# Magic-angle spinning NMR spectral editing of polysaccharides in whole cells using the DREAM scheme

Loic Delcourte, Mélanie Berbon, Marion Rodriguez, Kamalraj Subban, Alons Lends, Axelle Grélard, Estelle Morvan, Birgit Habenstein, Sven Saupe, Laurence Delhaes, et al.

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Loic Delcourte, Mélanie Berbon, Marion Rodriguez, Kamalraj Subban, Alons Lends, et al. Magic-angle spinning NMR spectral editing of polysaccharides in whole cells using the DREAM scheme. Methods, 2024, 230, pp.59-67. 10.1016/j.ymeth.2024.07.003. hal-04667178

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# Magic-angle spinning NMR spectral editing of polysaccharides in whole cells using the DREAM scheme

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Loic Delcourte<sup>a</sup>, Mélanie Berbon<sup>a</sup>, Marion Rodriguez<sup>b</sup>, Kamalraj Subban<sup>c</sup>, Alons Lends<sup>a</sup>, Axelle Grélard<sup>a</sup>, Estelle Morvan<sup>d</sup>, Birgit Habenstein<sup>a</sup>, Sven J. Saupe<sup>e</sup>, Laurence Delhaes<sup>b,f</sup>, Vishukumar Amanianda<sup>g</sup>, Asen Daskalov<sup>c,h,*</sup>, Antoine Loquet<sup>a,*</sup>

## ABSTRACT

Most bacterial, plant and fungal cells possess at their surface a protective layer called the cell wall, conferring strength, plasticity and rigidity to withstand the osmotic pressure. This molecular barrier is crucial for pathogenic microorganisms, as it protects the cell from the local environment and often constitutes the first structural component encountered in the host-pathogen interaction. In pathogenic molds and yeasts, the cell wall constitutes the main target for the development of clinically-relevant antifungal drugs. In the past decade, solid-state NMR has emerged as a powerful analytical technique to investigate the molecular organization of microbial cell walls in the context of intact cells. ¹³C NMR chemical shift is an exquisite source of information to identify the polysaccharides present in the cell wall, and two-dimensional ¹³C–¹³C correlation experiments provide an efficient tool to rapidly access the polysaccharide composition in whole cells. Here we investigate the use of the adiabatic DREAM (for dipolar recoupling enhancement through amplitude modulation) recoupling scheme to improve solid-state NMR analysis of polysaccharides in intact cells. We demonstrate the advantages of two-dimensional ¹³C–¹³C experiments using the DREAM recoupling scheme. We report the spectral editing of polysaccharide signals by varying the radio-frequency carrier position. We provide practical considerations for the implementation of DREAM experiments to characterize polysaccharides in whole cells. We demonstrate the approach on intact fungal cells of *Neurospora crassa* and *Aspergillus fumigatus*, a model and a pathogenic filamentous fungus, respectively. The approach could be envisioned to efficiently reduce the spectral crowding of more complex cell surfaces, such as cell wall and peptidoglycan in bacteria.

## 1. Introduction

Most microbes including bacteria, yeasts, filamentous fungi and pathogenic molds present an elaborated structural organelle at their surface called the cell wall (CW). The CW is found outside of the lipidic plasma membrane and helps the cell to withstand the osmotic pressure. As such, the CW possesses an intrinsic rigidity allowing the cell to maintain a mechanical integrity and avoid unwanted cell lysis [1]. This mechanical strength is crucial for microbe survival when cells are exposed to external environmental stress such as fluctuating chemical/ pH conditions or competing microorganisms or viruses (e.g.

bacteriophages for bacteria). Moreover, pathogenic microbes might directly encounter host immune defenses during the infection process, and the CW often constitutes the first component recognized by host receptors [2,3]. While conferring the required cell surface rigidity, the fungal CW possess a complex structure allowing some degree of dynamics, e.g. to undergo morphologically remodeling during cell growth [4,5] or to allow the diffusion of small molecules or extracellular vesicles [6]. Due to its critical role in survival and proliferation of pathogenic microbes, the CW constitutes a target of choice for the development of antimicrobial compounds. Because microbial CW biomolecules are absent in humans, the development of antibiotics and/or antifungals

<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, Bordeaux 33000, France</footnote>

<footnote><sup>c</sup> ImmunoConcEpT, CNRS, UMR 5164, University of Bordeaux, Bordeaux, France</footnote>

<footnote><sup>d</sup> Univ. Bordeaux, CNRS, Inserm, IECB, UAR3033, US01, Pessac, France</footnote>

