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Glycoconjugate Oxime Formation Catalyzed at Neutral pH: Mechanistic Insights and Applications of 1,4Diaminobenzene as Superior Catalyst for Complex Carbohydrates Mads Østergaard, Niels Johan Christensen, Christian T. Hjuler, Knud J. Jensen, and Mikkel B. Thygesen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00019 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Glycoconjugate Oxime Formation Catalyzed at Neutral pH: Mechanistic Insights and Applications of 1,4-Diaminobenzene as Superior Catalyst for Complex Carbohydrates Mads Østergaard, Niels Johan Christensen, Christian T. Hjuler, Knud J. Jensen* and Mikkel B. Thygesen* Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Correspondence to: [email protected] and [email protected]

Abstract The reaction of unprotected carbohydrates with aminooxy reagents to provide oximes is a key method for the construction of glycoconjugates. Aniline and derivatives serve as organocatalysts for the formation of oximes from simple aldehydes, and we have previously reported that aniline also catalyzes the formation of oximes from the more complex aldehydes, carbohydrates. Here we present a comprehensive study of the effect of aniline analogs on formation of carbohydrate oximes and related glycoconjugates, depending on organocatalyst structure, pH, nucleophile, and carbohydrate, covering more than 150 different reaction conditions. The observed superiority of

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the 1,4-diaminobenzene (PDA) catalyst at neutral pH is rationalized by NMR analyses and DFT studies of reaction intermediates. Carbohydrate oxime formation at pH 7 is demonstrated by the formation of a bioactive glycoconjugate from a labile, decorated octasaccharide originating from exopolysaccharides of the soil bacterium Mesorhizobium loti. This study of glycoconjugate formation includes the first direct comparison of aniline-catalyzed reaction rates and equilibrium constants for different classes of nucleophiles, including primary oxyamines, secondary N-alkyl oxyamines, as well as aryl and arylsulfonyl hydrazides. We identified 1,4-diaminobenzene as a superior catalyst for the construction of oxime-linked glycoconjugates under mild conditions.

Introduction The classical condensation reactions of oxyamines and hydrazides with aldehydes or ketones have become powerful methods for modifying or linking biomacromolecules.1-2 Among the advantages of this approach is the fact that oxime and hydrazone formation occur in aqueous medium with a high degree of chemoselectivity with unprotected biomolecules, for example peptides, proteins, carbohydrates or oligonucleotides. It is well established that anilines act as nucleophilic catalysts for these reactions, enhancing reaction rates by several orders of magnitude.3 The key step in the catalytic pathway is the formation of protonated iminium species.4 While imines are generally considered less reactive than their parent carbonyl compounds towards nucleophilic addition, protonated imines are several orders of magnitude more reactive.4 The use of aniline catalysts has broadened the scope of these reactions towards milder conditions, more suitable for conjugation of biomolecules.5-6


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In 2010, we reported the extension of nucleophilic catalysis to a different class of aldehydes, reducing carbohydrates.7 Designed glycoconjugates are important molecules, for example as drugs, or in biosensors, as well as for the study of fundamental aspects of glycobiology.8-11 Novel methods for glycoconjugate synthesis from unprotected carbohydrates is a rapidly developing field.12 Reducing carbohydrates act as electrophiles in oxime formation, and the masking of the aldehydo/keto species as hemi-acetal/ketal moieties causes a lowering of reaction rates (Fig. 1).7 Catalysis of carbohydrate oxime formation is complicated by the fact that protonated iminium intermediates may be trapped reversibly7 as cyclic N-aryl-glycosylamines by intramolecular addition-elimination reactions (Fig. 1). Catalytic rate enhancements (kcat/kuncat) are in the order of 20-fold for glycosyl (aldose) oximes at high catalyst concentration (100 mM) and pH 4.5.7 Such rate enhancements are particularly important for preparation of glycoconjugates from carbohydrates of low reactivity, for example oligosaccharides having 2-acetamido-2deoxyglucose (GlcNAc) at the reducing end. Catalyzed oxime formation has enabled the synthesis of a range of complex oligo- and polysaccharide conjugate oximes at pH 4-5,7,

13-19

exemplified by the labeling of complex influenza antigen tri-, tetra- and pentasaccharides.20


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Figure 1. Aniline-catalyzed glycoconjugate formation from α-nucleophiles (R1Z-NHR2) proceeds through a protonated iminium intermediate. Low degree of reversibility in additionelimination steps is indicated by single arrows. Z: Heteroatom, O (hydroxylamines) or NH (hydrazines).

Recent efforts towards optimizing nucleophilic catalysts with respect to efficiency at neutral pH, have built on the early discovery of 4-methoxyaniline (para-anisidine, PAN)3 as a useful catalyst for peptide5 and carbohydrate oxime7 formation at pH 7. This led to the discovery of 2amino-5-methoxybenzoic acid (5-methoxyanthranilic acid, PAN-C) and 3,5-diaminobenzoic acid (MDA-C) in model hydrazone reactions,21 as well as 1,4-diaminobenzene22 (paraphenylenediamine, PDA) for protein modification by oxime formation, each with 1–2 orders of magnitude rate enhancements over aniline (Chart 1). Recently, Langenhan et al.23 compared a panel of anilines for glycoconjugate formation with N-benzyl-N-methoxyamine and found PANC as the preferred catalyst at pH 4.5. However, this catalyst was found to be ineffective at neutral pH.


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Chart 1. Aniline catalysts, including acronyms, used in this study and their associated pKa values.

