University of Groningen Exploring and exploiting bacterial protein glycosylation systems Yakovlieva, Liubov

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(1)University of Groningen. Exploring and exploiting bacterial protein glycosylation systems Yakovlieva, Liubov DOI: 10.33612/diss.173544104 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2021 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): Yakovlieva, L. (2021). Exploring and exploiting bacterial protein glycosylation systems. University of Groningen. https://doi.org/10.33612/diss.173544104. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 21-07-2021.

(2) CHAPTER 6 Site-selective palladium-catalyzed oxidation of glucose in glycopeptides This Chapter describes a novel method of site-selective oxidation of glucose moieties on individual glycopeptides and on a mixture of tryptic glycopeptides. The organometallic catalyst [(neocuproine)PdOAc]2OTf2, previously shown to perform regioselective C3-oxidation of glucosides, was used in the scope of this work. The selectivity of the catalyst towards glucose and the sensitivity of specific amino acid residues to oxidation was explored by screening a select panel of glycopeptides in the oxidation reactions. The oxidation methodology was subsequently applied to the complex mixture of tryptic glucopeptides, generated from a fragment of the Haemophilus influenzae adhesin glycoprotein. The resulting ketogroup of the glucose was further transformed into an oxime functionality, which allows introduction of various groups of interest. The methodology outlined in this Chapter will allow to perform late-stage modification of glucopeptides as well as selective oxidation and functionalization of tryptic glucopeptides for proteomics analysis.. Reintjens, N. R. M.; Yakovlieva, L.; Marinus, N.; Hekelaar, J.; Nuti, F.; Papini, A. M.; Witte, M. D.; Minnaard, A. J.; Walvoort, M. T. C. Site-selective palladium-catalyzed oxidation of glucose in glycopeptides. ChemRxiv 2021, doi.org/10.26434/chemrxiv.14230244.v1. .

(3) CHAPTER 6. Carbohydrates form a diverse class of biomolecules with a myriad of roles in living cells. They form oligo- and polysaccharides or decorate proteins and lipids, thereby influencing the interactions and biological roles of these molecules.1 The presence of the carbohydrate moieties (glycans) confers a variety of properties to the resulting glycoconjugates, both biophysical (stability, solubility) and functional (transport, intra- and extracellular interactions).2 Importantly, aberrant glycosylation or altered levels and composition of the glycan structures are implicated in various diseases, including cancer.3,4 Bacterial cell surface glycoproteins frequently mediate motility, adherence, immune system evasion and biofilm formation, and as such constitute important virulence factors of pathogenic bacteria, and are considered interesting targets for antibiotic development.5,6 Interestingly, in recent years virulence-associated glycoproteins synthesized via unconventional glycosylation systems have been discovered and feature bacteria-specific rare sugars and glycosidic linkages.6 A notable example comprises the high molecular weight adhesin (HMWA) proteins from the Gramnegative human pathogen Haemophilus influenzae.7 Specifically, HMW1A is hyperglucosylated (i.e. modified with multiple glucose moieties) on 31 Nglycosylation sites by the action of the associated N-glycosyltransferase HMW1C in the cytoplasm.8 Extensive glucosylation is paramount for the stability of the adhesin protein and tethering to the cell membrane, where HMW1A is involved in adhering to the host cells during the initial stages of colonization.9 Studies of H. influenzae HMWA and other clinically relevant glycoproteins are complicated by the limitations of the available methods to selectively label these unusual glycoproteins. Glycoprotein synthesis is nontemplated, often features microheterogeneity (i.e. multiple glycoforms of the same protein) and glycans are difficult to reliably manipulate on a genetic level. Therefore, methodologies that allow to unambiguously identify and enrich the glycoprotein of interest are highly valuable tools in glycobiology. In this regard, development of methods that allow the introduction of selective glycan modifications amenable for further manipulations are needed. The metabolic oligosaccharide engineering (MOE) method, as pioneered by C. Bertozzi10,11 and W. Reutter,12 has introduced a multitude of possibilities for glycan manipulation. Through the development of unnatural sugar precursors bearing bioorthogonal reporter groups, metabolic pathways can be exploited to incorporate sugar analogues into the glycans of interest. The field of MOE has expanded over the recent years and now features multiple 144.

(4) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES bioconjugation reactions, reporter tags and monosaccharide-based probes for profiling glycans of interest.13,14 Nonetheless, there are various challenges associated with MOE, in particular in the context of bacterial glycoproteins, such as the design and synthesis of unnatural monosaccharides, cell permeability of the probes and promiscuity of the metabolic pathways. Selective modification of a glycan or glycoconjugate of interest remains a challenging task which requires fine-tuning and adapting for each individual system of interest. An alternative method to isolate glycoconjugates of interest is their derivatization with a bioorthogonal handle after their synthesis. This has been achieved enzymatically by oxidizing primary alcohols of the carbohydrate residues into their corresponding aldehydes (e.g. with oxidase enzymes) and chemically by oxidative cleavage of vicinal diols (e.g. with sodium periodate). The resulting carbonyl group can then be readily transformed into a hydrazone or oxime.15 Enzymatic oxidation16–20 has been reported for galactose, mannose, GalNAc, and GlcNAc, and has been applied to the diversification of glycopeptide antibiotics and the introduction of amines and amides.21,22 In addition, oxidation and subsequent oxime/hydrazone formation can also be used as a strategy to introduce biotin or fluorophores (also on-cell).19–20 Similar to the MOE methodology, which relies on the enzyme specificities of the metabolic pathways, enzymatic oxidation is limited to the range of suitable substrates for the available oxidases. Chemical oxidation of the glycan hydroxyl groups can be performed by sodium periodate, and this has been used extensively on galactose and sialic acid residues, also on glycopeptides.19,23–25 Although this type of oxidation is particularly effective for cis diols, the oxidation of trans diols such as O-GlcNAcmodified proteins can be achieved by using a high concentration of sodium periodate in combination with elevated temperatures.26 A drawback is that sodium periodate also acts on 1,2-aminoalcohols and rapidly cleaves N-terminal serine (Ser) and threonine (Thr) residues.23 In addition, sodium periodate may oxidize other residues in peptides as well, such as methionine, carbamidomethylated cysteine, and tryptophan, especially under the forcing conditions used in oxidative cleavage of vicinal trans-diols.23,26 Therefore, sodium periodate is not suitable to specifically modify glucosylated proteins and peptides. In this Chapter the possibility to modify glucopeptides using siteselective palladium-catalyzed oxidation of unprotected carbohydrates is described. This method has been extensively studied by the Minnaard group and the group of Waymouth,27–30 and has proven to be very useful for the 145.

(5) CHAPTER 6 selective oxidation of the C3 position in glucose-configured pyranosides.30,31 When applied to 1,4-linked glucans, the reaction is highly selective for the terminal residue, due to steric reasons (Figure 1).28 The resulting unprotected 3keto glucosides can be used to prepare rare sugars and to introduce click handles and reactive groups, as was recently shown by Marinus et al.32 Although transition metal-catalyzed reactions, like with palladium, are widely used in synthetic chemistry, their use in biological settings is met with difficulties such as low conversions, the requirement of high temperatures or loss of protein.33–37 These catalysts have the tendency to coordinate with functional groups, such as amides and amines. For example, Ourailidou et al. observed during the oxidative Heck reaction that the binding of Pd(II) to the protein hampered the reaction by limiting the availability of the catalyst.38 This effect can be counteracted by selecting an appropriate ligand for the Pd catalyst, as was also recently reported by Cao et al. for the Suzuki-Miyaura reaction in cell lysates.39 In this Chapter it is demonstrated that the palladium neocuproine catalyst system can be employed to selectively oxidize glucose residues in selected glucopeptide sequences, containing serine and threonine residues. The resulting keto function is used to attach useful tags and labels prior to proteomics experiments. The limits of this method are also described. Consequently, a new method is added to the toolbox for glycopeptide labeling. This method will be of use in the process of understanding the impact of specific glycoproteins in biological processes, with the glucosylation of HMWA adhesins as a prime example.. 146.