<footnote><sup>e</sup> CNRS, Université de Bordeaux, IBGC, UMR 5095, Bordeaux, France</footnote>

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

<footnote><sup>g</sup> Institut Pasteur, Université Paris Cité, Immunobiology of Aspergillus, Mycology Department, Paris, France</footnote>

<footnote><sup>h</sup> State Key Laboratory for Managing Biotic and Chemical Treats to the Quality and Safety of Agro-products, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China</footnote>

<footnote>* Corresponding authors at: ImmunoConcEpT, CNRS, UMR 5164, University of Bordeaux, Bordeaux, France and CBMN, CNRS, UMR5248, University of Bordeaux, France.</footnote>

<footnote>E-mail addresses: asen.daskalov@u-bordeaux.fr (A. Daskalov), a.loquet@iecb.u-bordeaux.fr (A. Loquet).</footnote>
targeting the biosynthesis of CW components remains one of the most promising route to fight the current global antimicrobial resistance [7,8].

Considering filamentous and yeast fungal pathogens, the CW presents a typical molecular composition, mainly dominated by glucans, chitins and glycoproteins [9]. Their complex organization into a semi-rigid cell wall allows fungi to deceive innate immune recognition and poses important challenges to control fungal infections. The most promising clinically-relevant treatment against fungal infections is associated with the targeting of the CW biosynthesis, e.g. echinocandins that target β-(1,3)-glucan synthase [10], while nikkomycin Z targets chitin biosynthesis and has been demonstrated to be effective against fungal infection in animal model [11]. Although the chemical composition and the global molecular organization of fungal CWs have been investigated for well-studied species such as *Aspergillus fumigatus* [12], the precise details of the spatiotemporal evolution during CW biosynthesis and how this organelle compensates for the effect of antifungal drugs are still under debate [13,14]. Hoenigl [15]. Analytical approaches based on harsh chemical solubilization using acids or alkali have been used to isolate polysaccharide-rich fractions and analyze them by gas-chromatography coupled with mass spectrometry or solution NMR [16,17] to decipher the chemical nature of fungal CW polysaccharides. However, these semi-destructive approaches are of limited use to understand the native organization of the CW and the structural and dynamic interplay between its different components [18].

In the past decade, solid-state nuclear magnetic resonance spectroscopy, especially performed under magic-angle conditions (MAS NMR), has emerged as a powerful analytical technique to study the molecular organization of CWs in the context of non-solubilized samples. MAS NMR is not limited by the solubility, the crystallinity, the size or the structural homogeneity of the sample. The technique can be applied to biomaterials such as organelles [19], cellular environment [20,21] as well as CWs in prokaryotic or eukaryotic cells [22]. For such samples, MAS NMR techniques present the advantages to be applicable directly on intact cellular preparation obtained directly after culture, without chemical or enzymatic pretreatments that alter the native CW organization.

Pioneered by the group of prof. Mei Hong on plant CWs [23], multi-dimensional ¹³C-detected MAS NMR techniques, such as two-dimensional (2D) ¹³C-¹³C correlation experiments, provides structurally-relevant information to identify the chemical nature of polysaccharides and quantify their molecular abundance within native samples. During the past decade, MAS NMR approaches have been applied to investigate the molecular organization of CWs of various cell types, including fungi [24–26], bacteria[20,27,28], plants [29], algae [30] or on extracted peptidoglycan samples[31–33]. Despite recent commercial release of few ultra-high NMR magnets (>1GHz) and MAS probes allowing very fast spinning frequencies (>100 kHz) and ¹H detection, examples of MAS NMR studies using such developments are still extremely scarce [30,34–37,83]. As a consequence, most laboratories working on polysaccharide analysis by MAS NMR usually employ pulse sequences based on direct and indirect ¹³C detection at moderate MAS frequencies (10–20 kHz). This approach has the advantage to readily give access to ¹³C polysaccharide chemical shifts, which constitute a highly valuable source of information to identify the chemical nature of the monosaccharide subunits, the branching patterns and the glycosidic linkages [38,18]. As an example, Wang and coworkers recently released a database of MAS NMR ¹³C chemical shifts to speed up the assignment process [39]. Two 2D experiments are routinely employed to obtain high-resolution correlation spectra and investigate the polysaccharide composition in CWs, i.e. the 2D single-quantum single-quantum (SQ-SQ) dipolar ¹³C-¹³C correlation experiment and the 2D double-quantum single-quantum (DQ-SQ) ¹³C-¹³C INADEQUATE [40,41]. Their combined use is particularly useful for biological samples containing polysaccharides, due to the relatively poor ¹³C chemical shift dispersion for carbohydrates and the absence of diagonal