In the present study, we have examined commercially available aniline derivatives from the above-mentioned new generation24-25 of nucleophilic catalysts (Chart 1) with carbohydrates as electrophiles in order to optimize conditions for glycoconjugate formation towards neutral pH. This is important because certain glycosidic linkages are prone to hydrolyze even under mildly acidic conditions, for example uronides (e.g., hyaluronic acids), ulosonic acids (e.g., sialic acids in mucins, or KDOs), and more.12 Furthermore, complex carbohydrate ligands often carry ‘decorations’ in the form of various substituents, for example sulfates, carbamates, acetates, or pyruvate ketals, which are highly susceptible to cleavage or rearrangement reactions under acidic conditions. Such substituents are often crucial for the biological activity of carbohydrate ligands in receptor binding events.18 Neutral conditions are often also preferable during protein glycomodification and in dynamic covalent chemistry based on oxime or hydrazone chemistry. In the present study, we find that PDA is a superior catalyst for aldose oxime formation at pH 7, and report NMR studies and DFT calculations to rationalize the extraordinary catalytic effect of PDA.


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Results and Discussion Aniline-based catalysis of reactions between aldehydes or ketones and α-nucleophiles rely on the formation of the intermediate imine with aniline being kinetically rapid, but thermodynamically unfavourable relative to the desired product.4 In general, intermediate imine species do not accumulate, or accumulate to a lesser extent,21 but once protonated they react rapidly with the αnucleophile to yield the products. When carbohydrates are used as electrophiles, reactivity towards α-nucleophiles differ markedly between monosaccharides as a result of different rates of pyranose ring-opening. The rate of formation for the reactive aldehyde species is generally governed by the thermodynamic stabilities of the pyranoses. Furthermore, various substitutions to the pyranose ring influence the rate of oxime formation. In particular, the 2-acetamido-2deoxy and 6-carboxy substitutions are known to decrease or increase reaction rates, respectively,7 and could potentially influence catalysis. These considerations led us to first study the catalytic effect for the three monosaccharides, D-glucose (Glc), 2-acetamido-2-deoxy-Dglucose (GlcNAc), and D-glucuronic acid (GlcA), in the pH range 3-7 for five different anilines (supporting information, Fig. S1), yielding products 1-11 (Chart 2).


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Chart 2. Products of model glycoconjugation reactions compared in this study.

Glycoconjugate formation rates for different nucleophiles. We initially examined reactions of the

three

monosaccharides

with

the

structurally

related

nucleophiles,

primary O-

benzylhydroxylamine (oxime products 1-3) and secondary, N-substituted O-benzyl-Nmethylhydroxyl-amine

(oxyamine

products

4-6)

and

N-benzyl-O-methylhydroxylamine

(oxyamine products 7-9) in the presence or absence of aniline. Importantly, the reactions of these secondary hydroxylamine nucleophiles proceed through a cationic intermediate,9 formally a quaternized oxime intermediate, which is unable to lose a proton in contrast to the primary hydroxylamine (Fig. 1). To the best of our knowledge, no direct comparison of glycoconjugation reaction rates and equilibrium constants between these different types of nucleophiles has been reported in the literature. We aimed to compare these different reactions and conditions, which displayed a rate span of > 10,000-fold between fast and slow reactions, as well as an equilibrium constant span of > 100-


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fold for different nucleophiles and pH values. For this we chose data treatment for second-order reversible reactions (Equation 1, and supporting information, supplementary methods). The derived kinetic rate equation was able to provide unique rate constants, k2, for (i) reactions performed under different starting conditions, as well as (ii) reactions that proceeded to an equilibrium position rather than to full conversion (reversible reactions); supporting information, Figs. S10-S52. In the following, k2 values denote the intrinsic second-order rate constant for the uncatalyzed equilibrium reactions, whereas k2,app values denote the apparent k2 for the corresponding catalyzed reaction at a nucleophile/carbohydrate/catalyst ratio of 1:10:10 (Table 1).

Table 1. Rate and equilibrium constants for different nucleophiles with D-glucose.a Nucleophile

pH

Keq

k2 (no cat.)

(M-1)

(M-1h-1)b

k2,app (AN) Product (M-1h-1)c

BnO-NH2

4

800

12.5 ± 0.09

43.6 ± 0.27 Oxime 1

BnO-NHMe

4

18

0.8 ± 0.01

7.6 ± 0.18 Oxyamine 4

MeO-NHBn

4

18

0.2 ± 0.01

2.7 ± 0.03 Oxyamine 7

BzNH-NH2

4

44

2.5 ± 0.05

20.3 ± 0.80 Hydrazine 10

TsNH-NH2

4

29

1.4 ± 0.02

3.8 ± 0.05 Hydrazine 11

BnO-NH2

7

3,600

0.1 ± 0.01

0.8 ± 0.07 Oxime 1

BnO-NHMe

7

∼40d

NDe

0.1 ± 0.01 Oxyamine 4

MeO-NHBn

7

∼40d

NDe

NDe Oxyamine 7

a

Conditions: 50 mM citrate buffer (pH 4.0), or 50 mM phosphate buffer (pH 7.0). bSecondorder rate constant in the absence of catalyst ± standard deviation for the fit (σ). cApparent second-order rate constant ± σ for nucleophile/D-glucose/aniline ratio 1:10:10. dThe reaction did


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not reach completion during the experiment. eRate constant below 0.01; See supporting information, Tables S1 and S2 for complete data set. Fast reactions were performed with 1 mM nucleophile, whereas slower reactions were performed with at 3 or 5 mM nucleophile. Our data show that uncatalyzed reactions of O-benzylN-methylhydroxyl-amine were approximately 20-fold slower than reactions with Obenzylhydroxylamine. Reactions performed in the presence of aniline showed a 4-15-fold enhancement of rate constants across pH 4-7 (supporting information, Table S1). Interestingly, we found that the rate constants for the O-benzyl-N-methylhydroxylamine nucleophile were more than 3-fold that of the isomeric N-benzyl-O-methylhydroxylamine (Table 1). This discrepancy is most likely a result of steric hindrance from the benzyl substituent, which is located closer to the reactive nitrogen in the latter case. Our comparison with hydrazone formation of benzhydrazide, or tosylhydrazide with D-glucose (products 10 and 11, respectively) showed that both of these nucleophiles reacted 2-10-fold faster than both secondary hydroxylamines. The equilibrium constants, Keq, for the different conjugates generally increased at higher pH (supporting information, Table S2). Furthermore, Keq was increased by more than one order of magnitude for reactions using the primary aminooxy nucleophile relative to all other nucleophiles studied. The exceptionally high Keq for oxime reactions with GlcA indicate that these oxime bonds are stable down to pH ∼ 3. The low Keq values for reactions using Nalkylhydoxylamine or hydrazide-type α-nucleophiles imply that very high (>0.3-0.5 M) concentrations of either nucleophile or carbohydrate are required for effective conversion in these cases, whereas oxime formation can be performed using low mM reactant concentrations (Table 1).