(6) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES. Figure 1. Previous and current scope of C3-selective oxidation of glucans and subsequent keto-group functionalization. Pd-cat = [(neocuproine)PdOAc]2OTf2, BQ = benzoquinone.. Oxidation takes place under dilute conditions To translate the oxidation protocol, developed for preparative carbohydrate synthesis, to glycopeptides and ultimately glycoproteins, first, the lower limits of substrate concentration were explored. The palladium-catalyzed oxidation of monosaccharides is generally performed in DMSO or methanol at relatively high concentrations (0.1-0.3 M).27,30,31 However, due to the low abundance of glycopeptides and glycoproteins in natural samples, these concentrations are not attainable, and the first goal was to screen for catalyst activity at dilute conditions. Both the α- and β-anomer of methyl D-glucopyranoside were using subjected to the oxidation reaction in DMSO-d6 [(neocuproine)PdOAc]2OTf2 as the catalyst and benzoquinone as the terminal oxidant. The reactions were performed in an NMR tube and monitored over time. As shown in the Table S1 (Supporting Information), full oxidation of the D-glucopyranosides was achieved at a concentration as low as 3 mM, and heating at 40 °C accelerated the oxidation. It has been shown earlier that the oxidation reaction is considerably accelerated in DMSO as the solvent.30,40 In line with this observation, the rate of oxidation decreased in DMSO/water mixtures, 147.

(7) CHAPTER 6 and increasing amounts of the Pd catalyst (0.1 eq vs of 0.05 eq) were required to obtain full conversion in a mixture of D2O/DMSO-d6 (9/1 v/v). Using qNMR it was revealed that oxidation of both the α- and β-anomer of methyl Dgalactopyranoside led to full consumption of the monosaccharides (Table S2), however, significant amounts of degradation were observed for both substrates due to overoxidation and subsequent rearrangement, as observed before.31 Interestingly, the dilute conditions as developed above significantly reduced the degradation after oxidation.. Chemoselective oxidation of model glycopeptides In the next step, the glucopeptide Ac-YEPN(Glc)GAS (1) was selected to investigate the chemoselective oxidation of the asparagine-linked glucoside residue (Figure 2A). This glucopeptide was designed in such a way that it would show a reasonable solubility and the amino acid side chains would not interfere with the reaction. The workflow of the glycopeptide oxidation experiments is depicted in Figure 2B. The oxidation reactions were performed at 3 mM (30 µL total volume), and the reactions were monitored using reverse-phase liquid chromatography-mass spectrometry (RP-LCMS) (Figure 2C). Initial experiments showed that the dilute conditions used in the NMR experiments on monosaccharide oxidation did not yield any oxidized glucopeptide (Table 1, entry 1). However, increasing the amount of [(neocuproine)PdOAc]2OTf2 to 0.5 eq (i.e. 1 equivalent of palladium, entry 2) led to the appearance of the peak corresponding to keto-product 2 (m/z = 939). The mass corresponding to the deglycosylated product 3 was also detected (m/z = 779). Comparison of the RPLCMS fragmentation profiles of peptides 1 and 2 proved that solely the glucose residue was oxidized (Figure 2D). To estimate the conversion, we compared the relative intensity of the peaks and corrected them for the isotope pattern, accounting for 88% conversion to 2 and 5% to 3 after 75 min reaction time (Figure 2C). For one sample, the conversion was also determined by calculating the relative ionization factor (Figure S1),41 which gave slightly higher conversions. Due to the minimal difference in the calculation methods, it was decided to calculate the conversions directly from the relative peak intensities.. 148.

(8) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES. Figure 2. Workflow and analysis of the oxidation of glucopeptide 1. A: Oxidation of glucopeptide 1 to keto-product 2 and deglycosylated product 3. B: Setup of the oxidation reaction. C: RP-LCMS analysis by comparison of the peak intensities. The mass values 941, 939, and 779 correspond to peptides 1, 2, and 3 respectively. D: MS/MS fragmentation of 2.. It was observed that longer reaction times led to increased formation of deglycosylated product 3. To understand the origin of this bond cleavage, first the stability of glucopeptide 1 in the presence of Pd(OAc)2 was investigated (entry 1, Table S3). Negligible formation of 3 was observed, suggesting that the deglycosylation reaction only occurs after oxidation, hence from 2. Addition of more Pd catalyst to a fully oxidized reaction mixture resulted in the increased formation of 3 (entry 2-3, Table S3). Moreover, removal of the Pd catalyst after the oxidation reaction by treatment with ammonium pyrrolidine dithiocarbamate (APDC) as a palladium scavenger suppressed the deglycosylation reaction (vide infra).42,43 It was therefore concluded that the deglycosylation reaction is caused by the Pd catalyst, either via an E1cB elimination or via overoxidation. In the first pathway, enolization of the carbonyl group is followed by E1cB elimination of the anomeric substituent (see Figure S2 for a proposed mechanism).44,45 In the second proposed pathway, C2-OH is oxidized, which can then enolize and after formation of an hemiaminal 149.

(9) CHAPTER 6 hydrolysis takes place. A small signal corresponding to the overoxidized product (m/z = 937) was observed (Figure 2). Similar to the monosaccharide oxidation reactions, the oxidation of 1 occurs at a higher rate at elevated temperatures (compare entries 2 and 3, Table 1). A higher Pd catalyst loading gave a slightly faster conversion but resulted, as may be expected, also in further deglycosylation (entries 4 and 5). Oxidation reactions in different solvent mixtures revealed that oxidation of 1 in DMSOd6/D2O mixture (entries 6-8) is possible, however, longer reaction times and more Pd catalyst are required. These results suggest that this method may have potential for the oxidation of glycoproteins in aqueous media, a perspective that will be studied in the near future. To assess whether the preference for glucose over galactose, as observed in monosaccharides oxidation reactions,31 was also present in the corresponding glycopeptides, the oxidation of the glycopeptide S9 containing a galactose moiety was investigated (Table S4). The oxidation occurred considerably slower and more Pd catalyst was required compared to the corresponding glucopeptide 1. This suggests that it may be possible to oxidize glucose residues in the presence of galactose residues in a glycopeptide.. 150.

(10) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Table 1. C-3 Oxidation of glucose in glycopeptide 1[a] Temp. (°C). Solvent. Pd-cat. (eq). Conversion (%) 2/3. 1. 40. DMSO-d6. 0.05. ON: 0/0. 2. 40. DMSO-d6. 0.5. 75 min: 88/5. 3. 22. DMSO-d6. 0.5. 150 min: 69/5 ON: 81/15. 4. 40. DMSO-d6. 1. 75 min: 85/9 ON: 35/58. 5[b]. 40. DMSO-d6. 3. 75 min: 87/12 ON: 10/90. 6. 40. DMSO-d6/D2O: 9/1 v/v. 1. 75 min: 82/6 ON: 51/45. 7[c]. 40. DMSO-d6/D2O: 1/1 v/v. 0.5. ON: 37/29. 8[b]. 40. DMSO-d6/D2O: 1/1 v/v. 3. 7 h: 54/19 ON: 14/83. [a] Reactions were performed at. 3 mM concentration in the presence of 3 eq benzoquinone. RP-LCMS samples were prepared by 10x dilution with CH3CN/tBuOH/H2O 1/1/1 v/v/v to obtain a 0.3 mM concentration. Samples of entries with more than 1 eq of Pd catalyst were treated with APDC before RP-LCMS analysis. Conversions were calculated by comparison of the relative ionization intensities of the corresponding molecular ions. [b] No benzoquinone was added. [c] A DMSO-d6/D2O mixture (1/9 v/v) resulted in slower oxidation.. Oxidation of complex glycopeptides Next, two peptide sequences 5 and 6 (Table 2) were selected of the bacterial high molecular weight adhesin protein HMW1A, known to be glucosylated on specific asparagine residues in Haemophilus influenzae.8 Compared to the nonglycosylated control peptide 4, glucopeptides 5 and 6 both contain one Glc moiety, in addition to serine, threonine, and lysine residues. These residues were selected to pose two challenges. The hydroxyl functions of serine and threonine side chains might be oxidized by the catalyst. The lysine residue might block the oxidation reaction, coordinating palladium and thus poisoning the catalyst.38 To get an indication of the sensitivity of threonine towards oxidation by the Pd catalyst, peptide 4 (containing two Thr) was treated with one eq of Pd 151.

