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OPEN Synthesis of glycoconjugates utilizing the regioselectivity of a lytic monooxygenase Bjørge Westereng 1*, Stjepan K. Kračun 2, Shaun Leivers 1, Magnus Ø. Arntzen 1, Finn L. Aachmann 3* & Vincent G. H. Eijsink 1

Polysaccharides from plant are the most abundant renewable chemicals on Earth and can potentially be converted to a wide variety of useful glycoconjugates. Potential applications of glycoconjugates include therapeutics and drug delivery, vaccine development and as fne chemicals. While anomeric hydroxyl groups of carbohydrates are amenable to a variety of useful chemical modifcations, selective cross-coupling to non-reducing ends has remained challenging. Several lytic polysaccharide monooxygenases (LPMOs), powerful known for their application in degradation, specifcally oxidize non-reducing ends, introducing carbonyl groups that can be utilized for chemical coupling. This study provides a simple and highly specifc approach to produce oxime- based glycoconjugates from LPMO-functionalized . The products are evaluated by HPLC, mass spectrometry and NMR. Furthermore, we demonstrate potential biodegradability of these glycoconjugates using selective enzymes.

Over the years, numerous methods for synthesis of glycoconjugates have been developed, mainly using traditional synthetic methods. An unprotected carbohydrate contains multiple hydroxyl groups with similar reactivity. Coupling ligands to a specifc position is therefore challenging and normally requires an exhaustive number of chemicals and synthetic steps­ 1–3. Tis is one of the major hurdles for efcient utilization of some of Nature’s most abundant molecules as building blocks for novel biomolecules. Glycoconjugates have a considerable potential as drugs, fne chemicals and in material science­ 4. Moreover, the efciency of enzymatic depolymerization of plant has improved tremendously in recent years, partly due to the inclusion of oxidative enzymes in ­mixtures5–7. Tis development enables more efcient production of plant derived carbohydrate-based “bulk chemicals”, including a wide variety of structurally diverse oligosaccharides that could be converted to glycoconjugates. Oxidative carbohydrate-active enzymes are involved in a multitude of biological processes, including biomass decay. Tese enzymes use diferent mechanisms to generate oxidations on mono-, oligo- and/or polysaccharides. Tese oxidation reactions produce either carbonyls or carboxylic acids and, for poly- and oligomeric substrates, these reactions involve enzymes such as LPMOs­ 5,8–10, dehydrogenase (CDH)11,12, galactose oxidase­ 13, pyranose dehydrogenase­ 14,15 and glucooligosaccharide oxidases­ 16. Together, these enzymes enable a range of oxidative ­functionalizations17 and ofer several possibilities for subsequent ­glycoconjugation18. Notably, there are very few enzymes that oxidize the non-anomeric hydroxyls in the sugar rings of oligo- and polysaccharides. LPMOs may play a special, albeit so far scarcely explored, role in glyco-functionalization, since they are active on polymers and can functionalize polymeric surfaces. Te initial step of the lytic action of LPMOs on cellulose entails breaking the energetically strong C–H ­bond19 at position C1 or C4, catalyzed by a triangularly shaped catalytic copper site termed the “histidine brace”9. Some LPMOs oxidize C1, thus generating a carboxylic acid that could also be generated in alternative ways, for example using CDH. Interestingly, other LPMOs have the

1Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, Chr. M. Falsens vei 1, Ås, Norway. 2Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. 3Department of Biotechnology and Food Science, NTNU-Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway. *email: [email protected]; [email protected]

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ability to specifcally oxidize C4, generating a ketone functionality that is hard to generate in other ways. Due to the discovery of an increasing number of C4-oxidizing LPMOs active on abundant polysaccharides such as ­cellulose20, ­glucomannan21, ­xylan22 and xyloglucan­ 21, several target substrates for non-reducing end function- alization by LPMOs are now available. A feasible way to synthesize heteropolymers is via polycondensation (analogous to the synthesis of well- known materials like polyesters and polyurethanes). Tis requires carbohydrates that are bi-functionalized. While functionalization and/or cross coupling to carbohydrate reducing ends is relatively straightforward, doing the same at the non-reducing end has proven more challenging, although elegant approaches to this using galactose ­oxidase23 and recently galactose oxidase/pyranose dehydrogenase and transaminidase have been ­shown24. Te possibility to specifcally functionalize C4 using LPMOs opens a possible route towards generating other bi- functional carbohydrate building blocks. Here we describe a highly selective chemo-enzymatic coupling procedure for the C4 position of oligosaccha- rides derived from one of the most abundant polysaccharides in Nature, cellulose (Fig. 1 sketches the diferent synthetic steps). We further demonstrate that this allows the production of enzymatically cleavable bi-functional glycoconjugates. Results and discussion Demonstrating the regioselectivity of NcLPMO9A. Several LPMOs characterized so far have mixed activities leading to a combination of C1- and C4-oxidized products, whereas some strict C4-oxidizers act on oligomeric substrates and thus produce short ­products25. Tese properties would interfere with the coupling chemistry and complicate analysis in this proof of concept study. Hence, we searched for an LPMO that is not active on oligosaccharides and only releases C4-oxidized products with a degree of polymerization (DP) that would provide oligosaccharides of a suitable range (3–6). Based on previous studies, the C4-oxidizing Neuro‑ spora crassa enzyme, NcLPMO9A (NCU02240)26,27, which is not active on soluble cellooligosaccharides, was selected. More detailed analysis, necessary to validate this enzyme’s suitability for the present study, showed that it releases longer, oxidized oligosaccharides (predominantly DP 3–5) (Fig. 2). Furthermore, MS/MS analysis displayed in Fig. S1 showed fragmentation patterns typical of C4-oxidized cello-oligosaccharides25. When NcLPMO9A was combined with a cellobiose dehydrogenase (MtCDH from Myrococcum thermophi‑ lum)28, the dominating ketone signal in the MALDI-ToF MS spectra shifed by 16 or 38, representing the forma- tion of an aldonic acid (+ 16), which subsequently has a tendency to form a double sodium adduct (M − H + 2Na; + 38) (Fig. 2). Since CDH oxidizes oligosaccharides in the reducing end, this observation confrms that NcLP- MO9A oxidizes in the non-reducing end only. Since it is challenging to determine the oxidative regioselectivity of LPMOs by HPAEC and ­MALDI29,30, we designed a precise and simple way for probing C4-oxidation on oligosaccharides, inspired by previous work by Beeson et al.20 who looked at . Reduction of the ketone at C4 results in a mixture of galactose and and hence reduction of a C-4 oxidized cello- will result in the formation of a mixture of oligosaccharides with either a glucosyl or a galactosyl at the non-reducing end and a glucitol (GlcOH) in the reducing end. To assess this in detail, we frst generated ­GalGlcn (n = 3 or 4) standards (Fig. 3) by using UDP-Gal (donor), cellotriose/tetraose (acceptor) and a galactosyltransferase (Fig. 3E). Tese oligosaccharides have considerably shorter elution times in HPAEC than their corresponding cello- oligosaccharides (Fig. 4A). Oligosaccharides generated by NcLPMO9A and our in-house prepared oligomeric standards were reduced to completion using ­NaBD4 and the resulting oligomeric products were analyzed directly by HPAEC and MALDI ToF MS (Figs. 2, 4B,C). Reaction products generated by NcLPMO9A indeed yielded a mixture of (Glc)nGlcOH and Gal(Glc)nGlcOH (n = 2–4) oligosaccharides confrming C4 oxidation. Tese results further prove that NcLPMO9A only oxidizes C4. Notably, this reduction approach, used here to prove C4 oxidation, also provides a relatively simple method for quantifcation of oxidized products, based on using ­NaBD4 for reduction. Afer reduction, oligosaccharides with oxidized C4 will possess two deuteriums whereas non-oxidized oligosaccharides will only contain one deuterium, thus yielding an easily detectable diference of m/z = 1 (Fig. 4C). Internal standards could thus be produced from non-oxidized oligosaccharides as described above, possibly using NaBH­ 4 for reduction, generat- ing a more pronounced diference of m/z = 2. Figures 1 and 3 further show that NcLPMO9A primarily releases oligosaccharides of DP3-5, which is a suit- able size for the purpose of this study. Several experiments were performed in order to evaluate the specifcity of NcLPMO9A. Screening of other substrates using MALDI-ToF MS for product detection showed that, in contrast to several other C4 oxidizing LPMO9s, NcLPMO9A is not active on curdlan [β-(1,3) glucan], pustulan [β-(1,6) glucan], β-chitin, glucomannan, xylan, xyloglucan, mixed linkage glucan [β-(1,3),(1,4)] and is thus highly specifc for cellulose. Furthermore, there was no activity on cello-oligosaccharides with a degree of polymeriza- tion (DP) of 2–6, which shows that this enzyme is not active on solubilized oligomeric products. Together the properties demonstrated above makes NcLPMO9A suitable for this proof of concept study on C4-functionalized cellooligosaccharides.

