in biosolid protein samples [41, 43–47]. First, to provide an efficient polarization transfer between the

nitrogen nucleus of chitin and its neighboring carbon nuclei, we considered the use of heteronuclear $^{15}$N-$^{13}$C double cross-polarization (DCP [48]). This pulse sequence consists of a first CP step between abundant $^{1}$H spins to $^{15}$N spins, followed by a heteronuclear CP between $^{15}$N to $^{13}$C spins. Only $^{13}$C spins coupled to $^{15}$N are observed, providing an efficient tool to indirectly detect $^{15}$N signals via a$^{13}$C detection. Baldus and Griffin [49] introduced a highly efficient frequency selective version of DCP, called “spectrally induced filtering in combination with CP” (SPECIFIC-CP), that employs low radio-frequency $^{13}$C and $^{15}$N field strengths to provide a spectral filtering in $^{15}$N, $^{13}$C spin pairs. In this experiment, the $^{13}$C carrier frequency is shifted to the center of the spectral area of interest, i.e. Cα or C' for proteins, to obtain a frequency selective $^{15}$N-$^{13}$C polarization transfer. The mixing period typically used for proteins is ~3–5 ms, and high power continuous wave decoupling is used [50] during the heteronuclear CP step. This polarization transfer step gives access to the so-called SPECIFIC-CP $^{15}$N-$^{13}$Cα and $^{15}$N-$^{13}$C' (Fig. 2A), that are routinely used to obtain $^{15}$N, $^{13}$C spectral fingerprints for biosolid peptides and proteins. For cell walls containing chitinous species, the use of such frequency selective transfer strategy can be envisioned to obtain selective polarization transfers between the $^{15}$N spin to C₂ and C' respectively (Fig. 2B). Both amino acids and chitin constitute strongly $^{13}$C-coupled spin systems surrounded by a dense $^{1}$H environment. To optimize the duration of the SPECIFIC-CP transfer step for chitin detection in whole cells, we recorded a build-up magnetization curve by varying the length of the heteronuclear $^{15}$N-$^{13}$C CP step (Fig. 2C). We found a fast build-up leading to a maximum intensity at ~3 ms for a spinning frequency of 11 kHz, a value similar to optimal transfer lengths for biosolid proteins. Fig. 2D presents 1D spectra of the ($^{1}$H)- [13]C CP experiment, and two SPECIFIC-CP ($^{1}$H)- [15]N-$^{13}$C designed to selectively detect carbonyl and aliphatic signals, by setting the $^{13}$C frequency during the heteronuclear CP step to ~ 177 ppm and ~56 ppm respectively. We observed a selective $^{15}$N to $^{13}$C transfer to chitin C' with a CP $^{13}$C carrier frequency set at ~177 ppm; and for C₂ for
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** $^{15}$N indirect detection of chitin with heteronuclear SPECIFIC-CP polarization transfers. (A) Polarization transfer pathways in protein residues by SPECIFIC-CP NCA (left) and NCO (right). (B) Polarization transfer pathway in chitin by SPECIFIC-CP NC₂ (left) and NC' (right). Magnetization build-up curves for $^{15}$N- $^{13}$C' (red) and $^{15}$N- $^{13}$C₂ (blue). (D) 1D $^{13}$C CP (black), SPECIFIC-CP ($^{15}$N- $^{13}$C') (red) and SPECIFIC-CP ($^{15}$N- $^{13}$C₂) (blue) spectra of *A. fumigatus* cells.</figcaption></figure>

a carrier set at ~55 ppm. In summary, the SPECIFIC-CP $^{15}$N- $^{13}$C experiment is well suited to indirectly detect chitin in the cell wall of whole cells, with respect to other polysaccharides that do not contain nitrogen spins.

Next, we investigated the addition of a$^{13}$C-$^{13}$C polarization transfer step to the SPECIFIC-CP $^{15}$N-$^{13}$C. Such multi-step polarization transfer experiments involving $^{15}$N-$^{13}$C-$^{13}$C correlations is particularly useful to discriminate $^{13}$C signals by adding a second carbon edition. Several $^{13}$C-$^{13}$C recoupling schemes have been proposed for biosolid proteins to set up $^{15}$N-$^{13}$C-$^{13}$C experiments at moderate MAS frequencies, e.g. proton-driven spin-diffusion (PDSD [51]), Dipolar Assisted Rotational Resonance (DARR [52]), CORD [32] or band-selective homonuclear CP (BSH-CP [53]). Mixing times of ~25–100 ms are usually employed to provide efficient polarization transfers between spatially close carbons. In proteins, such pulse sequences give access to Nᵢ-C'ᵢ₁-Cₓᵢ₁, Nᵢ-Cαᵢ-Cₓᵢ and Nᵢ-Cαᵢ-C'ᵢ experiments (Fig. 3A) that are particularly useful to connect backbone and side chain $^{13}$C spin system via their $^{15}$N frequencies [41]. In the case of chitin, the use of an additional $^{13}$C-$^{13}$C recoupling scheme after the SPECIFIC-CP is proposed (Fig. 3B) to obtain a$^{15}$N-$^{13}$C₂-$^{13}$C₁ and $^{15}$N-$^{13}$C₂-$^{13}$C₃ with a DCP carrier frequency set at