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Catalysts for aldose oxime formation towards neutral conditions. Next, we focused on oxime formation with variation in the aniline catalyst structure. Oxime formation with the three monosaccharides (1-3) was studied in the pH range 3-7 for the five different anilines,26 and second order rate constants for reactions with an equimolar catalyst to carbohydrate ratio were determined (supporting information, Table S1). We found that the highest reaction rates for all three monosaccharides were observed at pH 4.0. For Glc and GlcNAc, the PAN-C catalysed reaction was the fastest, while the aniline-catalysed reaction was the fastest for GlcA. At pH 3.0, we observed a decrease in reaction rate relative to pH 4.0, as well as a ∼ 10-fold decrease in the equilibrium constant. Generally, a decrease in reaction rate and increase in the equilibrium constant was observed as the pH increased from pH 4. At pH 5, PAN-C remained the most effective catalyst in reactions with Glc and GlcNAc, while PDA was superior in the reaction with GlcA. At pH 6 and 7, PDA provided the fastest reaction rates for all carbohydrates tested. PANC, which has a carboxylic acid moiety in the ortho position,21 was a superior catalyst at lower pH, except in reactions with GlcA, which carries a carboxylic acid moiety. At higher pH, PDA was a superior catalyst for all three monosaccharides. For each carbohydrate, we evaluated the performance of the five catalysts as a function of pH by considering the reaction rate of the catalyzed reaction relative to the uncatalyzed reaction (krel = k2,app/k2, Fig. 2). The relative catalytic rate enhancement, krel, generally increased with increasing pH, that is, the advantage of using a nucleophilic catalyst becomes greater at higher pH values. At pH 4, little variance was seen between the different catalysts, while at pH 6 and 7, PDA greatly outperformed the other catalysts tested. The PDA-catalyzed reaction at pH 7 offered a 34-fold increase in reaction rate for Glc, 50-fold for GlcA, and 75-fold for GlcNAc relative to the uncatalyzed reactions. The catalysts showed similar trends in catalytic rate enhancement


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across the pH values for Glc and GlcA. At pH 6 and 7, PAN was a better catalyst than PAN-C in reactions with Glc and GlcA. This relationship was however reversed in reactions with GlcNAc, for which PAN-C was a better catalyst than PAN across all pH values. Interestingly, PAN-C was an outstanding catalyst at pH 5 in reactions with GlcNAc. In addition, the increase in the catalytic efficiency of PDA from pH 6 to pH 7 was more prominent for GlcNAc, than for the other carbohydrates. The trends in catalytic rate enhancement by PDA as a function of pH seen here resemble those reported by Baca and co-workers22 for aldehydes introduced on proteins, except that we observed an increase in the catalytic rate enhancement by PDA as the pH increased from 6 to 7.

a Catalytic rate enhancement, krel

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b

80

c 80

80

1

60

2

60

40

40

40

20

20

20

0

0

0 pH 4

pH 5

pH 6

pH 7

3

60

pH 4

pH 5

pH 6

pH 7

pH 4

pH 5

pH 6

pH 7

Figure 2. Catalytic rate enhancements (krel = k2,app/k2, i.e. fold rate increase over the uncatalyzed reaction) for the five catalyst, aniline (red), MDA-C (orange), PAN (yellow), PAN-C (green), and PDA (blue), for O-benzyl oxime formation with Glc (a), GlcNAc (b), and GlcA (c) across pH 4-7. Error bars indicate standard deviations for the ratio of individual fits, (k2,app±σ)/(k2±σ). The structure of the oxime products are indicated in each panel.


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Catalytic profile of 1,4-diaminobenzene. The effective catalyst must encompass two opposing chemical characteristics: (i) It should be a good nucleophile for a fast kinetic reaction with the aldehyde, and (ii) it should provide a thermodynamically instable imine for substitution by the hydroxylamine. The latter implies that it should also be a good leaving group. For anilines, nucleophilicity as well as basicity of the intermediate imine are generally proportional to the aniline pKa value, while leaving group capability is inversely proportional to this pKa value. Thus, a pKa-dependent pH optimum exists for each catalyst at which point nucleophile and leaving group effects are balanced. PAN and PAN-C both have an optimum range, with respect to catalytic efficiency, peaking at ∼ pH 6 (Fig. 2). In reactions catalyzed by PDA, we observed that the catalytic efficiency was markedly higher at pH 7, as well as at pH ≤ pKa, compared with the other catalysts tested. The pKa value of PDA is slightly higher than that of the other catalysts, but its catalytic efficiency at pH∼pKa was dramatically increased compared to the other catalysts. PDA is a unique catalyst in that it contains a para-amino group with acid/base properties. It has been suggested that this second amino group statistically increases the likelihood of imine formation.27 However, our results contradict this hypothesis, as we found that the catalytic efficiency was significantly more than two-fold compared to aniline. Furthermore, it has been reported that 4-(methylamino)aniline (N-methyl-PDA), which can only form the imine at the primary amine position, has a catalytic effect similar to PDA in oxime formation.22 In search of an alternative explanation for the enhanced catalytic effect of PDA we compared N-aryl-glucosylamine formation at equilibrium for aniline, PAN-C, and PDA, by

13

C NMR

spectroscopy, as determined from the characteristic C-1 anomeric peaks for the α- and βpyranosyl forms (Fig. 3, and supporting information, Tables S3-S5). Interestingly, the glucosylamine levels for PAN-C were significantly lower than for aniline, indicating a