Generation of mono‑ and difunctional glycoconjugates. Oxidized products generated by NcLP- MO9A (C4-oxidized) or by a combination of NcLPMO9A and CDH (C1 and C4-oxidized; Fig. 1) were subjected to targeted carbonyl functionalization using a hetero-bi-functional linker (aminooxy-linker), 2-(aminooxy)- 1-ethanaminium dichloride, to generate oximes (Fig. 5). Product formation was confrmed by MALDI-ToF MS (Fig. 5) and porous graphitized carbon electrospray ionization mass spectrometry (PGC-ESI-MS) (Fig. 6) as well as by NMR (Fig. 7).

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Figure 1. A simple four step chemo-enzymatic procedure to generate a cello-oligosaccharide with FITC conjugated to C4 in the non-reducing end. Te starting point is cellulose. NcLPMO9A cuts the cellulose chain and generates C4-oxidized cello-oligosaccharides. CDH converts the reducing end to its corresponding aldonic acid, which protects the reducing end from oxime formation. Te oxime linker is coupled to the C-4 keto group prior to the last step in which FITC is cross-linked to the oxime linker.

Due to the presence of a carbonyl function in the reducing end, oxime formation takes place at both ends of the C4-oxidized oligosaccharide. Chromatography/tandem mass spectrometry (LC–MS/MS) with PGC yielded masses corresponding to three double-oxime functionalized oligosaccharides (m/z 619.25, 781.28 and 943.35). Furthermore, the obtained fragmentation patterns could be matched with the oxime functionalized oligosac- charides (Fig. 6). Notably, when analyzing the oxime functionality on PGC we observed a considerably increased retention of the oligosaccharides (Fig. 6), compared to the corresponding C-4 oxidized oligosaccharides­ 30. Tis may be due to charge efects which are well known to increase the retention of charged solutes on PGC. Replacing ammonium acetate with a stronger eluent like formic acid in the gradient was needed to enable elution. Since the oxime linkers will occur in an equilibrium between charged and neutral states, this may have given rise to the

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+ OH H OH H OH H OH O+Na] O 2

[M+Na] H O H O H O H R1 1 [M+Na]

[M+H O O O O+2Na-H] 2

4 HO HO HO 87. HO R 6 H OH H OH H OH H OH 2 703. 524 .9 [M+H H H H H H H H .] .u 4 R1= O R1= OH 5.

. [a OH O 2 72 2 R2= C R2= C 4 9. ens t 4 8 5 541. H OH .2 887. 5 . 63 9 865. Rel. In 11 .2 5 0

* 04 1 1

600 800 1000 1200 1400 m/z

Figure 2. MALDI ToF MS analysis of products generated from PASC by NcLPMO9A using ascorbic acid (red structure and spectrum) or CDH as reductant (black). Te reaction with CDH will lead to oxidation of reducing ends and thus generate double oxidized products. Te main peaks in the spectra correspond to (m/z values apply to sodium adducts of the tetramer): 687, mono-oxidized (keto-sugar; dominates in the red spectrum); 703, double oxidized (aldonic acid form at C1; dominates in the black spectrum); 725, double oxidized (sodium salt of the aldonic acid form; dominates in the black spectrum). Te following minor species are also visible: 685, double oxidized (keto at C4, lactone form at C1); 705, single oxidized (gemdiol of 4-keto sugar, indicated by arrows and *). Similar peak clusters were observed for other oligomers. Te spectra were generated with Flex analysis 3.4, https://www.bruke​ r.com​ .