peaks in the INADEQUATE experiment. 2D SQ-SQ ¹³C-¹³C experiments traditionally used to assign protein ¹³C resonances have been applied to CW analysis. These pulse sequences include proton-driven spin diffusion (PDSD [42,43]), dipolar-assisted rotational resonance (DARR [44]) or CORD [45], with examples for cell wall analysis reported by Wang [24,46] and us [25]. These sequences present the key advantage to be applicable at slow and moderate MAS frequencies (~5–20 kHz) [47,48] and, from a spectroscopic point of view, are straightforward to implement on a NMR spectrometer. In 1998, the group of prof. Beat H. Meier introduced the DREAM scheme (for dipolar recoupling enhancement through amplitude modulation) [73,74]. This adiabatic-passage technique employs a radio-frequency (RF) amplitude modulation, with a ¹³C irradiation matching conditions around half of the MAS frequency to obtain efficient polarization transfers covering a large chemical shift range in proteins [49–51]. The DREAM scheme has several advantages, such as (i) a moderate ¹³C RF-field strength requirement compared to other recoupling techniques, (ii) efficient polarization transfer and (iii) to be frequency selective at slow-moderate MAS. The DREAM scheme has been implemented in several 2D and 3D pulse sequences used for protein residue assignment, such as ¹⁵N-¹³Cα-¹³Cβ for intra-residue assignment [50], ¹⁵N(¹³CO)¹³Cα-¹³Cβ for sequential assignment or ¹³C-¹³C-¹³C experiments [52–54]. Moreover, Westfeld et al. [55] demonstrated that the chemical-shift selectivity of the DREAM scheme could be an advantage to create spectral editing in 2D ¹³C-¹³C correlation spectra of amino acids, in order to improve the process of residue resonance assignment.

Inspired by the work of Meier and coworkers on protein samples and small molecules,55], we here investigated the potential of the DREAM scheme to study polysaccharides in the context of the analysis of whole cells. We provide a detailed analysis of the spectral pattern obtained using the DREAM sequence in 2D ¹³C-¹³C experiments. We demonstrate that the tuning of the carrier frequency during the DREAM scheme (the pulse sequence is shown in Fig. 1 and Supplementary Fig. 1) could be used to provide a spectral editing and reduce the spectral crowding. Finally, we provide practical considerations for the implementation of the DREAM sequence for whole cell samples.

## 2. Material and methods

### 2.1. Sample preparation

Spores of *Neurospora Crassa* (FGSC2489) were spotted on solid Vogel's medium and then incubated for 2 days at 30 °C in dark condition and 2 days at room temperature. Fresh conidia were used to inoculate solid Vogel's medium containing ¹³C-labeled glucose (15 g/L). Tubes were then incubated for 2 days at 30 °C in dark condition and 2 days at room temperature to promote sporulation. Conidia were collected in milli-Q water by vortexing and filtered through Miracloth® filters. Final conidial suspension was washed in deionized water several times by centrifugation steps (5000 × g, 15 min) and the pellet was used to fill in a 4 mm NMR rotor.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Pulse scheme of the DREAM experiment used for two-dimensional mononuclear ¹³C-¹³C correlation spectroscopy. CP: cross-polarization. Dec: decoupling. CW: continuous wave.</figcaption></figure>
Spores of *Aspergillus fumigatus* (ATCC204305) 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, 500 µL of 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. Then paraformaldehyde (PFA) fixation [56] was performed as follows: the mycelium was incubated for 30 min at room temperature with 2.5 % PFA in PBS with a 1:1 mycelium to buffer ratio (v/v). After discarding the supernatant, the fresh fixation buffer was added, and the suspension was incubated at 4 °C overnight. The next day, residual PFA was quenched with three washes of 0.1 M ammonium chloride and washed twice with deionized water. The pellet was used to pack a 4 mm NMR rotor.

## 2.2. Solid-state NMR spectroscopy

All experiments were recorded at 600 MHz ¹H frequency (Bruker spectrometer) at 11 kHz MAS using a 4 mm HCN Bruker probe. A cross-polarization contact time of 1 ms and high-power (90 kHz) SPINAL-64 [57] proton decoupling were used. The temperature-dependent position of the water proton resonance was used to set the sample temperature at 5 °C [58].