~55 ppm and a$^{15}$N-$^{13}$C'-$^{13}$CH₃ for a$^{13}$C carrier at ~177 ppm. Fig. 3C presents 1D$^{13}$C-detected experiments recorded under the two conditions by using spectral spin diffusion achieved with the PDSD scheme with a mixing time of 50 ms and 11 kHz MAS frequency. For the $^{15}$N-$^{13}$C₂-$^{13}$C (Fig. 3C, in pale blue) the use of a PDSD recoupling provides a broadband $^{13}$C-$^{13}$C polarization transfer between the C₂ spin and spatially-closed carbon positions of chitin. The signal is spread between all carbon spins, which correspond to a well-known feature of the NCC experiment employing a PDSD scheme, which provide a broadband and unspecific polarization transfers between spatially close carbon spins [41]. For the $^{15}$N-$^{13}$C'-$^{13}$C (in green in Fig. 3C), an intense signal is observed at the CH₃ frequency, the carbon position that is chemically bonded to C'. In conclusion, the multi-step polarization transfer offered by the NCC experiment is suitable to detect chitin species.

The NCC-PDSD pulse sequence is a straightforward experiment to set up and does not require intense field strength, however it leads to a broadband $^{13}$C-$^{13}$C polarization transfer during the recoupling step. To implement a frequency selective $^{13}$C-$^{13}$C recoupling for indirect $^{15}$N chitin detection, we propose a homonuclear double-quantum transfer scheme based on the DREAM sequence (dipolar recoupling
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** $^{15}$N indirect detection of chitin with NCC polarization transfer. (A) Polarization transfer pathways in protein residues by NCACX (left) and NCOCX (right). (B) Polarization transfer pathways in chitin by $NC_2C_{1/3}$ (left) and $NC'CH_3$ (right). (C) $1D^{13}C$ CP (black), SPECIFIC-CP-SD $^{15}N$-$^{13}C'$-$^{13}CH_3$ (green) and $^{15}N$-$^{13}C_2$-$^{13}C_{1/3}$ (pale blue) experiments of *A. fumigatus* cells.</figcaption></figure>

enhancement through amplitude modulation [54]. In this NCC-DREAM experiment (Fig. 4), a radio-frequency amplitude modulation matching half of the MAS frequency is employed to provide an efficient polarization transfer between spatially close carbon spins [55,56]. At slow-moderate MAS frequencies, chemical-shift selectivity can be implemented by a careful optimization of the ¹³C carrier position during the DREAM scheme, as demonstrated for protein samples [56]. By incorporating the DREAM scheme after a SPECIFIC-CP, a ¹H-¹⁵N-¹³C-¹³C can be implemented and optimized with respect to the ¹³C carrier position during the SPECIFIC-CP and DREAM steps to select signals based on their chemical shift position. The NCC-DREAM experiment [41] has been designed to detect $N_i-C\alpha_i-C\beta_i$ correlations (Fig. 4A) in proteins and peptides, by setting the DREAM carrier frequency in the

middle of the $C\alpha$ and $C\beta$ amino acid resonances (i.e. ¹³C carrier frequency at ~50–60 ppm [41,57]). In a recent study, we reported the use of the DREAM scheme to obtain a spectral editing of polysaccharides in whole cells via $2D^{13}C-^{13}C$ experiments [58]. For chitin, two possibilities for NCC polarization transfer pathways can be envisioned. By implementing a ¹³C carrier position at ~55 ppm during the SPECIFIC-CP step, the polarization is mainly set on the $C_2$ spin, and could be theoretically transferred to one-bond $C_1$ (~105 ppm) or $C_3$ (~75 ppm) (Fig. 4B), by shifting the carrier around the middle of the two recoupled frequencies. Fig. 4C shows a $1D^{15}N-^{13}C_2-^{13}C$ obtained by setting the SPECIFIC-CP and the DREAM ¹³C carrier position at 55 ppm. In this condition, the $(^{15}N-^{13}C_2)^{13}C_3$ polarization transfer is favored. Note that the DREAM scheme creates a negative-intensity signal for $C_3$.
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** ¹⁵N indirect detection of chitin with NCC-DREAM polarization transfer. (A) Polarization transfer pathways in protein residues by NCACB-DREAM (left). (B) Polarization transfer pathways in chitin by NC₂C₃-DREAM. (C) 1D¹³C CP (black), SPECIFIC-CP ¹⁵N-¹³C₂ (blue) and SPECIFIC-CP-DREAM ¹⁵N-¹³C₂-¹³C₃ (pale blue) experiments of *A. fumigatus* cells.</figcaption></figure>