peak broadening observed. Upon viewing the associated fragmentation patterns there were no signs of isomer formation as the fragmentations were consistent throughout the elution times for each particular peak (Fig. S2). In double oxidized oligosaccharides, containing a CDH-generated carboxyl functionality that will block oxime formation at the former “reducing end”, only the carbonyl function at C4 in the non- reducing end will react. Indeed, for C4-oxidized oligosaccharides that were protected in their reducing end by CDH-catalyzed oxidation to an aldonic acid, only one oxime was formed, in the non-reducing end. Te efciency of the reaction was assessed by mass spectrometry (Figs. 4, 5) and the position of the oxime was verifed by NMR both for the reducing and the non-reducing end (Fig. 7, Fig. S3, Table S1). Obviously, these double oxidized oligosaccharides allow the formation of hetero-linked compounds by utilizing both carbonyl specifc (at the non-reducing end) and carboxyl-specifc coupling reactions in the down-stream (former reducing) end. Structural elucidation by NMR provided direct proof of the structure of the putatively bi-functionalized products generated by coupling aminooxy-linkers to either one or both ends oligosaccharides. Te individual monosaccharides were assigned by starting at the anomeric signal as well as from the primary alcohol group at C6 and then following 1H–1H connectivity using DQF-COSY, H2BC and ­[1H–13C] HSQC-[1H,1H] TOCSY (full assignment of shif values in Table S1). Most of the carbon chemical shifs were obtained from ­[1H–13C] HSQC (Fig. 7). Te ­[1H–13C] HMBC spectrum provided long range bond correlations that connect the monosaccharides to the aminooxy-linker. Te carbon chemical shif of the C4 oxidized end was determined by combining the correla- tions from a [­ 1H–13C] HSQC spectrum and a [­ 1H–13C] HMBC spectrum (see Fig. 7B). Both for the reducing end (Glc1–H/C-2) and for the C4 oxidized non-reducing end (C4ox-3 and C4ox-5) HMBC correlations are observed from proton signals 4.61 ppm and 5.01/4.27 ppm to a carbon signal at 155.4 ppm and 159.2 ppm, respectively (see Fig. 7B). Tese carbon chemical shifs ft well to the expected carbon chemical shifs for an aminooxy group, which would form upon coupling. Altogether, the NMR results suggest that the structure of the bi-functional products correspond with the structures depicted in Fig. 6C.

Enzyme degradability. To explore the potential of further expanding the functionality of the modifed cello-oligosaccharides we tested various treatments. Te aminooxy-functionalized oligosaccharides can indeed be enzymatically degraded using a cellulase. Tis indicates that glycoconjugates produced in this study may be biodegradable which is an important aspect in the applicability of such biobased chemicals, whilst it also allows for the formation of glycoconjugates with an unmodifed reducing end Tis efectively expands the application potential of the present technology and underpins the fexibility of these conjugates, by allowing for the potential of a multistep approach to labeling. Two diferent were used to demonstrate degradability of the functionalized oligosaccharides (Fig. 8). Both Cel6B from T. fusca and Cel5A from H. jecorina degraded the aminooxy-functionalized oligosaccharides.

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A) 3 E) O OH OH

689. H NH O OH H OH H O O H OH HO H H OH H O N O H O O P O P O H O H O O O OH HO HO -- HO H OH HO O O H OH H

3 H OH H H UDP-Gal H H H

527 .3 n=1/2 OH OH 05. Glc3/Glc4

7 galactosyl transferase B) OH H OH OH H OH H OH H O H O H O H O

3 O O O OH H HO HO H OH H OH HO 2. HO H OH H H H OH

9 H ] H H H H 3 H 6

u. n=1/2 0. a. 3 .[ 5 GalGlc3/Glc4 s en 4 nt C) I 851. Rel. .3 9 68

D) 4 3 854. 692.

500 600 700 800 900 1000 1100 m/z

Figure 3. MALDI ToF MS of GalGlc­ n standards, prior to and afer reduction with NaBD­ 4. Te non-reduced compounds shown in (A), GalGlc­ 3 (at m/z 689 with a little bit of unreacted Glc­ 3 at m/z 527), and (C), GalGlc­ 4 (at m/z 851 with a little bit of unreacted ­Glc4 at m/z 689), yield masses equivalent to cello-oligosaccharides. Te reduced compounds (B) and (D) show an expected m/z of + 3, refecting reduction and the incorporation of one D (see Fig. 4 for structures). (E) Shows route for generating GalGlc­ n (n = 3 or 4) standards. Te spectra were generated with Flex analysis 3.4, https://www.bruke​ r.com​ .

Both enzymes degraded bi-functionalized DP5 and DP6 completely, and Cel5A also degrade most of bi- functionalized DP4. Since the mass diference of degradation products with the oxime linker in the reducing end is m/z + 2 compared to products where the oxime linker occurs in the non-reducing end, the mass spectra reveal preferred sites for cellulase cleavage. For example, Cel5A primarily converted the double-functionalized pentamer to a trimer with C4-oxime linker (583.2) and a dimer with a reducing end linker (423.1), whereas conversion to a dimer with C4-linker (421.1) and a trimer with a reducing end linker (585.2) was less frequent. For Cel6B the degradation pattern for the double-functionalized pentamer was slightly diferent; here the main products were a trimer with a reducing end linker (585.2) and a dimer with a C4-linker (421.1). Tis exempli- fes a rational approach for generating defned functionalized oligosaccharides. Essentially, by playing with the initial reaction (one or two oxime functionalizations) and the use of cellulases, in principle any combination of a natural and a modifed chain end can be generated. More uniform products may be obtained by frst purifying the C4-oxidized cello-oligosaccharides, which could be carried out using Porous Graphitized Carbon columns or ­HILIC30. By combining C4-specifc LPMOs and CDHs, more complex glycoconjugates may be generated since one may employ chemistries pertaining to carbonyl groups, such as the method described above, and chemistries pertaining to carboxyl groups, for example using carbodiimide ­activation31. Notably, C1-specifc LPMOs, or even LPMOs with a mixed C1/C4 activity could also be considered for this purpose. Importantly, the use of LPMOs in principle allows partial surface oxidation of polysaccharide fbers­ 32–34, which may enable functionalization of fber surfaces without afecting the tensile strength of the fbers. Such applications could be of commercial interest.

Further derivatization of the amino groups introducing fuorescein isothiocyanate (FITC) han- dles. Te functional group introduced through aminooxy functionalization was an amino group, and the usefulness of this group for further functionalization of the glycoconjugate was demonstrated by labeling with fuorescein isothiocyanate (FITC) that is highly reactive towards amines. Indeed, afer FITC labeling, oligosac- charides with oxime functionalization in both ends carried either one (due to incomplete labeling) or two FITC molecules (Figs. 9A, 10).