1D ¹³C cross-polarization (CP) spectra (Fig. 2) of *N. crassa* and HET-s (218–289) were recorded with respectively 512 and 20 k scans.

2D ¹³C–¹³C PDSD spectrum of *N. crassa* was recorded with a mixing time of 50 ms. Acquisition time of 4.5 and 15 ms were used in indirect and direct dimension respectively for a total acquisition time of 10 h.

2D ¹³C–¹³C DREAM spectra of *N. crassa* were recorded at various sweep lengths as described in the manuscript. Acquisition time of 4.5 and 15 ms were used in indirect and direct dimension for a total

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** ¹³C chemical shift dispersion for amino acids in proteins and polysaccharides. (A) Averaged ¹³C chemical shifts for all amino acids [82]. Cα's are colored in red. (B) Representative set of ¹³C chemical shifts for the main polysaccharide units found in plants and fungal microorganisms, extracted from the CCMRD database [39]. (C) ¹³C cross-polarization spectrum of uniformly ¹³C-labeled HET-s fibrils (218–289). (D) ¹³C cross-polarization spectrum of ¹³C-labeled N. crassa. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

acquisition time of 11 h. An “up” tangential amplitude modulation was used 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 90 kHz was applied.

2D ¹³C–¹³C PDSD spectrum of *A. fumigatus* was recorded with a mixing time of 50 ms. Acquisition time of 11 and 15 ms were used in indirect and direct dimension respectively for a total acquisition time of 33 h.

2D ¹³C–¹³C DREAM spectrum of *A. fumigatus* was recorded with a mixing time of 3 ms. Acquisition time of 11 and 15 ms were used in indirect and direct dimension respectively for a total acquisition time of 33 h. A tangential amplitude modulation was used for the DREAM scheme as described for the *N. crassa* sample.

¹³C T₁ relaxation times were measured based on an inversion-recovery experiment with a CP excitation. The relaxation times were measured with a delay variation from 0.005 ms to 60 s.

## 3. Results and discussions

The variety of chemical moieties present in polysaccharides is higher than in side chains of protein amino acids, however their chemical shifts span a similar range. Fig. 2 presents typical ¹³C chemical shifts found in amino acids, with a distribution that spans between ~ 10 to ~ 185 ppm. Cα chemical shifts (colored in red in Fig. 2A) spans around ~ 55–65 ppm, with the exception of glycine Cα (~47 ppm). As illustrated in Fig. 2C with the 1D ¹³C CP spectrum of HET-s (218–289) amyloid fibrils, it leads for biosolid proteins to a broad distribution of Cα and side chain signals between ~ 10–68 ppm, a narrow distribution for carbonyl ¹³C' at ~ 178 ppm and few additional signals for aromatic side chains (~110–140 ppm) and Arginine side chain tails (~160 ppm). For polysaccharides found in microbial and plant cell walls, the chemical structure is often composed of 5- or 6-membered carbon heterocycles with oxygen, with a broad variety of side chain groups and branching. This leads to a chemical shift range mainly spanning ~ 55–110 ppm (Fig. 2B and D). In proteins, the DREAM scheme is usually employed to achieve efficient transfer between ¹³Cα (~55–65 ppm) and side chain ¹³C (~10–68 ppm), with a ¹³C carrier frequency optimized at ~ 50–60 ppm during the DREAM recoupling [54]. We therefore question whether the DREAM scheme, which is a polarization transfer technique that presents a strong dependence on the RF-irradiation frequency at moderate MAS frequencies, might offer spectroscopic advantages compared to broad-band recoupling techniques.

We selected *N. crassa* and *A. fumigatus* as two microbial species to investigate the use of the DREAM scheme to study CW polysaccharides in intact cells. *Neurospora crassa* (Sordariales) is a model organism, which has been at the forefront of fungal biology for more than a century [59]. Playing a crucial role during the early days of molecular biology [60], *N. crassa* has established as a useful model for the exploration of circadian clocks [61], genome defense [62], cell polarity [63], cell fusion [64], allorecognition and regulated cell death [65] and CW composition and biogenesis [66,67]. Like almost all fungi, *N. crassa* relies on a thick CW as a barrier between the fungal cell and the environment, its CW structure and composition have been investigated since the 1950 s [68]. Through a combination of biochemical and genetics analyses, the composition of Neurospora CW has been determined as consisting glucan and chitin [66]. We performed a culture of *N. crassa* in rich medium and fresh conidia were used to inoculate a minimal medium supplemented with ¹³C-labeled glucose to obtain *N. crassa* conidia uniformly ¹³C-labeled. Intact conidia were packed into a 4 mm rotor to perform MAS NMR. Fig. 3A presents the 2D ¹³C–¹³C PDSD experiment recorded with a mixing time of 50 ms. It reveals three sets of interconnected correlations spanning between ~ 56–108 ppm. The three spin systems were assigned to α-(1,3)glucan, β-(1,3)-glucan and chitin (see Fig. 3B for their chemical structure) based on our previous analysis of fungal CW of intact conidia of *A. fumigatus* [25].
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** NMR identification of the main cell wall polysaccharides in intact *N. crassa* conidia. (A) 2D proton-driven spin-diffusion correlation spectrum of *N. crassa*, recorded at 11 kHz MAS at 600 MHz ¹H frequency. (B) Chemical structure of α-(1,3)-glucan, β-(1,3)-glucan and chitin.</figcaption></figure>