12

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destabilizing effect on the imine with PAN-C, as also reported previously as a key catalytic driving force.21 Surprisingly, the equilibrium glucosylamine levels for PDA were dramatically larger than for both aniline and PAN-C (Fig. 3). From this data we conclude that the PDA catalyst provides increased stabilization of the intermediate imine species as compared to aniline and PAN-C. At neutral conditions, however, more than half of the PDA catalyst will exist in the uncharged state in which it will be a significantly stronger nucleophile than in the monoprotonated form (pKa of doubly protonated PDA = 3.1), leading to an increase in the rate of hemiaminal formation and in imine stabilization. By NMR, we determined the pKa value for the distal amino group of the PDA glycosylamine (pKa = 6.1, Fig. 4, and supporting information, Fig. S53). Clearly, the presence of the glucosyl moiety did not significantly alter the pKa of the distal amino group for this intermediate. Protonation of the distal amino group of corresponding PDA-imine species would presumably lead to destabilization of the imine and enhanced leaving group properties during substitution by the α-nucleophile. Hence, at pH 7, PDA would be capable of playing the dual role of a strong nucleophile and a good leaving group through proton exchange reactions. We hypothesized that this could be an important mechanism for enhancing the catalytic efficiency of PDA at neutral pH.

N-Aryl-D-[1-13C]glucosylamine (% of total 1-1 3C signal)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40

AN PAN-C

30

PDA

20

10

0 pH 4

pH 5

pH 6

pH 7


13


Figure 3. Equilibrium levels of glucosylamines for the catalysts, aniline (red), PAN-C (green), and PDA (blue) in the pH range 4-7. Determined from the labeled C-1 signals of N-aryl-D-[113

C]glucopyranosylamine in equilibrated solutions of D-[1-13C]glucose and anilines (50 mM

each) by 13C NMR.

pKa= 6.1

85.0 84.5 84.0

13

CC1,β-glucosylamine (ppm)

85.5

δ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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83.5 2

3

4

5

6 pH

7

8

9

10

Figure 4. Determination of pKa value for the distal amino group of N-(4-aminophenyl)-β-D-[113

C]glucopyranosylamine by 13C NMR in 50 mM aqueous citrate () or phosphate () buffers.

Corresponding values for N-phenyl-β-D-[1-13C]glucosylamine (), lacking the distal amino group, showed no pH dependency. * = 13C label.

Interestingly, small amounts of 1,4-bis(glucosylamino)-benzene were observed in

13

C NMR

experiments for PDA, demonstrating that the two amino groups may act in tandem (supporting information, Fig. S53). However, the amounts of this species relative to the mono-glucosylamine did not reach levels (∼ 3 % at pH 6, supporting information, Table S5) that could justify a stoichiometric advantage over mono-aminobenzenes, such as aniline. Various other anilines containing a second amino group may undergo proton exchange reactions, or form bis-substituted intermediates, as described for PDA above. This includes MDA-C, as well as 1,2-diaminobenzene (ortho-phenylenediamine, ODA), and 1,3-


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diaminobenzene (meta-phenylenediamine, MDA), which we also tested. However, these three anilines were found to be inferior catalysts to PDA at pH 7 (supporting information, Table S1). One explanation could be that MDA and MDA-C are poorer nucleophiles than catalysts with a para-donating group, and presumably react less rapidly with the aldose, while ODA has a markedly lower pKa value than PDA, indicating extensive proton sharing of the two proximate amino groups. Furthermore, the second amino group of ODA may react intramolecularly with the iminium intermediate thereby lowering the rate of oxime formation.

Mechanistic studies. In order to gain a better understanding of the superior catalytic properties of the PDA catalyst, we sought to analyze reactive intermediates during imine formation in the absence of α-nucleophile by

13

C-NMR, using [1-13C]-labelled D-glucose and aniline or PDA.

While N-aryl-glucosylamines were readily detected (Fig. 3 and 4), we were surprisingly unable to detect any imine, iminium ion, or hemiaminal species even at very high (1 M) reagent concentrations (data not shown). On this basis, we estimate the concentration of these species to be below 0.005% of the glucose concentration.28 The fact that neutral imine species were not detected indicates that aldose imine protonation is highly dynamic or is more prominent than with other aliphatic or aromatic imines, which are easily detected.21 We also examined the ‘lag phase’ (sigmoidal profile) for product formation observed by HPLC with several catalysts, including PDA, in catalyzed oxime formation at pH 7 (supporting information, Figs. S16, S24, and S26). Here, the rate of product formation was not highest at the start of reaction, but rather after a period of 1-4 h. This ‘lag phase’, which has also been reported previously,21 is in the present case presumably due to a build-up of a reservoir of glycosylamine species available for substitution by the α-nucleophile. A kinetic study of the rate of oxime


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formation from pre-equilibrated solutions of D-[1-13C]-glucose and catalysts, including aniline, PAN-C, and PDA, by NMR at pH 6.0, confirmed that a significant amount of the pre-formed glucosylamine rapidly disappeared upon addition of the nucleophile (supporting information, Fig. S54). This reveals that, under the conditions of the experiment, the initial rate for conversion of glycosylamine to imine (Fig. 5c) was faster than that of formation of glycosylamine from catalyst and carbohydrate. These findings indicate that the predominant reaction pathway at neutral pH for catalyzed glycoconjugate oxime formation proceeds via imine formation from ring-opening of glycosylamines.

a

b

HO H3C H

HO H N

H

n H 2O

H 3C H

H

N

HO HO HO

OH n H H

CH 3 -n H2O

H

N

R

c

R TS_Ia-d

O

O

H + H 2O

Ia: R=H Ib: R=NH2 Ic: R=NH 3+ Id: R=NH-β-D-glucosyl

HO HO

HO HO HO

HO H

H N

n H 2O

H

HO H

H

OH n

H N H

HO

OH -n H 2O

HO H

N

H

R R

IIa-d

I'a: R=H I'b: R=NH 2 I'c: R=NH 3+

R TS_I'a-c

R II'a-c

d

Figure 5. Calculated reaction free energy profiles as a function of distal R-group protonation state (-NH2, -NH3+) for imine formation by hemiaminal dehydration (a, b), or pyranosylamine ring-opening (c, d). Proton transfers were considered for n=0-2 participating water molecules (a,


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c). Resulting energy profiles are shown for n=0 (b, d). Blue arrows indicate lowered barriers for distally protonated PDA intermediates.