Considering the chitin spin topology (Fig. 4B), a second polarization transfer pathway is theoretically conceivable. If the ¹³C carrier position is shifted to ~175 ppm during the SPECIFIC-CP, the polarization is set to the carbonyl C' position, and one can envision a C'-CH₃ DREAM transfer with a carrier frequency shifted at ~100 ppm during the ¹³C-¹³C recoupling. However, such recoupling between carbonyl and methyl spins implies a large chemical shift difference of ~22.5 kHz on a 600 MHz spectrometer. This condition for a selective C'-CH₃ DREAM polarization transfer is therefore not compatible with the use of large volume NMR rotors (in this study a 4 mm rotor), because the maximum MAS frequency rate is limited to ~12 kHz, which is considerably lower than the isotropic chemical shift difference between the considered spins. Much faster MAS frequency rates, only achievable with NMR probe heads such as 1.3- and 0.7-mm probes, would be suitable to efficiently recouple C'-CH₃ using the DREAM scheme.

Next, to expand the use of multi-step heteronuclear polarization transfer experiments for indirect ¹⁵N detection of chitin, we considered CNC-type pulse sequences. In proteins, the backbone ¹⁵N spin is attached to Cα and C' spins and one can achieve ¹³C-¹⁵N-¹³C polarization transfer by using two consecutive SPECIFIC-CP steps. For proteins, this pulse sequence has been implemented by Rienstra and coworkers to set up the Cα-N-C' experiment [59], providing useful connectivity's to link sequential residues through their Cαᵢ-Nᵢ-C'ᵢ-1 correlations (Fig. 5A). Note that a C'NCα experiment can also be set up by adjusting ¹³C carrier

frequencies during the two DCP steps. Here we proposed to implement the CNC experiment to indirectly detect ¹⁵N chitin signals, by considering two polarization transfer pathways (Fig. 5B). By setting the first SPECIFIC-CP carrier frequency at 55 ppm, and then a second SPECIFIC-CP at 175 ppm, one can envision a C₂-N-C' polarization transfer pathway. In analogy, by reverting the choice of the carrier frequencies, a C'-N-C₂ experiment is amenable. We implemented 1D version of these two experiments (Fig. 5C). For the C₂-N-C' experiment (in red), the signal is detected at 175 ppm at the C' position, indicating a selective polarization transfer pathway during the CNC pulse sequence. For the C'-N-C₂ experiment (in blue), the C₂ signal is also selectively detected.

For the last 1D experiment considered (Fig. 6), we took advantage of the 4D (H)CNCC heteronuclear experiment developed by the Rienstra group for proteins [60]. In this experiment, two consecutive SPECIFIC-CP transfers are used with an additional ¹³C-¹³C recoupling using the DARR scheme to achieve Cαᵢ-Nᵢ-C'ᵢ-1-Cxᵢ-1 correlation experiments for proteins. The pulse sequence is particularly useful to disambiguate crowded spectral region in large proteins, typically when the nitrogen resolution is not enough optimal to separate all sequential correlations in the Nᵢ-Cαᵢ-Cxᵢ experiment. This experiment is also powerful to correlate Cα to sequential side chain ¹³C nuclei (Fig. 6A). In the case of chitin, such multi-step polarization transfer experiment would help to correlate the carbonyl position to either C₁ and/or C₃, via
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** $^{15}$N indirect detection of chitin with CNC polarization transfers. (A) Polarization transfer pathways in protein residues by CANCO (left) and CONCA (right). (B) Polarization transfer pathways in chitin by C₂NC (left) and C₄NC₂ (right). (C) 1D$^{13}$C CP (black), C₂NC (red) and C₄NC₂ (blue) experiments of *A. fumigatus* cells.</figcaption></figure>

$^{13}$C-$^{15}$N-$^{13}$C$_2$-$^{13}$C$_{1/3}$ correlations (Fig. 6B). In this study, we replace the broad band DARR $^{13}$C-$^{13}$C recoupling by the DREAM scheme, to provide more selectivity according to the $^{13}$C carrier frequency. We recorded a 1D CNCC-DREAM on *A. fumigatus* cells (Fig. 6C), using a$^{13}$C carrier frequency at 55 ppm, shifted to 95 ppm during the DREAM scheme. The resulting 1D C'NCC spectrum led to a negative-intensity peak at 105 ppm, encoding for a C$_2$-C$_1$ transfer during the DREAM step. Therefore, the proposed experiment confirms the 3-step polarization transfer between C' and C$_1$.