We implemented a 2D ¹³C–¹³C correlation experiment by replacing the PDSD mixing with a DREAM scheme. With initial CP transfers in both experiments, we only excited rigid components of the CW. We used a DREAM sweep duration of 3 ms, to match mixing periods used for proteins (see *vide infra* for its optimization). In analogy to protein samples for which the carrier frequency is set between ¹³Cα's and sidechain ¹³C, here we used a carrier frequency at 71 ppm, at the middle of the ¹³C chemical shifts detected for the three polysaccharides. The resulting 2D DREAM spectrum, exemplified in Fig. 4A for the C₅ correlations of β-(1,3)-glucan (in red and green), presents a distinct spectral pattern compared to the PDSD spectrum (in black). First, we observed a typical pattern of positive-intensity signals at the diagonal (in green in Fig. 4A) and negative-intensity cross-peaks (in red in Fig. 4A), reflecting the double-quantum properties of the DREAM recoupling scheme. In dipolar-based recoupling experiments, the ¹³C–¹³C polarization transfer is through-space mediated and correlated to the strength of the dipolar couplings. However, the presence of strong dipolar couplings between covalently attached carbons (e.g. C₅–C₆ and C₅–C₄ if we consider C₅ as the polarization source) can quench smaller dipolar couplings (e.g. C₅–C₁, C₅–C₂ or C₅–C₃) following the so-called “dipolar truncation effect” [69–72]. As a consequence, the resulting polarization pathway could not be simply considered as a sum of direct polarization transfers (Fig. 4B) and possible relayed transfers mediated by one-bond ¹³C–¹³C connectivity must be considered (Fig. 4C). In the case of the DREAM scheme performed here with a relatively short mixing period of 3 ms, we

observed negative-intensity cross-peaks with strong intensity for one-bond correlation C₅–C₄ and C₅–C₆, and negative-intensity cross-peaks with low intensity for C₅–C₂ and C₅–C₃. Note that the cross-peak encoding for the C₅–C₁ correlation was not observed. This observation reflects that the cross-peaks arise mostly from direct polarization transfers between the correlated carbon nuclei. In addition, the 2D DREAM experiment shows an important asymmetry (full spectrum showed in Supplementary Fig. 2). This effect has already been described [51] and is mainly caused by ¹³C spin pairs that recouple at different time during the DREAM sweep.

We inspected all possible polarization transfers within the three polysaccharides and found that this trend is not homogeneous. It is exemplified in Fig. 5 for the polarization transfer pathway ending at the C₄ position for the β-(1,3)-glucan. In a model assuming only direct polarization transfers during the DREAM scheme, all correlations associated with C₄ in the direct dimension (i.e. C₁–C₄, C₂–C₄, C₃–C₄, C₅–C₄ and C₆–C₄) should be encoded by negative-intensity cross-peaks. We observed negative cross-peaks for the one-bond ¹³C–¹³C correlations C₃–C₄ and C₅–C₄ (Fig. 5A–C). However, the cross-peaks are positive for correlations C₂–C₄ and C₆–C₄. This observation reflects the presence of relayed polarization transfers during the DREAM recoupling. Indeed, in such Cx-Cy-Cz spin systems, a Cx-Cz polarization transfer could be either direct and lead to a negative cross-peak, or be the result of two polarization transfers, first between Cx and Cy (leading to a negative contribution) followed by Cy to Cz (leading to a positive contribution because