(11) CHAPTER 6 catalyst (Table 2, entry 1). It turned out that the secondary hydroxyl group on Thr was indeed oxidized, with the singly oxidized compound as the major product compared to the twice oxidized product. The mass fragmentation pattern of this oxidized peptide indicates that the C-terminal Thr is oxidized first. Longer reaction times led to the formation of degradation products, probably as a result of overoxidation, in which the C-terminal Thr or the peptide fragment Thr-Ile-Thr (TIT) is split off. Proposed mechanisms are reported in Figure S3, wherein it is shown that either overoxidation can take place or, after oxidation of Thr, tautomerization to the corresponding enol occurs. Subsequent imine formation followed by water addition forms a hemiaminal product, which is readily hydrolyzed. This side-reaction has also been observed by Bose et al. in the synthesis of β-lactams46 and by Abeysinghe et al. when using oiodoxybenzoic acid as an oxidizing agent.47 NMR experiments with N-acetyl-threonine confirmed that the Pd catalyst oxidized the secondary hydroxyl group in threonine, although at least 0.5 eq of catalyst was required to obtain full conversion (entry 2, Table S5). Encouragingly, when N-acetyl-threonine and methyl D-glucopyranoside were combined in equal amounts in a competition oxidation experiment, a strong preference for glucose oxidation was observed at earlier time points (entry 3, Table S5).. 152.

(12) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Table 2. Oxidation of glucose in the presence of threonine and lysine[a]. #. 1. Substrate. 4. Pd-cat. (eq). 1Ox/2Ox/3Ox. 1. 15min: 44/8/n.a.. (%). Degradation products 75min: -T, -TIT. 45min: 69/10/n.a 75min: 63/26/n.a. 2. 5. 1. 75min: 25/1/0. ON: -T, -TIT. ON: 58/26/3. 3xON: -T, -TIT, Glc. 3. 5. 0.5. ON: 8/0/0. -. 4. 6. 0.5. No oxidation. -. 5[b]. 6. 3. ON: Mixture of 0Ox/1Ox/2Ox/3Ox/4Ox. -. Reactions were performed at 3 mM with 3 eq of benzoquinone in DMSO-d6 at 40 °C. RP-LCMS samples were prepared by 10x dilution with 10 mM APDC in CH3CN/tBuOH/H2O 1/1/1 v/v/v to obtain a final 0.3 mM concentration. After mixing, the Eppendorf tubes were centrifuged for 5 min at 12500 rpm, and the supernatant was used for LCMS analysis. [b] No benzoquinone was added to the reaction mixture.. [a]. Therefore, the oxidation experiments were continued with glycopeptide 5. The use of 1 eq of Pd catalyst led to 58% of a mono-oxidized product (Table 2, entry 2). Fragmentation showed that the glucose moiety is oxidized (based on the b6 fragment ion, peptide 5 spectra in the Supporting information). Unfortunately, it was not possible to determine the ratio between the oxidized glucose and threonine. Similarly to the oxidation of glucopeptide 1 and peptide 4, degradation products were formed upon prolonged reaction times. Treatment with APDC and charcoal proved instrumental in facilitating the LC-MS analysis of 5 by removing overlapping ligand peaks from the chromatogram. In an attempt to suppress these side-reactions, the amount of catalyst was reduced to 0.5 eq (Table 2, entry 3), which resulted in a low conversion (8%) to the monooxidized product, which slowly increased over time.. 153.

(13) CHAPTER 6 It was not possible to assign the oxidation site due to the presence of the different oxidation-prone residues. Therefore, it was decided to perform a set of competition experiments to investigate the kinetics of threonine and carbohydrate oxidation reaction. To this end, the glucopeptide 1 was combined with control peptide 4, and RP-LCMS was used to monitor oxidation progress (Table S6). In the presence of 0.5 eq Pd catalyst and 3 eq of benzoquinone, glucopeptide 1 was preferentially oxidized, albeit at a lower rate due to the presence of the threonine-containing peptide 4. Increasing the amount of 4 slowed down the oxidation of 1 even more, resulting in a decrease in turnover number (TON) by more than 50%. It is hypothesized that the catalyst can form a bidentate chelate with threonine via the hydroxyl group and the carbonyl amide in the peptide backbone. As such, threonine residues compete for chelation with the Pd with the vicinal diol units in glucose and thereby slow down the oxidation of glucopeptide 1. Notably, when a similar competition experiment was performed on an equal mixture of peptide 4 and galactosylated peptide S9, with the same peptide sequence as glucopeptide 1, no oxidation of either galactose or threonine was observed. This suggests that galactose, while being a poor substrate for oxidation, chelates well with the Pd catalyst and thereby inhibits threonine oxidation. Overall, these competition experiments highlight again the strong preference of the catalyst to oxidize gluco-configured moieties. Finally, glycopeptide 6 was subjected to the oxidation conditions (Table 2, entries 4 and 5). Despite the presence of an N-terminal amine and the amine of the lysine side chain, oxidation was observed with an increased catalyst loading (3 eq). Similar to glycopeptide 5, however, the multiple oxidationsensitive positions hampered the assignment of the oxidation products. Nonetheless, this shows that the oxidation reaction is possible in the presence of amines, setting the stage for even more complex peptides and mixtures (vide infra).. 154.

(14) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Oxidation of tryptic glucopeptides Following the successful oxidation of individual glycopeptides, the methodology was used in the oxidation of a complex tryptic digest of a glucoprotein. For this the glucosylated C-terminal fragment of the high molecular weight adhesin (HMWA) protein from Haemophilus influenzae was used. The hyperglucosylated C-terminal fragment of HMWA (termed GlcHMW1ct) was produced via co-expression with its cognate Nglycosyltransferase, as described previously and in Chapter 3.48 Glc-HMW1ct was purified using anion exchange chromatography and intact protein massspectrometry revealed the presence of 8-9 glucose moieties per adhesin protein.48 The glycoprotein sample was digested with trypsin to obtain shorter glycopeptides. There is a considerable degree of diversity among the peptide fragments with regard to length, number of glucose residues per peptide and number of Ser and Thr residues (Table 3, Figure S4). The workflow for the oxidation reactions on the tryptic glycopeptides is depicted in Figure 3. After tryptic digestion, the glucopeptides were purified via solid phase extraction (SPE) to remove buffer salts that could interfere with the reaction, and were dried under vacuum. The resulting residue was reconstituted in DMSO to prepare a stock solution for the oxidation reaction. The oxidation was performed at 2.8 mM concentration of glycopeptides (molarity based on glucose and calculated based on the input protein amount) at 40 °C with varying amounts of the Pd catalyst (0.5, 1, 5 and 10 eq calculated relative to Glc residues).. Table 3. Tryptic glucopeptides generated from H. influenzae HMW1ct. Glucosylated asparagines are shown in bold, threonines are shown in red, lysines are shown in blue. I. AHHHHHHVWTAN(Glc)SGALTTLAGSTIK. II. ATTGEAN(Glc)VTSATGTIGGTISGNTVN(Glc)VTANAGDLTVGNGAEI N(Glc)ATEGAATLTTSSGK. III. GQVN(Glc)LSAQDGSVAGSINAAN(Glc)VTLN(Glc)TTGTLTTVK. IV. GSNIN(Glc)ATSGTLVINAK. V. DAELNGAALGN(Glc)HTVVN(Glc)ATNAN(Glc)GSGSVIATTSSR. VI. VN(Glc)ITGDLITINGLNIISK. VII. FIEPN(Glc)NTITVDTQNEFATRPLSR. 155.