Scientific Reports | (2020) 10:13197 | https://doi.org/10.1038/s41598-020-69951-7 5 Vol.:(0123456789) www.nature.com/scientificreports/ + 1 A) 3 4 689. nC [M+Na] ] + u. 9 GalGlc GalGlc a. .[ [M+Na] 526. ns te 2 3 Rel. In + 4 + 2 Glc Glc 5 .1 Glc [M+Na] 9 851. 68 Glc [M+Na] 6 400 600 800 1000 m/z Glc

10.0 15.0 20.0 25.0 30.0 35.0 40.0 min B) GlcOH GlcOH 3 2 GlcOH GlcOH 4 3 Glc nC GlcOH 2 GlcGlcOH Glc GalGlc Glc GalGlc *

5.0 10.0 15.0 min

OH H OH H OH H OH D H O H O H O H OH Glc3GlcOH C) HO O O O OH HO HO HO HO OH OH OH OH D H H H H H H H H H H H H OH H OH H OH H OH HO H H H H O O O OH GalGlc2GlcOH O O O D OH HO HO HO HO OH OH OH OH D H H H H H H H H H H H H ] + + 4 u. + . 2 a. 4 .[ [M+Na] [M+Na] 693 531. ns [M+Na] te 855. OH H OH H OH H OH H

Rel. In H O H O H O H OH OH O O O OH + HO HO HO HO 0 OH

OH + OH OH D H

H 3 H H . [M+Na] 368. H H H H 2 H H H H + 1 [M+Na] 69 + .3 [M+Na] 530. 54 [M+Na] 8

400 500 600 700 800 900 m/z

Figure 4. Reduction of soluble products generated by NcLPMO9A. (A) Superposition of HPAEC chromatograms of various standards, showing that GalGlc­ 3 and GalGlc­ 4 have weaker retention than cello-oligosaccharides of the same DP. Te inset to the right shows the MALDI ToF–MS spectrum for ­GalGlc3 (pink, m/z 689 with a little bit of unreacted Glc­ 3 at m/z 527) and ­GalGlc4 (brown, m/z 851 with a little bit of unreacted ­Glc4 at m/z 689). (B) HPAEC chromatograms of oligosaccharides reduced with ­NaBD4: Glc­ 2-5 (green), GalGlc­ 3 (pink), GalGlc­ 4 (brown) and products generated by NcLPMO9A (black). GlcOH glucitol, Glc glucose, Gal galactose. (C) MALDI ToF MS of products generated by NcLPMO9A (black) and a DP2-5 mixture of cello-oligosaccharides (green) each reduced by NaBD­ 4. In the C4-oxidized products generated by NcLPMO9A one extra D is incorporated; expected m/z values for the tetrameric compound are: native, 689; C4-oxidized, 687; native reduced with NaBD­ 4, 689 + 2 + 1 = 692; C4-oxidized reduced with ­NaBD4, 687 + 2 + 1 + 2 + 1 = 693; Insets show the structures of the DP4 compounds.

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OH H OH H OH H OH H2N N O H O H O H O H OH O O O R = COOH HO HO HO HO R1 1 H OH H OH H OH H OH O H H H H H H H N NH2 R1=C + + H [M+Na] 803.5 [M+Na] + 965.6 + [M+Na] + + + [M+H] 641.5 [M+Na] + + ] [M+H] [M+H] 781.5 u. + [M+K] a. 127.7 1071.6 1 .[ + + [M+K] 943.6 619.4 ens [M+K] t [M+H] [M+K] + Rel. In + [M+Na] [M+Na] 761. 4 + + + + + 599. 3 + + + + + [M+Na] [M-H+2Na] + + [M+K ] [M-H+K+Na ] + [M+H ] [M+H ] 923. 5 [M-H+2Na] [M-H+K+Na ] [M-H+2Na] [M-H+K+Na ] [M+H ] [M+K ] [M+K ] 739. 5 577. 3 1085.6 901. 5

400 600 800 1000 1200 m/z

Figure 5. MALDI ToF MS spectra of oxime functionalized oligosaccharides. Te structure of a functionalized tetramer is shown above the spectra. Te upper spectrum shows bi-functionalized oligosaccharides obtained from a product mixture generated by NcLPMO9A from PASC. Te spectrum shows clear signals for a trimeric, tetrameric, and pentameric double-funtionalized compound and a lower signal for the hexameric product (m/z 641, 803, 965 and 1,127, respectively; all sodium adducts). Tere are also peaks corresponding to proton and potassium adducts denoted [M+H]+ and [M+K]+. Te lower panel shows functionalization of LPMO-generated C4-oxidized products that were C1-protected through oxidation by CDH (m/z values are 42 lower compared to panel (A); sodium adducts). Te latter spectrum also shows peaks corresponding to [M−H+2Na]+ and [M−H+K+Na]+ adducts, which are formed due to the presence of an (CDH-generated) aldonic acid group. Te spectra were generated with Flex analysis 3.4, https://www.bruke​ r.com​ .

On the other hand, labeling oligosaccharides oxidized by CDH and thus carrying only one oxime functionali- zation carried only a single FITC specifcally labeled at C4 in the non-reducing end (Figs. 1, 9B). Te aminooxy functionalization shown in this study demonstrated only one of many possibilities for functionalization. Other functional groups such as azides and alkynes are also feasible and will extend the potential of the approach described here. Conclusion and implications Potential applications of the C4-specifc functionalization demonstrated here, include regiospecifc immobili- zation of C4-linked oligosaccharides to solid surfaces such as microarrays, conjugation to protein carriers for immunization purposes, and C4-specifc conjugation to nano- or microparticles, or quantum dots­ 35. C4-specifc functionalization also provides a potential novel analytical tool for studying whether glycosidases act at the reducing end or non-reducing end of oligosaccharides, a feature that has proven challenging to access and may require multistep approaches to resolve­ 36. Generating oligosaccharides that are either blocked in the non-reducing end or the reducing end could, in a straightforward way, aid in providing unequivocal evidence of which end is targeted by a given glycosidase. Furthermore, FITC labeling has been shown useful for studying the uptake of specifc carbohydrates by microbes­ 37. Te labeling strategy presented here provides new tools for such studies and could advance these by enabling studies of the directionality of glycan uptake.

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A 1x107 m/z 619.25

m/z 781.28

5x106 Intensity m/z 943.35 1x106 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) B b3 b 543.17 100 + m/z 2 e [M+H] = 619.27 381.11

y1 y 80 b1 2 219.02 239.06 401.14

60 b2-(H2O)2 b3-H2O 345.08 525.18 c c2 40 1 z2 237.02 383.14 399.14 b3-(H2O)2 z1 0,2 507.16 M-(H O) 20 x1 317.08 2 2 221.09 281.09 583.18 M

Relative Abundanc 0 b3 543.14 100 +

e [M+H] = 781.28 m/z b2 b4

nc 381.07 80 b2-(H2O)2 705.23 345.06 y2 60 b -(H O) b4-H2O bunda 401.13 3 2 2 A y1 z2 507.13 y3 687.21 40 563.20 239.03 383.10 c2 z3 b4-(H2O)2 399.18 b1 317.08 545.24 669.20 M-(H2O)2 20 0,2 489.17 219.07 x1 745.25 281.13 M Relative 0 b5 867.29 100 + [M+H] = 943.33 m/z b4