To investigate our hypothesis for the catalytic mechanism of PDA at neutral pH, we performed DFT calculations enabling comparison of key reaction barriers for the neutral and the distally protonated form of PDA (Fig. 5 and 6, R=NH2, and NH3+, respectively). Reference calculations were done for aniline (Fig. 5 and 6, R= H). In these calculations we used D-glucose in ringopening steps (Fig. 5c). Truncated electrophiles and α-nucleophiles, acetaldehyde and methoxyamine, respectively, were used in all other reaction steps (Fig. 5a, 6a). The DFT results reported here for aniline closely match the results reported for the same model compounds at nearly the same level of theory by Kirmizialtin et al.29 Our inclusion of the D3 empirical dispersion correction30 to B3LYP may account for the small discrepancies at some points in the reaction free energy profile. DFT calculated free energies were determined for a series of important reaction steps. Proton transfers were generally calculated in the absence of explicit water, with reference calculations performed with one or two water molecules acting as proton shuttles (supporting information, Figs. S57 and S58). Calculated free energies for the dehydration of the protonated N-arylhemiaminals (Fig. 5a-b), and the analogous ring-opening of the N-aryl-glucosylamines (Fig. 5cd), showed that the barriers were approximately 2-2.5 kcal/mol higher for ring-opening (TS_I’) compared to dehydration (TS_I). Otherwise, the relative differences in barriers between aniline, PDA, and distally protonated PDA intermediates were highly similar for dehydration and ringopening.


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As seen in Fig. 5 (and supporting information, Fig. S55), distally protonated PDA gave noticeable lower barriers for dehydration of ∆G‡ = 30.3 kcal/mol (TS_Ic) relative to both aniline and PDA with identical barriers of ∆G‡ = 32.4 kcal/mol (TS_Ia and TS_Ib). For ring-opening, the corresponding barrier for distally protonated PDA was ∆G‡ = 32.5 kcal/mol (TS_I’c) compared to aniline and PDA with similar barriers of ∆G‡ = 34.2 and 35.0 kcal/mol (TS_I’a and TS_I’b, respectively). Based on the Eyring equation,31 the lower barrier for dehydration for distally protonated PDA corresponds roughly to 35-fold and 40-fold rate enhancements relative to aniline and PDA, respectively. For ring-opening, the corresponding rate enhancements for distally protonated PDA are 18-fold and 68-fold relative to aniline and PDA, respectively. Dehydration in the presence of a distal glucosylamino group was calculated in order to investigate any catalytic role of the bis-substituted PDA observed in 13C NMR experiments (Fig. 5a). The calculated barrier for this species (TS_Id, ∆G‡ = 33.0 kcal/mol) was higher than that of any of the other intermediates. Thus, the presence of 1,4-bis(glycosylamino)benzenes are not expected to contribute significantly to the catalytic effect of PDA. Comparing the barriers (∆G‡ values) across catalysts for reactions steps in imine to oxime conversion shows that PDA gave the smallest barrier to proton transfer of 22.8 kcal/mol (TS_IIa-c, Fig. 6), followed by aniline (24.0 kcal/mol) and distally protonated PDA (25.8 kcal/mol). Thus, a 3.0 kcal/mol penalty was observed for distal protonation of the PDA intermediate in this step. The reverse order holds for the barriers for the catalyst leaving group step, i.e. departure of distally protonated PDA (4.9 kcal/mol), aniline (6.0 kcal/mol), and PDA (7.1 kcal/mol); supporting information, Fig. S56.


18

Page 19 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a H3C O H H3C

H 3C H N

H N

H

R IIIa: R=H IIIb: R= NH 2 IIIc: R= NH3+

n H2O

H O H +N OH δ H H n H 3C N H δ+

R TS_IIa-c

H3C O -n H 2O

H H3C

N

H H N H

R IVa-c

b

Figure 6. Calculated reaction free energy profiles as a function of distal R-group protonation state (-NH2, -NH3+) for the key proton transfer during imine to oxime conversion. Proton transfers were considered for n=0-2 participating water molecules (a). Resulting energy profiles are shown for n=0 (b). The blue arrow indicates a lowered barrier for the neutral PDA intermediate in this step.

Overall, the calculated energies are consistent with the qualitative trends expected from the inductive effects of the catalyst substituents: Relative to aniline, electron withdrawal by -NH3+ in distally protonated PDA should destabilize transition states where the transferred proton is located close to the ring, while electron donation by -NH2 in PDA should stabilize the corresponding transition states. The same inductive effects are expected to increase and decrease the barrier to catalyst expulsion in PDA and distally protonated PDA, respectively: The breaking


19


Page 20 of 45

C-N bond is strengthened by electron donation for R=NH2, and weakened by electron withdrawal by R=NH3+ in the distally protonated PDA. Collectively, these results support the hypothesis, that the pair, PDA and distally protonated PDA, provides a catalytic advantage by being able to switch distal protonation state at different intermediates and before key barriers in the reaction path (Fig. 7).

Figure 7. Switching of distal protonation state (blue: neutral, and red: protonated) for important intermediates in PDA-catalyzed carbohydrate oxime formation. The fastest route, based on our DFT calculations, is indicated by the grey arrow. Intermediates are shown in the charged forms preceding key reaction steps.