(15) CHAPTER 6. Figure 3. Workflow of the oxidation reaction and oxime formation on tryptic glycopeptides. (SPE = solid-phase extraction).. The highest Pd catalyst loadings (5 eq and 10 eq) led to a substantial decrease in general peptide levels (Figure S5A). In contrast, 0.5 and 1 eq of Pd did not result in significant changes in peptide levels compared to the nontreated control sample (Table S7, Figure S5B) and therefore the oxidation of the tryptic glycopeptides was performed with either 0.5 eq or 1 eq of Pd catalyst. The reactions were analyzed after 8 and 24 h by diluting reaction aliquots with an aqueous solution of the Pd catalyst scavenger APDC (vide supra). Following another SPE purification to remove traces of DMSO, ligand, and BQ, the aliquots were subjected to RP-LCMS/MS analysis. Several glucopeptides were identified in the oxidized and the control samples and these glucopeptides could be assigned to one of the three categories: non-oxidized glucopeptides (so-called hexose fraction, “Hex”), glucopeptides containing both glucose and oxidized glucose moieties (mixed fraction, “Mix”) and glucopeptides in which all glucose residues were fully oxidized (oxidized fraction, “OxHex”). To illustrate the different categories, possible peptide forms based on peptide V (Table 3) that could be observed in the reaction mixture are presented in Figure 4. The peak areas of the observed peptides were summed and plotted as depicted in Figure 5A, B.. 156.

(16) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Hex fraction. Mixed fraction. OxHex fraction. Figure 4. Possible forms of peptide V that are selected for the hexose (Hex), mixed (Mix) or oxidized (OxHex) fractions. Blue circle = hexose, purple circle = oxidized hexose.. Figure 5. Oxidation of tryptic glycopeptides from Glc-HMW1ct after 8 h and 24 h oxidation reaction with 0.5 eq (A) and 1 eq (B) of Pd catalyst. The Y-axis represents the peptide peak areas of the glycopeptides detected in LC-MS/MS. Representative peptide spectra can be found in the Supporting information.. 157.

(17) CHAPTER 6 As shown in Figure 5A, after 8 h with 0.5 eq of Pd catalyst already significant oxidation of glycopeptides was observed. After 24 h of oxidation with 0.5 eq of Pd catalyst, the mixture contained oxidized glycopeptides as the major fraction, but the total level of peptides decreased. For the oxidation reaction with 1 eq Pd catalyst (Figure 5B), a fully oxidized fraction was already dominant after 8 h, and longer reaction times led to an even higher extent of oxidation. Therefore, 1 eq of Pd catalyst and 8-24 h of incubation were found to be the optimal oxidation conditions to afford high levels of oxidation without compromising the peptide levels. Differences in the protein sequence coverage can be found in the Supplementary Information (Table S7). A certain degree of Thr (and Ser) oxidation was detected alongside glucose oxidation, however, it was difficult to reliably quantify as it typically coincided with other modifications in the peptides.. Introduction of (affinity) tags via oxime formation The selective introduction of affinity or fluorescence labels to glycopeptides is both highly valuable and difficult to achieve.49–52 The tag allows to identify, pulldown, enrich and profile glycoproteins of interest that are typically present in low amounts in biological samples. For the purpose of expanding the oxidation methodology presented in this work towards biological applications, it was investigated whether the oxidized mixture of glycopeptides can be further modified with a tag of interest via oxime ligation (Figure 5). First, the process of oxime formation on pure glycopeptide 1 was investigated. After the oxidation reaction, a sample was diluted with a 100 mM NaOAc/AcOH buffer (pH = 5) and subjected to a treatment with APDC to remove Pd species and prevent deglycosylation. After the addition of hydroxylamine, the reaction mixture was kept at 40 °C overnight. Under these conditions, oxime 7 was formed in 52%, which could not be increased by a longer reaction time or more hydroxylamine. It should be noted that only a slight increase of 5-10% of deglycosylated peptide 3 was observed overnight. Encouraged by this result, 3-keto peptide derivative 2 was treated with a biotinylated alkoxyamine derivative. LC-MS analysis revealed the formation of oxime 8, but we were unable to accurately estimate the conversion due to the differences in ionization of the peptides.. 158.

(18) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES. Figure 5. Transforming the 3-keto Glc into an oxime.. Next, these reaction conditions were applied to the mixture of oxidized tryptic peptides. The results after incubation with hydroxylamine are presented in Figure 7. Compared to the oxidation reaction, two additional fractions were identified: peptides that contained both an oxime and hexose/oxidized hexose modifications (mixed oxime fraction) and peptides that contained only oxime modification (oxime fraction). To simplify data analysis, mixed and fully oxidized peptides were grouped together as “OxHex” fraction, while peptides bearing at least one oxime modification were designated as “Oxime” fraction (possible peptide forms are shown in Figure 6). As is shown in Figure 7A, oxime formation from the 0.5 eq reaction aliquot after 8 h (and 24 h) of oxidation resulted in only small amounts of oxime product. Importantly, the aliquot after 24 h reaction with 1 eq of catalyst contained a large portion of hydroxylamine oxime product (Figure 7B). When alkoxyamine-biotin was used, a small amount of oxime product was detected in all samples (Figure 7C, D) with 0.5 eq 24 h aliquot containing the most significant oxime fraction. Interestingly, whereas multiple tryptic glycopeptides were identified in the non-treated adhesin protein sample (Table 3, Figure S4), reaction aliquots from the oxidation and oxime formation typically contained three major glycopeptides (peptides III-V, Table S8). This “enrichment” may reflect the preference for length and amino acid composition, or a higher stability of certain glucopeptides under the reaction conditions for oxidation and oxime formation.. 159.

(19) CHAPTER 6 OxHex fraction. Oxime fraction. Figure 6. Possible peptide forms that are selected for the oxidized hexose (OxHex) or oxime (Oxime) fractions. Blue circle = hexose, purple circle = oxidized hexose, green circle = oxime.. 160.

(20) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES. Figure 7. Oxime formation with hydroxylamine after oxidation with 0.5 eq (A) and 1 eq (B) of Pd catalyst. Biotin oxime formation after oxidation with 0.5 eq (C) and 1 eq (D) of Pd catalyst. Representative peptide spectra can be found in the Supporting information.. Site-selective Pd-catalyzed oxidation, developed for unprotected carbohydrates, has successfully been applied to glycopeptides. This is a significant step forward, as the tendency of transition metals, including palladium, to chelate to peptides and proteins is an inherent bottleneck for such an approach. The conditions for the oxidation reaction were optimized for 3 mM substrate concentration, to make these suitable for glycopeptides and glycoproteins that are typically obtained in low amounts. Oxidation of single glycopeptides demonstrates that it may be possible to selectively perform the oxidation of a glucose moiety in the presence of a galactose moiety, since the latter is far less reactive and requires higher catalyst loadings. Similar to sodium periodate oxidation, the Pdcatalyzed oxidation has some limitations, as Thr residues were found to be oxidized, and Pd-mediated deglycosylation was observed. Fortunately, 161.