705.24 b5-H2O 80 b2-(H2O)2 849.30 345.07 b3 b3-(H2O)2 b2 543.17 60 507.15 381.10 y4 y3 b4-(H2O)2 40 z2 725.27 b5-(H2O)2 y2 563.21 669.21 383.11 z3 831.27 327.07 401.13 c M-(H O) 20 545.22 3 651.20 c4 2 2 297.10 489.13 561.17 523.26 907.34 M

Relative Abundance 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950

m/z 0,2 y2 z2 x1 y1 z1

C OH H OH H OH + N [M+H] = 619.27 m/z H2N O H O H O H OH O O O N NH HO HO HO 2 H OH H OH H OH H H H H H H

b1 c1 b2 c2 b3

y z y z 0,2 y [M+H]+ = 781.32 m/z 3 3 2 2 x1 1 OH H OH H OH H OH H2N N O H O H O H O H OH O O O O N NH HO HO HO HO 2 H OH H OH H OH H OH H H H H H H H H

b1 b2 c2 b3 b4 + [M+H] = 943.37 m/z y4 y3 z3 y2 z2

OH H OH H OH H OH H OH H2N N O H O H O H O H O H OH O O O O O N NH HO HO HO HO HO 2 H OH H OH H OH H OH H OH H H H H H H H H H H

b2 b3 c3 b4 c4 b5

Figure 6. PGC-MS analysis of bi-functionalized cello-oligosaccharides. Te oxime bi-functionalized oligosaccharides were separated by porous graphitized carbon (PGC) chromatography using an LTQ Velos Pro mass spectrometer (Termo) for detection. (A) Overlay of three extracted ion chromatograms for m/z-ratios 619.25, 781.28 and 943.35, showing separation of the bi-functionalized products with DP 3, 4 and 5, respectively (note that these are proton adducts). Te dashed line shows the time of recording of the MS/MS spectra. (B) MS/MS spectra of the three products show a dominant cleavage of the glycosidic bonds followed by multiple water losses (annotated as B-(H2O)2). A cleavage of the reducing-end amine linker bond can also be seen resulting in the following fragments; 543.17, 705.23 and 867.29 for the mother ions 619.25, 781.28 and 943.35, respectively. (C) Structures of the three bi-functionalized products, with theoretical masses and the observed fragmentation patterns (using the nomenclature of Domon and Costello)49.

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60 A) H/C6 H/C6* 65

NH2-CH2-CH2O-N=C-R

R’-CH=N-O-CH2-CH2NH2 70 H/C2*

H/C5* Chemical shift H/C3 H/C3* 75 H/C5 H/C2

H/C4* 80

ppm B) H/C2* 85 [ppm] H/C5 80 H/C3 90 90 100 110 120 95 130 NH -CH -CH O-N=C-R 2 2 2 140 100 150 105 R’-CH=N-O-CH -CH NH 160 H/C1 2 2 2 5.0 4.3 4.7p4.6 pm 110 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 Chemical shift [ppm]

Figure 7. NMR analysis of bi-functional cello-oligosaccharides. (A) [­ 1H–13C] HSQC spectrum; the sample was in 99.9% D­ 2O and the spectrum was recorded at 25 °C. Peaks in the proton/carbon signals of the C4ox residue in the non-reducing end are marked by H/C#, where # indicates the carbon number in the residue. Peaks in the proton/carbon signals of the Glc1 (= the former reducing end) spin system are marked by H/C#*. Chemical groups giving rise to signals for the 2-aminooxy group are in bold and underlined. For the sake of simplicity, peaks related to internal residues are not marked (a full assignment of the chemical shifs is provided in Table S1). (B) Correlations between a ­[1H–13C] HSQC spectrum and a ­[1H–13C] HMBC spectrum recorded for the bi-functionalized product. Te lef and the middle insert show correlations (indicated by vertical lines) from H/C-3 and H/C-5 peaks in the HSQC (blue) for the C4-oxidized end to peaks (indicated by a horizontal dotted line) with a common carbon chemical shif of 159.2 ppm in the HMBC (red). Te right insert shows a correlation from H/C-2* in the HSQC (blue) for the reducing end to a carbon peak with a carbon chemical shif of 155.4 ppm in the HMBC (red). Both carbon chemical shifs obtained from the HMBC spectrum correspond well to the (expected) presence of an imine (–C=N–) group, which is expected to be formed during the coupling reaction (Fig. 6), thus confrming the structure of the bi-functionalized product. R and R′ represent the non-reducing and the reducing end of the bi-functionalized cello-oligosaccharides, respectively. Further confrmation of the successful coupling can be found in the DOSY spectrum, Figure S3. Te spectra were recorded, processed and analyzed using TopSpin 3.2 sofware (Bruker BioSpin AG), https://​ www.bruker.com​ .

Chemo-enzymatic protocols enable synthesis of glycoconjugates with unprecedented precision and are easier than the multistep procedures that are needed when using conventional carbohydrate chemistry. Much progress has been made in recent years on regioselective modifcation of unprotected sugars­ 38, for example through selec- tive chemical ­oxidation39, allowing increasingly efcient chemical synthesis of oligosaccharide-based glycoconju- gates. Te current study provides a novel chemo-enzymatic route towards glycoconjugates from oligo- and even polysaccharides, providing an attractive alternative to synthesis starting from monosaccharides­ 40. We envisage that technologies such as the one presented in this study will help pave the way towards environmentally friendly valorization of biomass and production of useful biochemicals. Materials and methods Materials. Cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose were from Megazyme (Bray, Ireland). Sodium cacodylate, manganese (II) chloride tetrahydrate, uridine diphosphate galactose (UDP-Gal), fuorescein isothiocyanate (isomer I) and galactosyl transferase from bovine milk were from Sigma. Alex- aFluor488 C5-aminooxyacetamide, and bis(triethylammonium) salt were from Invitrogen (Nærum, Denmark). 2-(aminooxy)-1-ethanaminium dichloride was from ABCR GmbH (Karlsruhe, Germany). 2,5-Dihydroxy-ben-

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DP2 DP3 DP3 DP4 DP5 DP6 single ox single ox + + + 803.5 [M+Na] + [M+Na] + + + + [M+Na] 965.6 [M+Na] [M+K] [M+H] + + [M+K] + + [M+H] 641.5 127.7 [M+H] 1 [M+K] [M+K] [M+H]

OH OH OH OH OH

O O O O O O O N O O O H2N O N NH2 HO HO HO HO HO OH OH OH OH OH H .] .u + + [a + s. en [M+Na] t [M+Na] [M+Na] + + 583.2 + 423.1 585.2 641.5 Rel. In + [M+K] + [M+Na] + + + + [M+Na] 421.1 [M+K] 803.5 [M+H] [M+K] [M+H] [M+K] [M+H] 747.3 + + + 585.2 + + [M+Na] [M+Na] [M+Na] + + [M+Na] 803.5 [M+Na] 583.2 + + 421.1 423.1 [M+K] [M+K] + + + 745.3 747.3 [M+K] [M+H] [M+K] [M+H] [M+H]