Oxime formation with bioactive octasaccharide at pH 7. To study the applicability of PDA as an efficient catalyst for oxime formation at pH 7, we first investigated different catalyst concentrations. Formation of the Glc oxime at pH 7 under PDA catalysis showed a concentration-dependent increase in the rate of product formation with increasing PDA


20

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concentration in the range of 10-200 mM (supporting information, Fig. S18), similar to that previously observed for aniline at pH 4.5.7 The obtained k2,app value of 25.7 M-1h-1 at 100 mM PDA corresponds to a 250-fold rate enhancement over the uncatalyzed reaction at pH 7. At very high PDA concentrations (≥ 200 mM), however, the reaction equilibrium for product formation was shifted slightly, indicating that PDA no longer behaved as a true catalyst under these conditions. Finally, the optimized conditions for oxime conjugation were applied to the synthesis of a glycoconjugate of a complex and chemically sensitive oligosaccharide. Here, we utilized the exceptionally mild conditions, pH 7, enabled by the PDA catalyst. We recently reported on the regulatory role of

exopolysaccharide (EPS) fragments,

octasaccharides.15,

Through covalent conjugation of isolated EPS octasaccharides to

18

in

the

form

of bioactive

biosensors,19 it was shown that the exopolysaccharide transmembrane receptor kinase (EPR3) from the plant Lotus japonicus binds bacterial EPS from the soil bacterium Mesorhizobium loti directly, and disruption of this perception causes a suppression of infection thread development, which in turn inhibits formation of infected root nodules and nitrogen fixation. Direct carbohydrate-protein interaction between EPS octasaccharide and EPR3, as well as kinetic measurements, were ultimately determined using biolayer interferometry (BLI). The purified EPS octasaccharide18 contains two uronic acid units and is decorated with a non-stoichiometric acetylation pattern, which is believed to be important for correct recognition by the EPR3 receptor. As the complex carbohydrate was available only in minute quantities, we performed these experiments with a nucleophile/carbohydrate ratio of 2:1 in the absence or presence of 50 mM aniline catalyst. The octasaccharide was coupled via oxime formation to a heterobifunctional OEG linker (supporting information, Fig. S59),19, 15 which we have used previously


21


for further conjugation reactions after unmasking of a trityl (Trt)-protected thiol group.19 The oxime reactions were performed with PDA at pH 7, and with PAN-C at pH 4.5, with aniline as a control. Ample formation of the desired oxime product (Fig. 8a) was observed with PAN-C relative to aniline at pH 4.5 (Fig 8b). Remarkably, the PDA-catalyzed reaction at pH 7 approached the rate of the aniline-catalyzed reaction at pH 4.5. Near quantitative conversion could be reached after 2 days at pH 7 at room temperature using PDA as catalyst under the experimental conditions (Fig. 8b).

a

b pH 4.5

pH 7.0 100

80

80

Conversion (%)

100

Conversion (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 45

60 40 20 0

60 40 20

0

6

12

18 24 Time (h)

30

36

42

0

0

6

12

18 24 Time (h)

30

36

42

Figure 8. Oxime formation with EPS octasaccharide. Structure of resulting glycoconjugate oxime (a). HPLC data showing oxime formation at pH 4.5 (b), or pH 7.0 (b) in the absence of catalyst (), or catalyzed by aniline (), PAN-C (), or PDA (). Trt= trityl.


22

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Conclusions In summary, we have conducted a systematic study of glycoconjugate formation with different nucleophiles, carbohydrate electrophiles, and aniline catalyst, and with variation of the pH of the reactions. By application of second-order kinetic data treatment for equilibrium reactions, rate constants for the individual reactions, which could be compared across all of the different reaction conditions, were obtained. Primary aminooxy nucleophiles, which provide glycoconjugate oximes, were clearly superior to all other nucleophiles, both in terms of reaction rates and equilibrium constants for product formation. For glycoconjugate oxime formation, five catalysts, including aniline, p-anisidine (PAN), 5methoxyanthranilic acid (PAN-C), 3,5-diaminobenzoic acid (MDA-C), and 1,4-diaminobenzene (PDA), were meticulously compared at pH 4-7. Surprisingly, MDA-C did not show any significant improvement over aniline across pH 4-7 for carbohydrate oxime formation. PAN-C was generally a superior catalyst at pH 4-5, but at pH 7 it was in many cases not better than PAN, indicating a decline in the effect of the ortho-carboxy group with increasing pH for carbohydrate electrophiles. PDA was superior to all other catalysts at pH 7 for all tested carbohydrates. At elevated PDA concentrations, rate enhancements of up to 250-fold over the uncatalyzed reaction could be reached at pH 7. Based on the experimental data, as well as mechanistic studies by

13

C-NMR

spectroscopy and DFT calculations, we propose that switching of protonation state for the distal amino group of covalent PDA-carbohydrate intermediates provides a catalytic advantage by altering the electronic substituent effects along the reaction pathway. In a demonstration of the potential of the optimized conditions, they were applied for oxime coupling of a bioactive octasaccharide, derived from exopolysaccharides of nitrogen-fixing


23


Page 24 of 45

bacteria, at pH 7. Importantly, the PDA-catalyzed reaction at pH 7 approached that of an anilinecatalyzed control reaction at pH 4.5. Near quantitative conversion could be reached after 2 days at pH 7 at room temperature using PDA as catalyst with only 5 mM octasaccharide.

Experimental Procedures Materials and methods. All chemicals were purchased from Sigma-Aldrich (Denmark) and used as received. MilliQ water was used for aqueous preparations. HPLC measurements were carried out on a Dionex UltiMate 3000 HPLC system, using a C18 column (FEF Chemicals Daiso (ODDMS) 10 µm, 200 Å, 250 × 20 mm or Phenomenex Gemini 5 µm, 110 Å, 50 × 4.6 mm). Samples were eluted by applying a gradient of CH3CN in H2O containing 0.1 % HCOOH or TFA with a flow rate of 1 mL/min for 15 minutes. High-resolution mass spectrometric (HRMS) analyses were carried out on a Dionex UltiMate 3000 UHPLC+ focused system equipped with a Bruker Impact HD UHR-QqTOF mass spectrometer. NMR spectra were recorded on a Bruker Avance 300 MHz equipped with a BBO probe or a Bruker 500 MHz instrument equipped with a cryoprobe at 300 K. Chemical shift values were referenced to the residual solvent signal. Assignments were aided by H,H COSY, gHSQC, and APT experiments. Data analysis, fitting and plotting was performed using GraphPad software (GraphPad Prism version 6.07 for Windows, San Diego, CA, USA). O-Benzyl-N-methylhydroxylamine. The compound, previously described by Gudmundsdottir et al.,32 was synthesized by a modified procedure. Briefly, O-benzylhydroxylamine hydrochloride (2.0 g, 12.5 mmol) was dissolved in 1 M NaOAc (50 mL), and pH adjusted to 4.6. Formaldehyde (37 % w/w in H2O, 2.8 mL, 37.6 mmol) was added, and the solution was stirred for 24 h at 25 °C. The solution was extracted twice with CH2Cl2 (50 mL), and the combined