(21) CHAPTER 6 deglycosylation can successfully be suppressed by tuning the reaction conditions and treatment with a Pd scavenger. In addition, the substrate scope of the Pd-catalyzed oxidation has been expanded to tryptic glucopeptides from the immunologically relevant H. influenzae adhesin protein. Sufficient levels of glucose oxidation were obtained to further exploit the introduced keto group in a bioconjugation reaction with hydroxylamine to form the desired oxime. Based on this, the widely used affinity tag biotin was incorporated in low to moderate levels. Taken together, Pdcatalyzed oxidation allows the selective modification of glucoproteins and this observation can now be further developed for proteomics identification and pulldown.. Dr. Niels R. M. Reintjens performed the NMR experiments and the oxidation of individual glycopeptides. Johan Hekelaar and Dr. Franz Ho are acknowledged for their help with the proteomics analysis. Gemma Sturt and Dr. Chiara Testa are acknowledged for their technical assistance in the glycopeptide synthesis.. 162.

(22) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES.

(23) Table S1. Regioselective oxidation of methyl glucoside[a]. #. Substrate. Solvent. 1. S1. DMSO-d6. Temperature. Concentration. (°C). (mM). 22. 27. Conversion (%) 30 min: 55% 70 min: 78% 12 h: 88% 5 h: 97% 11 h: 100%. 2. S1. DMSO-d6. 40. 27. 5 min: 17% (RT) 50 min: 95% 1 h 25 min: 100%. 3. S1. DMSO-d6. 40. 3. 15 min: 16% 1 h: 60% 7 h: 88% 23 h: 100%. 4. S2. DMSO-d6. 40. 3. 10 min: 10% 1.5 h: 65% 8 h: 100%. 5[b]. S1. DMSOd6/D2O 1/9 v/v. 40. 27. 5 min: 0% 50 min: 8% 1 h 50 min: 13% 4 h: 32% 23 h: 66%. Reaction conditions: 0.05 eq [(neocuproine)PdOAc]2OTf2, 3.5 eq benzoquinone. [b] 0.1 eq [(neocuproine)PdOAc]2OTf2 was used instead of 0.05 eq, since the latter only gave 66% conversion after 23 h. [a]. 163.

(24) CHAPTER 6 Table S2. Regioselective oxidation of methyl galactoside[a]. #. Substrate. Concentration (mM). Conversion S5/S6, (% yield S7/S8). 1. S5. 3. 2 h: 98 (61). 2. S5. 245. 163 h: 100 (43) 2.5 h: 84 (41) 21 h: 100 (0) 3. S6. 3. 2 h: 80 (52). 4. S6. 245. 2.5 h: 100 (38). 163 h: 97 (53) 21 h: 100 (10) [a] Reaction conditions:. 0.05 eq [(neocuproine)PdOAc]2OTf2, 3.5 eq benzoquinone, DMSOd6, 40 °C. qNMR was performed using dimethyl sulfone as internal standard. Table S3. Deglycosylation[a]. #. Substrate. Added. Temperature. Concentration (mM). Time. Conversion to 2/3 (%). 40. 3. ON. 3/8. (°C) 1[a]. 1. Pd(OAc)2 (0.5 eq). 2[b]. 2. DMSO. 40. 0.25. 7h. 71/27. 3[b]. 2. Pd-cat. 40. 0.25. 7h. 42/56. (1.5 eq) [a] DMSO. was used as solvent which was diluted 10x with H2O/tBuOH/CH3CN (1/1/1 v/v/v) prior to RP-LCMS analysis. [b]After 75 min with 0.5 eq Pd-cat, 3 eq benzoquinone, 3 mM of peptide in DMSO-d6 at 40 °C the 2/3 ratio was: 84/6. DMSO or a solution of Pdcat in DMSO was added to obtain a final concentration of 0.25 mM and the mixture was kept at 40 °C for 7 h.. 164.

(25) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Table S4. C-3 Oxidation of galactose on a glycopeptide[a]. #. Pd-cat. (eq). Benzoquinone (eq). Conversion S10/3 (%). 1. 5. 3. ON: 2/6. 2. 10. 0. 75 min: 28/8 ON: 47/37. Reactions were performed at 3 mM in DMSO-d6 at 40 °C. RP-LCMS samples were prepared by the addition of APDC (100 eq) and dilution with CH3CN/tBuOH/H2O 1/1/1 v/v/v to obtain a 0.3 mM concentration. After mixing, the Eppendorf tube was centrifuged for 5 min at 12500 rpm, after which the supernatant was transferred to a RPLCMS vial. [a]. Table S5. Oxidation of Threonine. #. Substrate. Conc., [Temp.] (mM, °C). 1[a]. 2[b]. 3[a]. S11. S11. S1/S11 1/1. 27 [22]. 27 [40]. 3 [40]. Yield (%) S12. S13. S3. 1.5 h: 5. 1.5 h: 4. -. 7.5 h: 17. 7.5 h: 22. -. 26 h: 22. 26 h: 35. -. 2.5 h: 19. 2.5 h: 48. -. 5.5 h: 12. 5.5 h: 78. -. 27 h: 0. 27 h: 100. -. 2.5 h: 0. 2.5 h: 0. 2.5 h: 58. 6 h: 21. 6 h: 6. 6 h: 81. 23 h: 31. 23 h: 9. 23 h: 100. Reaction conditions: 0.05 eq [(neocuproine)PdOAc]2OTf2, 3.5 eq of benzoquinone, DMSO-d6. [b] 0.5 eq [(neocuproine)PdOAc]2OTf2 was used instead of 0.05 eq.. [a]. 165.

(26) 166 75 min: 3/29 4 h: 9/41 ON: n.d. 2ON: n.d.. 45 min: 45/5. 75 min: 56/4. 4 h: 81/3. ON: 83/17. 2ON: 74/24. (1/1). 2[e]. 45 min: 0/25. 15 min: 17/6. 1/4. 45 min: 0/2 75 min: 0/3 4 h: 0/5 ON: 0/12 2ON: 0/16. 45 min: 4/5. 75 min: 4/5. 4 h: 8/4. ON: 23/5. 2ON: 38/5. (1/5). 15 min: 0/0. 15min: 2/6. 1/4. 15 min: 0/10. (%) 2Ox/1Ox. Conversion. 1. 2/3 or S10/3. (%). Conversion. Substrates (ratio). #. 2ON: 19.6 [43]. ON: 12.6 [28]. 4 h: 5.6 [9]. 75 min: 4.1 [9]. 45 min: 4.1 [9]. 15 min: 3.3 [7]. 2ON: 35.4 [16]. ON: 26.5 [12]. 4 h: 10.8 [5]. 75 min: 6.6 [3]. 45 min: 4.4 [2]. 15 min: 0 [0]. 2ON[d]: n.d.. ON[d]: n.d.. ON: 44.8 [100] 2ON: 44.1 [98]. 4 h: 26.9 [51]. 4 h: 37.9 [84]. 45 min: 11.2 [25]. 45 min: 22.4 [50] 75 min: 15.9 [32]. 15 min: 4.5 [10]. 15 min: 10.4 [23]. 75 min: 27.1 [60]. Oxidized 4[c], [conversion] (nmol, %). Oxidized 1[b], [conversion] (nmol, %). 2ON: 1.2. ON: 0.9. 4 h: 0.4. 75 min: 0.2. 45 min: 0.2. 15 min: 0.1. 2ON: n.d.. ON: n.d.. 4 h: 1.6. 75 min: 0.9. 45 min: 0.7. 15 min: 0.3. (nmol ox/nmol Pd). TON. CHAPTER 6. Table S6. Competition experiments between glucose/galactose and threonine[a].