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Polarization transfer pathway in the DREAM experiment. (A) Excerpts of the 2D PDSD spectrum (in black) and DREAM (positive signal in green, negative signal in red) of *N. crassa*. Polarization transfer pathways for the C₅ position of β-(1,3)-glucan (schematically presented), using a PDSD scheme and assuming no relayed contacts (B), assuming relayed contacts (C) and using a DREAM scheme (D). The source polarization (C₅ position of β-(1,3)-glucan) is colored in purple. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Polarization transfer pathway and relayed polarization transfer. (A) Excerpts of the 2D PDSD spectrum (in black) and DREAM (positive signal in green, negative signal in red) of *N. crassa*. Polarization transfer pathway ending at the C₄ position of β-(1,3)-glucan, using a PDSD scheme assuming no relayed polarization transfers (B); using a DREAM scheme assuming one-bond transfers (C); and using a DREAM scheme assuming relayed transfers (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

of the signal inversion). We therefore hypothesize that the positive cross-peaks encoding C₂-C₄ and C₆-C₄ are the results of relayed transfers as schematized in Fig. 5D. Starting on C₂, the inversion of magnetization will lead to a negative C₂-C₃ cross-peak, followed by a C₃-C₄ polarization transfer leading to a positive C₂-C₄ cross-peak. The same pathway might also explain the positive C₆-C₄ cross-peak. Such Cx-Cy-Cz spin systems exemplified here for glucans are virtually similar to three-spin systems described by Ernst, Böckmann, Meier and coworkers [55] in proteins and named “Cα-Cβ-CX”. The main difference between the two spin systems is the chemical shift range, spanning only ~ 25 ppm for glucans and ~ 50 ppm (e.g. for valine Cα-Cβ-Cγ₁/₂) in proteins. For a micro-crystalline Ubiquitin sample, such relayed transfers also led to positive Cα-CX cross-peaks (e.g. Cα-Cγ for Valine), at the expense of direct (and therefore negative) Cα-Cγ transfer [55], depending on the DREAM recoupling conditions. As a side note, the possibility to generate relayed contacts in the 2D ¹³C–¹³C DREAM spectra could be envisioned as an advantage during the process of spin system identification. Indeed, the presence of a positive cross-peak would necessarily imply a relayed topology (Cx-Cy-Cz), and as a consequence that the negative-intensity cross-peak encodes for a closer Cx-Cy contact compared to the positive

Cx-Cz. It is particularly useful for spin systems observed in polysaccharides compared to their counterparts in amino acids, because several carbohydrates often present a topology made of consecutive and chemically bonded carbons of very close chemical shifts, i.e. C₂, C₃, C₄, C₅ and C₆ in α-(1,3)-glucan and ββ-(1,3)-glucan.

Next, to understand the polarization transfer dynamics during the DREAM scheme, we derived magnetization build-up curves. Fig. 6 presents the build-up curves (Fig. 6A), obtained by varying the sweep duration (from 0.5 to 6 ms), for 4 correlations considering C₄ of β-(1,3)-glucan as the starting polarization source, namely C₄-C₁, C₄-C₂, C₄-C₃ and C₄-C₅ (Fig. 6B). Although such detailed analysis of build-up curves is not available for solid proteins, Verel et al. presented experimental build-up magnetization data on sodium propionate, alanine and zinc acetate [51,73]. Here, we observed that for one-bond ¹³C–¹³C contacts (i.e. for C₄-C₅ and C₄-C₃), the optimized sweep duration is ~ 0.5–2 ms. For two-bond (C₄-C₂) and three-bond (C₄-C₁) transfers, we observed slower build-up and a maximum intensity reached respectively at ~ 1 ms and ~ 6 ms. Overall, these optimal durations are shorter compared to those reported for powder and crystalline samples, although the semirigidity of the cell wall analyzed here (i.e. ¹³C T₁ is ~ 3 sec for β-(1,3)-glucan) should be considered. We therefore recommend a DREAM sweep duration of ~ 2–4 ms to analyze CW polysaccharides in whole cells. Note that a detailed analysis of the polarization transfer dynamics, as for instance simulated for canonical amino acids by Meier and coworkers [55] would require extensive numerical simulations, considering that polysaccharide exhibit very strongly dipolar-coupled ¹³C spin topologies.