400 500 600 7009800 00 1000 1100 m/z

Figure 8. MALDI-ToF-MS analysis of a mixture of bi-functionalized aminooxy-cello-oligosaccharides and products formed afer cellulase treatment of this mixture. Te spectra shown are for an untreated mixture (magenta), a Cel5A treated mixture (red spectrum and red arrows; enzyme reaction 3) and a Cel6B treated mixture (green spectrum and green arrows; enzyme reaction 4). Te insert shows the structure of the bi-functionalized pentamer, with solid and dotted arrows indicating the major and minor cleavage activity of Cel5A (red) and Cel6B (green), respectively; see text for details. Adducts of proton, sodium and potassium are labelled as [M+H]+, [M+Na]+ and [M+K]+, respectively. Te spectra were generated with Flex analysis 3.4. https​ ://www.bruker.com​ .

zoic acid was from Bruker Daltonics (Bremen, Germany). Te LPMO used in this study, from Neurospora crassa (NcLPMO9A), and cellobiose dehydrogenase from Myrococcum thermophilum (MtCDH) were produced and purifed according to Petrovic et al. 2019­ 26 and Flitsch et al. 2019­ 28, respectively. Te endocellulase Cel5A from Hypocrea jecorina was produced according to Saloheimo et al. 1988­ 41 and the exocelluase Cel6B from Termobi‑ fda fusca was produced according to Vuong and Wilson ­200942.

Methods. Enzyme reactions.

(1) NcLPMO9A (1 µM), ascorbic acid (4 mM), phosphoric acid swollen cellulose (PASC, 1% w/v), TrisHCl (10 mM, pH 8.0), 50 °C, 1,000 rpm, 12 h. (2) MtCDH (1 µM), NcLPMO9A (1 µM), ascorbic acid (1 mM), phosphoric acid swollen cellulose (PASC, 1% w/v), 50 °C, 1,000 rpm, 12 h. MtCDH was used to form double oxidized products. (3) Cel5A (1 µM, endocellulase from Hypocrea jecorina), oxime functionalized oligosaccharides (1 mg/mL), 50 °C, 1,000 rpm, 12 h. (4) Cel6B (1 µM, exocellulase from Termobifda fusca), oxime functionalized oligosaccharides (1 mg/mL) 50 °C, 1,000 rpm, 12 h.

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OH A) OOO H OH O O OH R N N O O HO OH H H OH HO O OH HO N N R COOH O n OH b .7 .7 .6 H = R c 1354 1192 or + + .] 7 HN = R 1170 .u C

332. f . [a [M+Na] 1 [M+Na] ns S .8 + te In 559 g e 1 a

d + [M+Na] Rel. + 7 . + 721 .9 + 8 1 . 397 5 1 6 [M+Na] .

[M+Na] h 494 43 0 1 . [M+Na] 008 [M+Na] 4 1 628. 88 1

B) O O OH

k + 6 j . COOH 50 5 . [M+Na] 11 88 9 + ] . H = R .u + or [a . [M+H ] + HN = R s C [M+H ] 5 en + 6 nt i . S OH 966. OH Rel. I 1128 [M+2Na-H] O O .4

.6 OH 7 R-N 5 N [M+2Na-H] O 72 73

5 O . 1 H l HO 804. 0 1 OH

7 OH

. HO O 01 1 12

.8 OH

3 HO

1 COOH m n OH 1474

600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 9. MALDI-ToF-MS analysis of the results of FITC labeling. (A) FITC labeling of double oxime- functionalized oligosaccharides with possible structures shown above the mass spectrum. Peak clusters representing single labeled products are a (n = 1), b (n = 2), c (n = 3) and d (n = 4), and peak clusters representing double labeled products are e (n = 1), f (n = 2), g (n = 3), and h (n = 4). Tis fgure shows that labeling with FITC was incomplete, meaning that also singly labeled products were present. Tis is most likely due to the use of a short reaction time (1 h) and a low amount of FITC equivalents. (B) FITC labeling of single oxime- functionalized oligosaccharides with possible structures shown to the lower right. Te signal clusters represent i (n = 0), j (n = 1), k (n = 2), l (n = 3) and m (n = 4). Note the double sodium which is typical for saccharides containing carboxylic acids­ 50,51. Adducts of proton, sodium and sodium salts of sodium adducts are labelled as [M+H]+, [M+Na]+ and [M+2Na−H]+, respectively. R corresponds to FITC or H. Te spectra were generated with Flex analysis 3.4, https://www.bruke​ r.com​ .

Enzyme reactions (3) and (4) were used to demonstrate that oxime functionalized oligosaccharides were enzymatically degradable.

Production of galactosyl‑cello‑oligosaccharide standards. Galactosyl-cello-oligosaccharides were synthesized using a modifed procedure from ­literature43. Briefy, 1 mg of cellotriose or cellotetraose was dissolved in 500 µL of 40 mM sodium cacodylate bufer pH 6.8 containing 40 mM MnCl­ 2, 5 mg of UDP-Gal and 0.5 mg of galactosyl transferase from bovine milk (Sigma-Aldrich). Te galactosyl transferase will add one galactose to the non- reducing end of the cello-oligosaccharides, forming a β-(1–4) ­ 44. Te reaction was lef to proceed for 3 days at 37 °C with shaking at 1,400 rpm. Te reaction mixture was lef to cool down to room temperature and was directly applied to a CarboGraph column (Grace, Columbia, USA) for desalting of the oligosaccharides, essentially according to a previously published ­protocol45.