(27) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Table S6 (continued). Competition experiments between glucose/galactose and threonine[a] #. (%). Oxidized 1[b], [conversion]. Oxidized 4[c], [conversion]. 2Ox/1Ox. (nmol, %). (nmol, %). ON: 2/7. ON: 0/0/0. -. -. -. ON: 2/7. ON: 0/0/0. -. -. -. Substr.. Conv.. Conv.. (ratio). (%) 2/3 or S10/3. 3. S9/4 (1/1). 4. S9/4 (1/5). TON (nmol ox/nmol Pd). [a] All. reactions were performed with 0.5 eq Pd-cat, 3 eq benzoquinone, 3 mM in DMSOd6 of 1 or 4. at 40 °C. 45 nmol of 1 or 4 was used in each entry. [b] Oxidized 1 = 2 + 3. [c] nmol oxidized 4 = 2× nmol 2Ox + nmol 1Ox. [d] After ON and 2xON, -T and -TIT peptides were observed and the conversion was not determined. [e] No -T and -TIT fragments were observed after ON and 2ON. Table S7. HMW1ct sequence coverage in various samples. Sample. Sequence coverage. Non-treated (8 h). 91%. Non-treated (24 h). 91%. 0.5 eq (8 h). 89%. 0.5 eq (24 h). 74%. 1 eq (8 h). 91%. 1 eq (24 h). 73%. 0.5 eq 8 h hydroxylamine oxime. 73%. 0.5 eq 24 h hydroxylamine oxime. 74%. 1 eq 8 h hydroxylamine oxime. 72%. 1 eq 24 h hydroxylamine oxime. 73%. 0.5 eq 8 h biotin oxime. 91%. 0.5 eq 24 h biotin oxime. 73%. 1 eq 8 h hydroxylamine oxime. 72%. 1 eq 24 h hydroxylamine oxime. 74%. 167.

(28) CHAPTER 6 Table S8. Peptides identified in the proteomics search with hexose, oxidized hexose and oxime modifications. Asparagines with modification are shown in red. Number. Sequence. III. GQVNLSAQDGSVAGSINAANVTLNTTGTLTTVK. IV. GSNINATSGTLVINAK. V. DAELNGAALGNHTVVNATNANGSGSVIATTSSR. Figure S1. Calculation of the yield with the relative ionization factor.. Relative intensity 2. %I (Re). 0,93. 1. 2. 1. %I (Mix). 0,41. 88. 7. RIF (P to S). 0,79. % yield 2. 94. 75 min. 100. 8. After addition of 1. 64. 91. 168. Yield (%).

(29) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Figure S2. Proposed mechanisms of deglycosylation.. Figure S3. Proposed mechanisms of threonine overoxidation and peptide cleavage.. Figure S4. Glycopeptides generated from the tryptic digest of Glc-HMW1ct. Blue circle – hexose.. 169.

(30) CHAPTER 6 Figure S5. General peptide levels in the oxidation reactions with 5 eq and 10 eq Pd catalyst (A), oxidation reactions with 0.5 eq and 1 eq Pd catalyst (B). The Y-axis represents the peptide peak area in the chromatogram (based on the parent ion). NT = non-treated tryptic glucopeptides mixture.. %

(31). % . . %

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(37) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES LC-MS spectra of glycopeptides and oxidation products * *Due to the strong ionization of the compunds, the MS spectra from the LC-MS show fragmentation. Glycopeptide 2 (oxidized). [M+H]+. y5 c4. y6. b6 c5. z7. Deglucosylated peptide 3. [M+H]+. b6. y5 b4. y6. b5. c6. b7. 171.

(38) CHAPTER 6 Peptide 4: 1x and 2x oxidation after 75 min. y6 1ox y6 2ox. + [M+H]+ [M+H] 1ox 2ox. y7 1ox. z6 1ox. y8 1ox c8 1ox. Peptide 4: Oxidation after 75min showing -T*. 658.30. 100. 1089.11. 90. Relative Abundance. 80 70 60. 641.24 427.19. 50 367.19. 432.02. 40. 858.19. 522.20 303.02. 326.18. 10. 770.27. 409.20. 670.29. 623.21. 477.19. 368.23. [M+H]+. 787.29. 556.27. 30 20. 669.22. 540.26. 671.22. 607.24. 788.31 752.27 729.03. 840.20 859.21 900.33 789.35. 988.08 953.13. 0 300. 350. 400. 450. 500. 550. 600. 650. 700. 750. 800. 850. m/z. *The -T fragment has the same retention time as peptide 4.. 172. 900. 950. 1111.52. 989.15 1072.17 1016.30 1000. 1050. 1143.15 1100. 1150.

(39) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Peptide 4: Oxidation after 75min showing -TIT. [M+H]+. y3 y4. b6. y5. Glycopeptide 5: oxidation after ON, showing b6 ion of keto-Glc*. 45. 303.03. 432.10. 587.29. 818.44. b3. 40. Relative Abundance. 35. 717.38. 30. 698.35. 25. 433.15 503.27. 20. 509.80. 10 5 284.59. 304.09 415.32 372.23 434.17 368.33. 300. 400. y6 2ox 816.42 801.41. 652.42. 569.33. 718.40. 588.34. 15. 0. y6 1ox. 700.38. 589.37 641.41. [M+H]+ [M+H]+ 1ox. 819.44 829.37 830.37. 757.34. 831.42 846.40. 917.12 918.19. 847.44 500. 600. 700. 800. 2ox. b6 1ox. 900. b7 1ox 1018.35 1000. c8 1ox 1090.23 1100. 1148.25. 1249.28 1263.25 1233.37 1200. m/z. *Structure only shown for the keto-Glc peptide. 173.

(40) CHAPTER 6 Glycopeptide 5: Oxidation after ON showing -T, showing b5 ion of keto-Glc. *The -T fragment has the same retentiontime as glycopeptide 5 and 1Ox and 2Ox. 432.11. 60. Relative Abundance. 587.31. 717.38 700.39. 40. 818.44 816.41. 698.34. 819.43. 30 433.17 486.33. 20 10. b5 1ox. 303.04. 50. 569.33 588.34 509.75. 304.10. 415.35. 718.41 682.33 719.44. 589.36. 801.42. 830.37. [M+H]+. 832.30. 757.32. 846.40. 472.01. 847.46. 947.52. 1018.32. 0 300. 400. 500. 600. 700. 800 m/z. 900. 1000. 1100. Glycopeptide 5: Oxidation after ON showing -TIT. [M+H]+ 1ox y3 1ox [M+H]+ 0ox. y3 0ox y4 1ox. 174. 1250.26. 1148.24 1090.21. 1233.36 1200. 1286.11 1300.

(41) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Glycopeptide 6: Oxidation after ON showing multiple oxidations. [M+3H]3+ Multiple oxidations. Glycopeptide S9. 941.04 80. [M+H]+. 70. Relative Abundance. 60 50. 942.02. y5. 40. 607.19. 30. c5. 20 725.96. 10 0. 323.07 374.20. 269.07 250. 300. 350. 392.15. 445.25. 400. 450. 502.19 500. 608.23 563.88 605.16 609.31 550. 600. 650 m/z. 674.05 700. 782.99 762.00 784.00 750. 800. b6 835.97 837.02 850. z7. b7. 943.04. 940.48 883.12 923.07 900. 964.40 950. 1028.90. 1000. 175.

(42) CHAPTER 6 Glycopeptide S10 (oxidized). [M+H]+. y5 y6. b6 z7. LC-MS/MS spectra of a selection of tryptic digest peptides .

(43) . . • •. Peptide detected: GSNIN(+160.03)ATSGTLVINAK Calculated mass: 1718.8615 Da; Detected mass: 860.4292 Da, 2+ charge. 372.09. 176. b5 646.01. 1074.38. 1348.35.

(44) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES . . .

(45)

(46) . . • •. . Peptide detected: GQVNLSAQDGSVAGSINAAN(+162.05)VTLN(+160.03)TTGTLT(-2.02)TVK Calculated mass: 3521.7148 Da; Detected mass: 1174.8911 Da, 3+ charge. y10 1193.25 b22 1109.32 y9 919.27. y11 1306.70 b25 1302.12. Oxime formation (hydroxylamine).

(47) . . • •. . . Peptide detected: GSNIN(+175.05)AT(-2.02)SGTLVINAK Calculated mass: 1731.8658 Da; Detected mass: 867.9319, 2+ charge. y12 [2+] 681.01. b7 831.11. y11 1072. 19. 177.