4. Conclusions and outlook

Chitin is the most important nitrogen-containing biopolymer and
understanding its structural role in the molecular organization of fungal
or insect cell wall is of prime interest. MAS solid-state NMR, together
with infrared spectroscopy [61] and elemental analysis [62] is one the

main analytical tools to detect, quantify and distinguish various struc-
tural forms of chitin. ¹³C chemical shifts constitute an exquisite exper-
imental information to investigate chitin in complex matrices such as
whole cells and extracted cell walls. Here we presented the application
of several MAS NMR pulse sequences, commonly used by the NMR
community to perform resonance assignment in biosolid proteins, to
investigate nitrogen-containing polysaccharide species in whole cells.
We discussed the use of ¹⁵N indirect detection to improve the ¹³C NMR
detection of nitrogen-containing chitin. Our study was carried out at
moderate MAS frequency (11 kHz) and on a medium NMR field (600
MHz, 14.1 T), this instrumental set up being accessible to numerous
research laboratories working on biopolymers and carbohydrates. We
first employed multi-transfer polarization transfers via the (¹H-¹⁵N)-¹³C
experiment to perform a¹³C-detected spectral edition of ¹⁵N chitin
resonance. The use of the SPECIFIC-CP pulse sequence, widely used in
protein NMR to obtain heteronuclear ¹⁵N-¹³C fingerprint correlation
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** ¹⁵N indirect detection of chitin with CNCC-DREAM polarization transfers. (A) Polarization transfer pathways in protein residues by CONCACC-BREAM (left). (B) Polarization transfer pathway in chitin by C'NC₂C₁. (C) ¹D¹³C CP (black), C'NC₂ (blue) and C'NC₂C₁ (magenta) experiments of *A. fumigatus* cells.</figcaption></figure>

spectra, is advantageous as it provides a frequency-selective polarization transfer to design either ¹⁵N-¹³C' or ¹⁵N-¹³C₂ experiments depending the choice of the ¹³C carrier frequency. We extended the SPECIFIC-CP by introducing additional polarization transfers either after the heteronuclear step (i.e. NCC-PDSD and NCC-DREAM) or before (i.e. CNC) to improve ¹³C chemical shift assignment by providing respectively C'-CH₃/C₂-C₁/C₂-C₃ and C₂-N-C' and C'-N-C₂ transfer steps. Ultimately, additional polarization transfers can be implemented before and after the SPECIFIC-CP block in the ¹³C-¹⁵N-¹³C-¹³C pulse sequence to establish C'-C₁ transfers. All pulse sequences presented in our study are efficient at moderate MAS frequency and do not suffer too much from dipolar truncation effects [63,64]. They also present the advantage to be straightforward to implement and not demanding in terms of amplifier power levels. The fungal system of interest presented in our study is rather simple with the presence of only one main nitrogen-containing polysaccharide in its rigid cell wall. We anticipate that bacterial cells, that contain sophisticated peptidoglycan structures, might lead to more complex ¹⁵N signal network, challenging to identify solely based on their ¹⁵N chemical shift values. For instance, in Gram-positive bacteria D-Alanine from teichoic acid is challenging to quantify, and seminal work by the group of Cegelski demonstrated that composite MAS NMR pulse sequences involving indirect ¹⁵N detection provided an efficient tool to investigate and quantify D-Alanine in the context of whole bacterial cells [65,66]. In the same way, several fungal and yeast species,

such as *Cryptococcus neoformans*, contain an unusual high level of deacetylated chitin (chitosan) [67,68], a biopolymer that exhibit slightly different ¹³C chemical shifts when compared to the acetylated form (chitin) [31]. The use of multi-step polarization transfers involving indirect ¹⁵N detection, as presented in our study or in previous work by Cegelski and coworkers [65], will provide convenient MAS NMR approaches to tackle such microbial cell surfaces.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This work was supported by the PSGAR-MIE "EMERG" (to L.D. and A.L). We thank Estelle Morvan for technical assistance. This work has benefited from the Biophysical and Structural Chemistry Platform at IECB, CNRS UAR3033, INSERM US001, University of Bordeaux.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ssnmr.2025.102002.

## Data availability

Data will be made available on request.

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