The DREAM scheme depends on the frequency of the RF-irradiation (i.e. the ¹³C carrier position), and the sign of the cross-peaks as well as their intensities are directly dependent on the choice of the ¹³C carrier position during the DREAM sweep. This effect can be investigated by varying the ¹³C position used during the DREAM recoupling. For proteins, the carrier position is set at the middle of the recoupled resonances: in 2D ¹³C–¹³C DREAM, a carrier position set at ~ 40–45 ppm is typically used [49,50] to cover the full range of Cα's and side chain protein resonances at moderate MAS frequencies, with few exceptions (carbonyls, aromatic side chain and few side chain resonances for Arginine). In the case of a NCαCβ and N(CO)CαCβ experiments, a carrier position set at ~ 55–60 ppm [54] is optimal because the initial ¹³C polarization transfer before the DREAM is already set to Cα due to the use of a frequency-selective specific ¹⁵N–¹³C CP. For polysaccharides, the chemical shift range differs compared to amino acids. We tested four carrier positions (i.e. at 58, 71.5, 86.5 and 103 ppm) to cover a large part of the ¹³C resonances of the glucose unit (Fig. 7A). We observed that the four conditions led to four distinct chemical shift patterns (Fig. 7B, C), suggesting a strong dependence of the polarization transfer efficiency to the choice of the carrier position. For a carrier position set to 103 ppm,

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Polarization transfer dynamics using the DREAM scheme. (A) Build-up magnetization curves for C₄-C₃ (dashed curve), C₄-C₅ (black curve), C₄-C₂ (blue curve), and C₄-C₁ (red curve), correlations. (B) Polarization transfer pathways assuming a direct polarization transfer. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Spectral editing by varying the carrier position during the DREAM scheme. (A) 1D CP spectrum of *N. crassa*. The four ¹³C carrier positions tested in this study are highlighted in yellow. (B) Excerpt of the 2D ¹³C–¹³C PDSD spectrum (α-(1,3)-glucan (green), β-(1,3)-glucan (purple) and chitin (orange)). (C) Excerpt of 2D ¹³C–¹³C DREAM spectra obtained for the four carrier positions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

down-field signals are favorably recoupled (e.g. C₃–C₁ for α- and β-(1,3)-glucan)). Likewise, at a carrier frequency of 58 ppm, up-field signals show strong intensity cross-peaks (e.g. C₆–C₃ for α- and β-(1,3)-glucan). Overall, a similar effect has been observed in proteins [55], this effect being reinforced in our experimental conditions since the moderate MAS frequency used (11 kHz) is not optimal to cover the full range of chemical shifts. Used in combination with the PDSD experiment (Fig. 7B), the spectral editing provided by the choice of a particular carrier position in the DREAM experiment can be advantageous to intentionally favorize particular spin system topologies according to their chemical shift offsets. Such principle of discriminating spin systems based on their chemical topology was already proposed by R.R. Ernst [74]. For MAS NMR, spectral editing can be achieved through multiple techniques, including depolarization [75], chemical shift filtering [76] or dipolar dephasing [77]. Such approaches have been optimized to provide efficient spectral editing techniques to simplify congested protein spectra [78,79]. In the case of whole cells of *N. crassa*, the efficiency of the spectral editing is noticeable, with several correlations not observed at all carrier positions. A rational understanding of cross-peak intensities as a function of the carrier position is extremely challenging in our case, because the three *N. crassa* CW polysaccharides exhibit strongly dipolar-coupled spin system topologies. As already observed by Meier and coworkers for a microcrystalline protein, the presence of 3- and even 4-carbon multi-spin systems led to complex polarization

pathways [55]. Nevertheless, a clear and distinct spectral decongestion of the spectra is observed at various carrier position, facilitating the spectral inspection of such whole cell samples.

Finally, we question whether the results obtained on *N. crassa* could be generalized to other fungal whole cell analysis. We produced a ¹³C/¹⁵N-labeled sample of *A. fumigatus*, an airborne fungal opportunistic pathogen causing invasive aspergillosis [80]. *A. fumigatus* is phylogenetically distant from *N. crassa*, the species have diverged approximately 300 mya, which is comparable to the bird/mammal divergence time [81]. The evolution of the CW architecture during the germination process has been thoroughly scrutinized by MAS NMR methods [25], and CW polysaccharide ¹³C chemical shift assignments are available [24,25]. We compared 2D ¹³C–¹³C correlation experiments obtained using a PDSD mixing (Fig. 8A and C) and a DREAM scheme (Fig. 8B and D). Although the resulting ¹³C spectral fingerprints differ between the two species due to variable carbohydrate compositional complexity, we observed that the use of the DREAM scheme also enables a spectral editing with the presence of negative- and positive-intensity cross-peaks for *A. fumigatus*.