24

Page 25 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


organic phases were washed with brine (50 mL), dried over Na2SO4 and concentrated under reduced pressure. The resulting liquid was dissolved in glacial acetic acid (50 mL), and NaBH3CN (1.77 g, 28.2 mmol) was added. The solution was stirred for 1 h at 25 °C, and acetic acid was removed under reduced pressure. Sat. NaHCO3 (50 mL) was added, and the solution was extracted twice with CH2Cl2 (50 mL), and the combined organic phases were then extracted twice with 0.1 M HCl (50 mL). The combined aqueous phases were washed 3 times with CH2Cl2 (50 mL), basified by dropwise addition of 4 M NaOH and extracted twice with CH2Cl2 (50 mL). The combined organic phases were dried over Na2SO4 and carefully concentrated under reduced pressure (note that the product as the free base is volatile) to give the pure product (1.11 g, 65%) as a colorless liquid. The analytical data were in agreement with previously published data.32-33 N-Benzyl-O-methylhydroxylamine. The compound, previously described by Gantt et al.,34 was synthesized by a modified procedure. Briefly, methoxyamine hydrochloride (5.0 g, 59.9 mmol) and benzaldehyde (4.36 mL, 42.8 mmol) were dissolved in 60 mL CH2Cl2, and pyridine (7.58 mL, 94.1 mmol) was added. The solution was stirred overnight at 25 °C. 200 mL CH2Cl2 was added, and the organic phase was washed with 1 M HCl (100 mL) and brine (100 mL), dried over Na2SO4, and concentrated under reduced pressure. The resulting liquid was mixed with glacial acetic acid (30 mL), and NaBH3CN (5.37 g, 85.5 mmol) was added. The solution was stirred for 1 h at 25 °C, and acetic acid was removed under reduced pressure. The residue was dissolved in 0.05 M HCl (50 mL) and washed 3 times with CH2Cl2 (50 mL). The aqueous solution was basified (pH > 7.0) by addition of sat. Na2CO3, and extracted three times with CH2Cl2 (50 mL). The combined organic phases were washed with sat. Na2CO3 (100 mL) and brine (100 mL), dried over Na2SO4, and carefully concentrated under reduced pressure (note that


25


Page 26 of 45

the product as the free base is volatile) to give the pure product (2.36 g, 40%) as a colorless liquid. The analytical data were in agreement with previously published data.34 D-Glucose

O-benzyloxime (1). The analytical data were in agreement with previously published

data.7 HR-MS (ESI-TOF): calcd. for C13H19NO6, [M+H]+: m/z 286.1285; found: 286.1285. 2-Acetamido-2-deoxy-D-glucose

O-benzyloxime

(2).

HR-MS

(ESI-TOF):

calcd.

for

C15H22N2O6, [M+H]+: m/z 327.1551; found: 327.1552. 1H NMR (300 MHz, CD3OD, (E)oxime/(Z)-oxime/β-pyranosyloxyamine tautomeric ratio 7:2:1): δ 7.50 (d, J=5.9 Hz, 0.7H; (E)oxime H-1), 7.42-7.23 (m, 5H; Ph), 6.76 (d, J=6.3 Hz, 0.2H; (Z)-oxime H-1), 5.17 (dd, J=5.8 and 6.0 Hz, 0.2H; (Z)-oxime H-2), 5.12 (s, 0.4H; (Z)-oxime CH2Ph), 5.05 (s, 1.4H; (E)-oxime CH2Ph), 4.76-4.62 (m, 0.8H; (E)-oxime H-2 + β-pyranosyloxyamine CH2Ph), 4.24 (d, J=9.7 Hz, 0.1H; β-pyranosyloxyamine H-1), 4.12 (dd, J=2.6 and 5.5 Hz, 0.2H; (Z)-oxime H-3), 4.02 (dd, J=2.0 and 6.7 Hz, 0.7H; (E)-oxime H-3), 3.88-3.25 (m, 4.2H), 1.99-1.95 (3s, 3H; NAc).

13

C

NMR (75 MHz, CD3OD, (E)-oxime/(Z)-oxime/β-pyranosyloxyamine tautomeric ratio 7:2:1): δ 173.3 (C=O), 151.4 ((Z)-oxime C-1), 150.0 ((E)-oxime C-1), 139.1 (Ph ipso), 130.1 (Ph), 129.4 (Ph), 129.3 (Ph), 129.3 (Ph), 129.1 (Ph), 128.8 (Ph), 91.4 (β-pyranosyloxyamine C-1), 79.3, 78.1 (β-pyranosyloxyamine CH2Ph), 77.2 ((Z)-oxime CH2Ph), 77.0, 76.9 ((E)-oxime CH2Ph), 73.4, 73.0, 72.9, 72.2, 71.9, 71.4, 70.6, 64.9, 64.8, 63.0, 53.7 (β-pyranosyloxyamine C-2), 53.5 ((E)oxime C-2), 50.6 ((Z)-oxime C-2), 23.0 (β-pyranosyloxyamine NAc), 22.7 ((E)-oxime NAc), 22.6 ((Z)-oxime NAc). 6-Oxo-D-Glucose O-benzyloxime (3). HR-MS (ESI-TOF): calcd. for C13H17NO7, [M+H]+: m/z 300.1078; found: 300.1077. 1H NMR (300 MHz, CD3OD, (E)-oxime with trace (Z)-oxime (80% β-oxyamine, other forms not assigned) δ 176.7 (C=O), 138.5 (Ph ipso), 130.3 (Ph), 129.3 (Ph), 129.1 (Ph), 95.4 (C-1), 79.0 (C-3), 77.9 (C-5), 76.4 (CH2Ph), 73.4 (C-4), 71.5 (C-2), 39.4 (NCH3). N-Benzyl-N-(D-glucosyl)-O-(methyl)hydroxylamine (7). The analytical data were in agreement with previously published data.35 HR-MS (ESI-TOF): calcd. for C14H21NO6, [M+H]+: m/z 300.1442; found: 300.1441. N-Benzyl-N-(2-acetamido-2-deoxy-D-glucosyl)-O-(methyl)hydroxylamine (8). The analytical data were in agreement with previously published data.23 HR-MS (ESI-TOF): calcd. for C16H24N2O6, [M+H]+: m/z 341.1707; found: 341.1705.