Functionalization of C4‑oxidized cello‑oligosaccharides with a hetero‑bi‑functional linker and labeling with a fuo‑ rophore, forming oxime linked products. 5 mL of the supernatant from the reactions with PASC (10 mg/mL)

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a eb f c g d h OOO H

A ce 100 n a. m/z 1008.57 c. m/z 1332.76 D e. m/z 1397.87 g. m/z 1722.09

bunda b. m/z 1170.70 d. m/z 1494.88 3-7 50 y z 1-5 y 2 y 1 COOH A f. m/z 1560.00 h. m/z 1884.24 OH H OH H OH N H2N O H O H O H OH O 0 O O H Relative N N NH HO HO HO C 0 5 10 15 20 25 30 OH OH OH OOO H H H H Time (min) H H H H H H S n M-FITC b B 100 4 1008.69 b1-5 c 770.46 [M+H]+ = 1397.87 m/z 1-5

e b 80 b1 / y1 3 COOH c n=1 (a), 2 (b), 3 (c), 4 (d) n 608.33 390.12 y3 y 1-5 1-5 60 z 628.38 M-FITC-( H O) bunda 972.67 M-FITC-( H O) M-(H O) A 40 H OH H OH H OH 990.67 1362.04 HN N N

ve b / y 2 2 y4 C O H O H O H OH 20 433.18 790.51 O S O O N NH Relati M HO HO HO 2 543.38 1050.69 OH OH OH 0 1235.79 b b H H H M-FITC 1 2 H H H H H H n 100 b4 1170.84 + 770.47 [M+H] = 1560.00 m/z b e 3-7 c1-5 80 b3 nc

a 608.34 b1 / y 1 60 y3 E 390.17 b5 100

bund 628.38 + A 932.60 [M+H] = 1008.57 m/z , 40 ² ³a e e 1

c 80 v n

ti 1052.77

b2 / y2 a a y 5 el 20 433.23 1234.85 1396.89 60 R 952.62 M bund

543.41 A 0 b3 e 40 y 3 M-(H O) b b4 4 M-FITC iv b2 / y 2 608.37 628.42 972.68 100 770.48 1332.96 + 770.49 y 4 [M+H] = 1722.09 m/z elat 20 433.22 790.53 R b3 M 80 0 543.40 872.59 1205.06 1354.301484.38 1817.93 b3 b b1 / y 1 60 608.34 5 y 932.59 100 390.16 3 [M+H]+ = 1170.70 m/z 40 628.39 b6

ve Abundance y 4 1705.72 80 790.57 1094.72 20 1374.99 1560.09

Relati M 60 b 0 489.40 563.45 1234.91 b3 4 770.47 y 5 M-FITC 40 608.37 y 3 ve Abundance 952.64 M-(H O) 100 1495.11 + b2 / y 2 628.40 y 4 [M+H] = 1884.24 m/z b 1134.80 b4 20 433.22 790.50 5 Relati b3 932.61 M

e 80 770.55 c b5 543.39 1295.3911472.36 1655.35 951.20

n 0 y3 a b3 932.69 b y 60 628.45 M-2FITC 1 / 1 608.36 100 390.13 + bund 1105.94 [M+H] = 1332.76 m/z A

40 b4 e e y4 y 5 c v 80

i 770.47

t b

7 n

a b3 la 20 790.68 952.76 1816.08 e 1256.89 M 608.35

R 60 574.43 1396.03 1559.03 0 bund A

y 3 M-(H O) 400 600 800 1000 1200 1400 1600 1800 2000 40 b5 628.39 b6 m/z tive b2 / y 2 y 5 1296.89 OOO H 932.60 1094.73 C 20 433.19 y 4 952.64 Rela b3 790.52 OOO H M 0 543.38 1418.67 n=1 (e), 2 (f), 3 (g), 4 (h) b4 100 770.48 COOH [M+H]+ = 1494.88 m/z

y y 80 b3 y 3-7 z 1-5 2 1 COOH 608.33 60 H OH H OH H OH HN N N b5 C O y 3 H H H Abundance O O OH 40 932.61 M-FIT C M-(H O) O e y H b2 / y 2 628.39 4 S O O N NH 1105.91 1458.88 N 790.55 y 5 HO HO HO ativ 433.25 C l 20 b H OH H OH H OH 952.69 7 Re 1256.90 M b1 b 2 H H H H H H S n 0 419.16 1744.49 2,3 400 600 800 1000 1200 1400 1600 1800 2000 a1 m/z b3-7 c1-5

Figure 10. HILIC-FLD-MS analysis of the results of FITC labelling. (A) Overlay of extracted ion chromatograms corresponding to single (blue) and double (red) labelled FITC oligosaccharides for m/z-ratios a (n = 1) 1,008.57, b (n = 2) 1,170.70, c (n = 3) 1,332.76 and d (n = 4) 1,494.88 and e (n = 1) 1,397.87, f (n = 2) 1,560.00, g (n = 3) 1722.09 and h (n = 4) 1,884.24 respectively. (B) MS/MS spectra of the double labelled FITC oligosaccharides. (C) Structures of the four double FITC labelled products, with theoretical masses and the observed fragmentation patterns (using the nomenclature of Domon and Costello)49. (D) Structures of the possible single FITC labelled products arising from incomplete labelling, with theoretical masses and the observed fragmentation patterns (using the nomenclature of Domon and Costello)49, with the label located at the reducing end (red) or the non-reducing end (green). (E) MS/MS spectra of the single labelled FITC oligosaccharides.

prepared as described in enzyme reaction 1 above were used in further experiments. Te oligosaccharides were functionalized by reacting carbonyl groups with 2-(aminooxy)-1-ethanaminium dichloride to form oximes, using a modifed procedure from ­literature46. Te reaction was conducted in 0.1 M NaOAc bufer, pH 4.9, with ca. 50 molar equivalents of the aminooxy linker based on the amount of oxidized oligosaccharides, over a course of 6 days at 37 °C with shaking at 1,400 rpm. Te products were then purifed using active carbon, essentially according to ­literature45. Oxime-functionalized oligosaccharides were labeled with fuorescein isothiocyanate (FITC, ca. 6 molar equivalents with respect to the amino-group content of the oligosaccharide) by incubation in 10 mM sodium bicarbonate bufer, pH 9.5, for 1 h, with shaking at 1,400 rpm, at room temperature and in the dark. Te reaction mixture was then freeze-dried.

Reduction of soluble products generated by NcLPMO9A. Reduction of oligosaccharides was conducted by mix- ing 10 µL of oligosaccharide sample from reaction 1 with 65 µL H­ 2O (MilliQ) containing 700 µg NaBH­ 4 or NaBD­ 4. Te reduction reaction was incubated over night at ambient temperature and then quenched by adding 20 µL 25 mM sodium acetate. Chromatographic analysis by HPAEC (see below) was done with the sample as is, whereas a further sample preparation for MALDI-ToF-MS was done as follows. Approximately 10 µL of a 1:1 (w/w) suspension of ­H2O:Supelclean ENVI Carb (Sigma-Aldrich) was packed in a pipette tip containing a C8-disc that was trapping the Supelclean material. Te bed was conditioned with 50 µL H­ 2O. Te sample (5 µL) was applied, rinsed with 50 µL ­H2O and then eluted with 20 µL acetonitrile. Te eluate was then analyzed by MALDI ToF-MS.