(48) CHAPTER 6 Oxime formation (biotin) . . . . .

(49)

(50)

(51) . • •. . Peptide detected: DAELN(+.98)GAALGN(+162.05)HTVVNATN(+160.03)AN(+473.16)GSGSVIA TTSSR Calculated mass: 3964.7664 Da; Detected mass: 1322.5986 Da, 3+ charge. y12 1122.43 b20 1128.55. b21 1422.88. y13 1709.63. .

(52) . . • •. . . Peptide detected: GSN(+.98)IN(+473.16)ATSGTLVINAK Calculated mass: 2032.9755 Da; Detected mass: 1017.4941 Da, 2+ charge. b5 960.32. 178. b6 1031.00 b7 1132.22.

(53) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES. Solvents and Reagents All solvents used for reactions were of commercial grade, and used without further purification. [(Neocuproine)PdOAc]2OTf2 was prepared according to the literature procedure.53 Fmoc-L-Asn[β-D-Glc(OAc)4]-OH and Fmoc-L-Asn[β-DGal(OAc)4]-OH were synthesized as previously described.54 General procedure for the synthesis of model glycopeptides The glycopeptides were synthesized by microwave-assisted solid-phase synthesis (MW-SPPS) following the Fmoc/tBu strategy, using the Liberty BlueTM automated microwave peptide synthesizer (CEM Corporation, Matthews, NC, USA) following the protocol previously described.55 The glycopeptides 1, S9, and 6 were obtained starting from Fmoc-Ser(tBu)-Wang resin (0.6 mmol/g, 0.167 g, 0.1 mmol), while the glycopeptides 4 and 5 were obtained starting from Rink Amide TentaGel® S RAM resin (0.23 mmol/g, 0.423 g, 0.1 mmol). Swelling step was performed in DMF for 30 min. The MW-SPPS protocol was performed repeating the following cycles for each amino acid: Fmoc-deprotection with a solution of piperidine in DMF (20% v/v, 2 M); washings with DMF (3 times); coupling step with Fmoc-amino acids (5 eq, 0.2 M), DIC (5 eq, 0.5 M) and Oxyma Pure (5 eq, 1 M); washings with DMF (3 times). MW coupling cycles used for elongation of glycopeptides are listed in Table S9. Couplings of Fmoc-L-Asn[β-D-Glc(OAc)4]-OH and Fmoc-L-Asn[β-DGal(OAc)4]-OH were performed using the protected N-glycosyl amino acid (2.5 eq), HATU as activator (2.5 eq), and DIPEA (3.5 eq), at 75 °C, 30W, in 5 min. After the last Fmoc deprotection, N-terminal function of the glycopeptides 1, S9, 4, and 5 were acetylated using a solution of acetic anhydride (20 eq) and DIPEA (20 eq) in DMF for 2 h. Cleavage from the resin and side-chain deprotection of glycopeptides were performed using a mixture of TFA/TIS/H2O/ (95/2.5/2.5 v/v/v). After 2.5 h the resin was filtered off and the solution was concentrated flushing with N2. The peptides were precipitated from cold Et2O, centrifuged and lyophilized. For the deprotection of the hydroxyl functions, the glycopeptide was dissolved in methanol and a methanolic NaOMe solution (0.1 M) was added until pH 12 was reached. After 3 h, conc. HCl until pH 7 was added to the reaction for quenching, the solvent was evaporated under vacuum and the residue lyophilized.. 179.

(54) CHAPTER 6 Table S9. Microwave Cycles Used for Elongation of glycopeptides Step Deprotection Standard Coupling Arg-Coupling His-Coupling. Temperature (°C) 70 90 70 90 25 75 25 50. Power (W) 125 30 150 30 0 30 0 35. Hold Time (sec) 25 65 30 120 1500 120 120 240. Carbohydrate hydroxyl functions deprotection: A methanolic solution of NaOMe (0.1 M, pH 12) was added to a solution of each glycopeptide to deprotect the hydroxyl functions of the sugar moiety. After 3 h, conc. HCl was added to the reaction until pH 7 for quenching, the solvent was evaporated under vacuum and the residue lyophilized. Purification of all synthetic glycopeptides was performed by semipreparative RP-HPLC on a Waters instrument mod 600 (Separation Module 2695, diode array 2996 detector) using a Phenomenex (Torrance, CA, USA) Jupiter column C18 (10 μm, 250×10 mm), at 4 mL/min. Solvent systems A (0.1% TFA in H2O) and B (0.1% TFA in CH3CN). Analytical characterization of glycopeptides was performed by analytical HPLC using a Waters ACQUITY HPLC coupled to a single quadrupole ESI-MS (Waters® ZQ Detector, Waters Milford, MA, USA) supplied with a BEH C18 (1.7 μm 2.1× 50 mm) column at 35 °C, at 0.6 mL/min with solvent system A (0.1% TFA in H2O) and B (0.1% TFA in CH3CN). Gradient elution was performed with a flow of 0.6 mL/min and started at 10% B, with a linear increase to 60% B in 5 min (Table S10).. 180.

(55) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Table S10. Analytical characterization of model glycopeptides. Peptide. Sequence. (tR, min)a. ESI-MS (m/z) (calcd) found b. HPLC purity. Quantity (mg). Yield, %. HPLC. 1. Ac-YEPN(Glc)GAS. 1.5. (941.92) 941.55. 95%. 20. 5. S9. Ac-YEPN(Gal)GAS. 2.17. (941.55) 941.42. >95%. 14. 3. 4. Ac-FIEPNNTITNH2. 3.93. (1090.22) 1089.94. >95%. 40. 0. 5. AcFIEPN(Glc)NNTITNH2. 3.82. (1252.22) 1252.20. 95%. 5. 2. 6. YANVTLNTTGTL TTVKGSNIN(Glc) ATS. 4.2. (1302.00) 1302.35. 95%. 7. 3. Solvent system A: 0.1% TFA in H2O, B: 0.1% TFA in CH3CN. Analytical HPLC gradients at 1 mL min-1: a10-60% B in 5 min. ESI-MS: detected as b[M+H]+.. General procedure for the NMR experiments The following stock solutions were prepared in DMSO-d6: 1) substrate (30.9 mM); 2) benzoquinone and [(neocuproine)PdOAc]2OTf2 (796 mM and 11.4 mM, respectively). To a solution of substrate in DMSO-d6 was added a solution of benzoquinone and [(neocuproine)PdOAc]2OTf2 to obtain, for example, the ratio of 1/3.5/0.05. The solution was further diluted with DMSO-d6 to obtain a 3 mM reaction mixture and transferred to an NMR tube. The NMR tube was heated to 40 °C, while NMR measurements were taken at several timepoints. General procedure for the qNMR experiments The following stock solutions were prepared in DMSO-d6: 1) substrate (67.1 mM); 2) dimethylsulfone (107.3 mM); 3) benzoquinone and [(neocuproine)PdOAc]2OTf2 (respectively 796 mM and 11.4 mM). A mixture of substrate and dimethylsulfone (1/1 ratio) in DMSO-d6 was prepared and transferred to an NMR tube. At T0 an NMR measurement was taken to determine the ratio of starting material:dimethylsulfone. A solution of benzoquinone and [(neocuproine)PdOAc]2OTf2 were added to the NMR tube to obtain for example the ratio of 1/1/3.5/0.05 and a 3 mM concentration. The NMR tube was mixed well and heated to 40 °C, while NMR was taken at several timepoints.. 181.