On the whole, a prototypical set up, based on our results, to achieve an efficient DREAM scheme under moderate MAS would consist of: (1) a MAS frequency at least larger to 7–8 kHz to cover most of the ¹³C chemical shifts observed in glucose-based carbohydrates, (2) a carrier position around 70–90 ppm to cover a large number of chemical shift
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Excerpts of 2D PDSD spectra of *N. crassa* (A) and *A. fumigatus* (C) and DREAM spectra of *N. crassa* (B) and *A. fumigatus* (D).</figcaption></figure>

offsets and (3) a sweep period of ~ 2 ms. Finally, the recording of a conventional ¹³C–¹³C recoupling experiment using either the PDSD, CORD or DARR pulse sequences will provide an optimal set up to compare with the DREAM experiment and (i) distinguish direct to relayed polarization transfers based on the cross-peak sign and (ii) fully exploit the spectral editing provided by the DREAM. To document the practical implementation of the DREAM transfer and optimize spectroscopic parameters such as the carrier position, the choice of the sweep shape and pulse optimization, we present in Fig. 9 the comparison of 1D ¹³C-detected CP, CP DREAM (i.e. CP followed by a ¹³C–¹³C DREAM) and DCP DREAM (i.e. a specific ¹⁵N-¹³C CP followed by a ¹³C–¹³C DREAM). While the CP DREAM offers a 1D experiment for which the experimental parameters used in the 2D ¹³C–¹³C set up can be directly implemented, it suffers from the complexity of the 1D spectral profile, due to a distorted baseline resulting from the partial superposition of positive and negative signals. We recommend the use of a ¹H-¹⁵N CP followed by a specific ¹⁵N-¹³C CP prior to the DREAM transfer. In proteins, the 1D NCαCβ DREAM experiment allows to filter out most of the signals due to the specificity of the specific DCP transfer that only selects Cα signals to be transferred during the (Cα-Cβ) DREAM. For polysaccharides, such

frequency selectivity based on chemical shift is more challenging to obtain due to the narrower ¹³C chemical shift distribution. Nevertheless, we achieved a relatively clean 1D spectral profile after the ¹⁵N-¹³C DCP filter (in blue in Fig. 9), and the resulting negative signal at ~ 70–78 ppm (C₃ of chitin) and ~ 106–107 ppm (C₁ of chitin) could be used to monitor and optimize the effect of DREAM parameters such as the sweep pulses. Note that because only chitin contains nitrogen atoms, the sensitivity of the 1D NCC DREAM is lowered by a factor of ~ 15 compared to ¹³C-detected experiments, due to the low abundance of chitin compared to glucans in *A. fumigatus* [24,25].

## 4. Conclusions

We have demonstrated that the DREAM scheme can be implemented to investigate intact whole cell samples. The use two-dimensional ¹³C–¹³C correlation experiments employing the DREAM scheme provides selective polarization transfer according to the carbohydrate unit topology. In addition, varying the ¹³C carrier position during the DREAM sweep provides a spectroscopic tool to access the spectral editing of polysaccharide signals. The DREAM scheme will constitute an additional tool for increasing the repertoire of solid-state NMR techniques to identify and structurally characterize polysaccharides present in cell walls of model and medically-relevant pathogenic microbes. Our results have been obtained under experimental conditions (i.e. medium field NMR magnet, moderate MAS frequency) available to most laboratories working on whole cells analysis by MAS NMR. The study presented here was performed at moderate MAS frequency, and further experiments will be required to investigate how the combination of fast MAS (>60 kHz) and ultra-high magnetic fields could be beneficial to increase the sensitivity and selectivity of the DREAM scheme for CW analysis. By combining MAS NMR and the DREAM scheme, the present study paves the way to develop new approaches to decipher microbial CW at the molecular level, especially to promote the development of CW-targeted antifungals.

## 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.

<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Optimization of the DREAM scheme for cell wall analysis. 1D spectra obtained on *A. fumigatus*, with (¹H)-¹³C cross-polarization (CP) recorded with 64 scans, (¹H-¹³C)-¹³C DREAM recorded with 64 scans and (¹H-¹⁵N-¹³C) DCP DREAM recorded with 512 scans.</figcaption></figure>
## Data availability

Data will be made available on request.

## Acknowledgments

This work was supported by the ANR TransKingAmyloid (ANR-21-CE11-0007 to A.Lo and S.J.S), the PSGAR-MIE "EMERG" (to L.Delh. and A.Lo) and an ATIP-Avenir funding (to A.D.). This work has benefited from the Biophysical and Structural Chemistry Platform at IECB, CNRS UAR 3033, INSERM US001.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymeth.2024.07.003.

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