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HPLC analysis of oligosaccharides. Oligosaccharides were analyzed using three diferent chromatographical principles. Te frst principle, high-performance anion-exchange chromatography (HPAEC) at high pH and coupled with pulsed amperometric detection (PAD), was conducted on an ICS3000 system from Dionex (Sun- nyvale, CA, USA), using previously described conditions and ­gradients30,47. Te system was set up with PAD using disposable electrochemical gold electrodes. Two µL samples were injected on a CarboPac PA1 2 × 250 mm analytical column equipped with a CarboPac PA1 2 × 50 mm guard column and columns were kept at 30 °C. Te second principle, porous graphitic carbon (PGC) chromatography, was applied using an Ultimate 3000RS (Dionex) UHPLC system, which was connected in parallel (10:1 split) to an ESA Corona Ultra charged aerosol detector (ESA Inc.,Dionex, Sunnyvale, USA) and a Velos pro LTQ linear ion trap (Termo Scientifc, San Jose, CA USA). Te PGC column (Hypercarb, 3 µm, 2.1 × 150 mm) including a guard column (Hypercarb, 3 µm, 2.1 × 10 mm) was operated at 70 °C using previously described conditions and gradients­ 30. In some cases, 10 µM formic acid was used instead of ammonium acetate, to improve resolution of the oxime coupled products. Two µL samples were injected. Te third principle, hydrophilic interaction chromatography (HILIC), was applied using an Agilent 1290 Infnity (Agilent Technologies, Santa Clara, CA, USA) UHPLC system, which was connected in parallel (1:10 split) to an Agilent 1260 fuorescence detector—Ex 490 nm, Em 520 nm (Agilent Technologies, Santa Clara, CA, USA) and a Velos pro LTQ linear ion trap (Termo Scientifc, San Jose, CA, USA). Te HILIC column (bioZen Glycan, 2.6 µm, 2.1 × 100 mm) including a guard column (SecurityGuard ULTRA with bioZen Glycan cartridge, 2.1 × 2 mm) was operated at 50 °C, running at 0.3 mL/min, and using 50 mM ammonium formate pH 4.4 (eluent A) and 100% acetonitrile (eluent B). Samples were eluted using the following gradient: initial starting ratio of 85% B and 15% A, gradient to 75% B and 25% A from 0 to 5 min, gradient to 60% B and 40% A from 5 to 25 min, gradient to 40% B and 60% A from 25 to 28 min, isocratic from 28 to 32 min, gradient to 85% B and 15% A from 32 to 35 min. Two µL of the samples were injected.

MALDI‑ToF analysis of oligosaccharides. Two µL of a 9 mg/mL solution of 2,5-dihydroxybenzoic acid in 30% (v/v) acetonitrile was applied to an MTP 384 target plate ground steel TF (Bruker Daltonics). One µL of the sample was then mixed into the DHB droplet followed by drying under a stream of hot air. Te samples were analyzed with an Ultrafex MALDI-ToF/ToF instrument (Bruker Daltonics GmbH, Bremen, Germany) with a Nitrogen 337 nm laser beam. Te instrument was operated in positive acquisition mode and controlled by the FlexControl 3.3 sofware package. Te acquisition range used was from m/z 200 to 7,000. Te data were collected from averaging 250 laser shots, with the lowest laser energy necessary to obtain sufcient signal to noise ratios. Peak lists were generated from the MS spectra using Bruker FlexAnalysis sofware (Version 3.4).

Mass spectrometry of oligosaccharides, direct injection MS, PGC‑MS and HILIC‑FLD‑MS. For direct injection, oligosaccharides were analyzed using an LTQ-Velos Pro linear ion trap mass spectrometer (Termo Scientifc, San Jose, CA, USA) connected to an Ultimate 3000RS HPLC (Dionex, Sunnyvale, CA, USA). Te setup was used for direct injection without a column; the pump delivered 200 µL/min of 0.03 µM formic acid in 70% acetonitrile and data was acquired for 24 s afer injection. For the MS, the capillary voltage was set to 3.5 kV and the scan range was m/z 150–2,000 using two micro scans. Te automatic gain control was set to 10,000 charges and the maximum injection time was 20 ms. For fragmentation of selected precursor ions by MS/MS, the normalized collision energy was set to 37 and three micro scans were used. For PGC-MS, the same MS-parameters were used as for direct injection with the exception that the scan range was m/z 250–2,000. For HILIC-FLD-MS the instrument was operated in positive mode with an ionization voltage of 3.5 kV, auxiliary and sheath gas set- tings of 5 and 30 respectively (arbitrary units) and with capillary and source temperatures of 300 °C and 250 °C, respectively. Te scan range was set to m/z 110–2,000 and MS/MS analysis was performed with CID fragmenta- tion with helium as the collision gas. All data were recorded with Xcalibur version 2.2 (Termo Scientifc).

NMR sample preparation. Samples for NMR analysis were lyophilized, then dissolved in 1,000 µL 99.9% ­D2O (Chiron, Trondheim, Norway), frozen and lyophilized. Te dried samples were then dissolved in 300 µL 99.9% ­D2O and transferred into a 4 mm Shigemi tube (Shigemi, Allison Park, PA, USA).

NMR data acquisition and analysis. All homo- and heteronuclear NMR experiments were carried out on a BRUKER Avance III HD 800 MHz spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) equipped with a 5 mm TXI z-gradient probe. For chemical shif assignment, the following spectra were recorded: 1D 1H, 2D double quantum flter cor- related spectroscopy (DQF-COSY), 2D total correlation spectroscopy (TOCSY) with 70 ms of mixing time, 2D­ 48 heteronuclear single quantum coherence (HSQC) with multiplicity editing, 2D­ 48 heteronuclear 2-bond corre- lation (H2BC), 2D ­[1H–13C] HSQC-[1H,1H] TOCSY with 70 ms of mixing time on protons, and 2D ­[1H–13C] heteronuclear multiple-bond correlation (HMBC) with BIRD (bilinear rotation decoupling) fltering to suppress frst order correlations. Tese spectra were all recorded at 25 °C. Difusion-ordered spectroscopy (DOSY) was used to measure the difusion of the coupled products. A 2D DOSY was set up using a Bruker BioSpin stimulated echo pulse sequence with bipolar gradients (STEBPGP). Gradient pulses of 2 ms duration (δ) and 32 diferent strengths varying linearly from 0.03 to 0.57 T⋅m−1 were applied and the difusion delay (Δ) was set to 80 ms. Te DOSY spectrum was recorded at 25 °C. Te spectra were recorded, processed and analyzed using TopSpin 3.2 sofware (Bruker BioSpin AG).

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Received: 4 April 2020; Accepted: 10 July 2020

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Competing interests Te authors declare no competing interests. Additional information Supplementary information is available for this paper at https​://doi.org/10.1038/s4159​8-020-69951​-7. Correspondence and requests for materials should be addressed to B.W. or F.L.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access Tis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/.

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