(56) CHAPTER 6 General procedure for the oxidation of single (glyco)peptides The following stock solutions were prepared in DMSO-d6: 1) substrate (6 mM); 2) benzoquinone (231 mM); 3) [(neocuproine)PdOAc]2OTf2 (19.1 mM). A mixture of substrate/benzoquinone/Pd-cat in DMSO-d6 was prepared in an Eppendorf tube to obtain for example the ratio of 1/3/0.5 and a 3 mM concentration. The reaction mixture was mixed well before placed in a 40 °C water bath. RP-LCMS samples were taken at several timepoints. General procedure for the oxime formation of single glycopeptides After oxidation, the reaction mixture was diluted 9x with the buffer solution (NaOAc/AcOH pH 5) of APDC (Pd-scavenger). After mixing, the Eppendorf tube was centrifuged for 5 min at 12500 rpm, after which the supernatant was transferred to a new Eppendorf tube. Solutions of hydroxylamine in buffer (500 mM, 25 eq) and NaOAc in buffer (75 mM, 25 eq) were added to obtain a final concentration of 0.3 mM. The reaction mixture was mixed well before placing in a 40 °C water bath and stirring overnight. For the biotin oxime formation, the mixture with the biotin reagent was added as a DMSO solution (40 mM, 25 eq) with a final concentration of 0.21 mM. RP-LCMS The runs were performed on a UPLC-MS instrument (Thermo Scientific LCQ Fleet, equipped with HSS T3 column (Acquity UPLC, 150x2.1 mm, 1.8 µm) and eluents A (H2O, 0.1% FA) and B (ACN, 0.1% FA). Injection volume was 1 µL and the flow was 0.3 mL/min. Method for the experiments with glycopeptides 1 and S9: The gradient started at 0% B and was increased to 30% in 10 min, followed by immediate increase to 100% where it stayed for 2 min with subsequent decrease back to 0% in 4min. Method for the experiments with (glyco)peptides 4, 5, and 6: The gradient started at 15% B and was increased to 60% in 10 min, followed by immediate increase to 100% where it stayed for 2 min with subsequent decrease back to 0% in 4 min. Samples preparation: 2.5 μL of the 3 mM reaction mixture was diluted with 22.5 μL of CH3CN/tBuOH/H2O (1/1/1 v/v/v). After mixing, the Eppendorf tube was centrifuged for 5 min at 12500 rpm, after which the supernatant was transferred to an RP-LCMS vial.. 182.

(57) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES Scavenger treatment 2.5 μL of the 3 mM reaction mixture was diluted with 20 μL of APDC in CH3CN/tBuOH/H2O (30.4 mM, 1/1/1 v/v/v) and 2.5 μL CH3CN/tBuOH/H2O (1/1/1 v/v/v). After mixing, the Eppendorf tube was centrifuged for 5 min at 12500 rpm, after which the supernatant was transferred to a RP-LCMS vial. Optional: a spatula tip of charcoal can be added to the diluted reaction mixture to remove neocuproine ligand. Tryptic digest of Glc-HMW1ct To prepare tryptic glycopeptides for oxidation reaction, typically 600 µg of GlcHMW1ct was split into six samples of 100 µg. To each sample 100 mM ammonium bicarbonate (ABC) was added to reach 32 µL. 8 µL 8 M urea was added to obtain a concentration of 1.6 M urea. Furthermore, 1 µL of 0.2 M TCEP was added. The sample was mixed and incubated at 37 °C for 1 h. After the incubation, the sample was cooled to room temperature. Alkylation of cysteines was performed by adding 1 µL of freshly prepared 0.4 M iodoacetamide and incubated at 25 °C for 30 min in the dark. The pH was checked to be around 8-9 and if required adjusted using 1 M ABC. Trypsin (Promega, V5113) was added at a ratio of 1:50 w/w trypsin/protein and incubated overnight at 37 °C. Sample clean-up by solid phase extraction was performed with Pierce® C18 tips (Thermo, 87784) according to the supplier’s manual. The eluate fraction was dried under vacuum. Oxidation of tryptic glycopeptides Vacuum-dried tryptic glycopeptides were reconstituted in DMSO-d6 and combined into one stock. The concentrations were calculated based on starting concentration of the Glc-HMW1ct protein (600 μg) and glycosylation levels (avg 8Glc). Typically, stocks of 5.6 mM were prepared to be used in oxidation reactions. Solutions of benzoquinone (231 mM) and [(neocuproine)PdOAc]2OTf2 (19.1 mM) were prepared in DMSO-d6. Reaction mixtures were prepared in DMSO-d6 and substrate, BQ and Pd-cat were added from stock solutions to desired concentrations. Typically, final concentration of 2.8 mM of substrate was used in the oxidation reaction. The reaction mixture was mixed well and incubated at 40 °C in the water bath. Reaction aliquots were taken after 8 h and 24 h of incubation. Quenching: 2.5 μL of the oxidation reaction mixture was diluted with 22.5 μL of APDC in water (or acetate buffer, pH 5 for oxime formation). After mixing, the quenched aliquot was centrifuged for 15 min at 13000 rpm, after which the supernatant was transferred to a new tube and kept at -20 °C until SPE purification. 183.

(58) CHAPTER 6 Oxime formation from oxidized tryptic glycopeptides To prepare oxime from oxidized glycopeptides the quenched aliquot in acetate buffer was used in the subsequent reaction. Typically 25 eq of hydroxylamine was added from a 500 mM stock (40 mM stock for alkoxyamine biotin) to the quenched reaction aliquot. The reaction mixture was mixed well and incubated at 40 °C in the water bath overnight. Next day the SPE purification was performed before LC-MS/MS, as described above. LC-MS/MS SPE-purified samples were reconstituted with 50 µL 2% acetonitrile, 0.1% formic acid. Peptide separation was performed with 2 µL peptide sample using a nanoflow chromatography system (EASY nLC II; Thermo) equipped with a reversed phase HPLC column (75 µm, 15 cm) packed in-house with C18 resin (ReproSilPur C18–AQ, 3 µm resin; Dr. Maisch) using a linear gradient from 95% solvent A (0.1% FA, 2% acetonitrile) and 5% solvent B (99.9% acetonitrile, 0.1% FA) to 28% solvent B over 45 min at a flow rate of 200 nL/min. The peptide and peptide fragment masses were determined by an electrospray ionization mass spectrometer (LTQ-Orbitrap XL; Thermo) in data dependent data acquisition mode. Data processing Thermo raw files were imported into the Peaks Studio software (Bioinformatics Solutions) analyzed against forward and reverse peptide sequences of the expression host E. coli K12 and the over-expressed construct HMW1ct. The search criteria were set as follows: specific tryptic specificity was required (cleavage after lysine or arginine residues but not when followed by a proline); three missed cleavages were allowed; carbamidomethylation (C) was set as fixed modification; oxidation (M) and deamidation (NQ) as variable modification. Variable modification was set to hexose (162.05 Da), oxidized hexose (160.03 Da), oxidized Ser/Thr (-2.02) and if oxime samples were analyzed additionally 175.05 Da (hydroxylamine) or 473.16 Da (alkoxyamine biotin). The mass tolerance was set to 20 ppm for precursor ions and 0.5 Da for the fragment ions. Global data analysis Raw data files were processed with PEAKS X Plus software and a search for the modifications (listed above) on asparagines and serine/threonine was applied. Manual analysis: Peptides were sorted according to the quality of the spectra and FDR values (always set to 0%) and manually selected based on the presence of signature b and y ions that unambiguously confirmed the presence of 184.

(59) SITE-SELECTIVE PD-CATALYZED OXIDATION OF GLUCOPEPTIDES modification (examples of the supporting peptide spectra can be found in the SI). Peptides that did not contain sufficient number of signature ions to confirm the presence of the modification were discarded. The peak areas of the peptides bearing the same modification were summed for specific time point of the reaction. Peptide lists used for analysis can be found in the supplemented Excel sheet. Peak area values were plotted against time to show the changes in the product profile at different time points and for different equivalents of Pd catalyst. Plotted graphs were prepared with GraphPad Prism 9 software.. 185.

(60) CHAPTER 6.

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