27


Page 28 of 45

N-Benzyl-N-(6-oxo-D-glucosyl)-O-(methyl)hydroxylamine (9). HR-MS (ESI-TOF): calcd. for C14H19NO7, [M+H]+: m/z 314.1234; found: 314.1233. 1H NMR (300 MHz, CD3OD, >80% βoxyamine, other forms not assigned): δ 7.49-7.22 (m, 5H; Ph), 4.22 and 4.07 (2d, J=12.8 Hz, 2H; CH2Ph), 4.01 (d, J=8.9 Hz, 1H; H-1), 3.75 (t, J=8.9 Hz, 1H; H-2), 3.54 (d, J=9.2 Hz, 1H; H-5), 3.46 (t, J=8.9 Hz, 1H; H-4), 3.40 (s, 3H; OMe), 3.37 (t, partly overlapped, J=8.8 Hz, 1H; H-3). 13

C NMR (75 MHz, CD3OD, >80% β-oxyamine, other forms not assigned) δ 76.2 (C=O), 138.7

(Ph ipso), 131.1 (Ph), 129.2 (Ph), 128.4 (Ph), 93.4 (C-1), 79.1 (C-3), 78.0 (C-5), 73.3 (C-4), 71.3 (C-2), 62.6 (OMe), 57.6 (CH2Ph). N-Benzoyl-N’-(D-glucosyl)hydrazine (10). The analytical data were in agreement with previously published data.36 HR-MS (ESI-TOF): calcd. for C13H18N2O6, [M+H]+: m/z 299.1238; found: 299.1239. N-(D-Glucosyl)-N’-(4-methylbenzenesulfonyl)hydrazine (11). The analytical data were in agreement with previously published data.32 HR-MS (ESI-TOF): calcd. for C13H20N2O7S, [M+H]+: m/z 349.1064; found: 349.1064. Exopolysaccharide octaose conjugation. A purified exopolysaccharide-derived octaose (EPS octasaccharide)18, 37 from the soil bacterium Mesorhizobium loti was used to assess the feasibility of using 1,4-diaminobenzene as a catalyst of oxime formation at pH 7.0. A hetero-bifunctional aminooxy-tetra(ethylene glycol)-thiol linker (OEG linker)19 was used as the nucleophile. Separate solutions of the EPS octasaccharide (2.2 mg, 1.5 µmol), and the OEG linker (1.4 mg, 3.0 µmol) were prepared in MeCN-H2O (v/v 1:1 ratio) at 20 mM and 40 mM, respectively. These solutions were combined (v/v 1:1 ratio) and diluted with buffer solution (100 mM sodium acetate (pH 4.5), or 100 mM NaH2PO4 (pH 7.0), both MeCN-H2O (v/v 1:1 ratio)) to produce a final concentration of 5 mM EPS octasaccharide, 10 mM OEG linker, 50 mM buffer, MeCN-


28

Page 29 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H2O (v/v 1:1 ratio). For catalyzed reactions, the buffers were prepared with the further addition of 100 mM aniline, PAN-C, or PDA catalyst. Final concentrations were thus 5 mM EPS octasaccharide, 10 mM OEG linker, 50 mM buffer, 50 mM catalyst, MeCN-H2O (v/v 1:1 ratio). Product formation was monitored by HPLC. Samples were injected 15 minutes after mixing of reactants, and every 2 hours thereafter for 26-58 hours depending on the reaction rate. The conversion was determined from the absorbance at 254 nm by relating twice (due to the 2:1 ratio of nucleophile to EPS octasaccharide) the integrated absorbance of the product to the sum of the integrated

absorbance

of

product

and

OEG

linker

(i.e.,

conversion

=

2*Absproduct/(Absproduct+Abslinker)). The oxime conjugate product had HR-MS (ESI-TOF), triacetylated oxime conjugate: calcd. for C80H113NO48S, [M+H]+: m/z 1888.6226; found: 1888.6297, and calcd. for C61H99NO48S, [M-trityl+2H]+: m/z 1646.5130; found: 1646.5084. HRMS (ESI-TOF), diacetylated oxime conjugate: calcd. for C78H111NO47S, [M+H]+: m/z 1846.6120; found: 1846.6165, and calcd. for C59H97NO47S, [M-trityl+2H]+: m/z 1604.5024; found: 1604.5013. Kinetic HPLC Measurements. The rate of glycoconjugate formation was determined by HPLC, by calculating the integrated absorption of the product peak relative to the total integrated absorption of nucleophile and product peaks at 215 nm. As the extinction coefficient at 215 nm of the products differed from that of the corresponding nucleophiles, the absorption integrals were corrected to match the absorption at 254 nm. Generally, a volume of 10 µL was injected every 30 minutes for 13 hours. Solutions containing α-nucleophile, carbohydrate electrophile and aniline derivative catalyst were prepared individually in 50 mM buffer solutions. The pH and volume of each solution was adjusted individually to reach the desired conditions immediately upon mixing. Reactions were started, after prior equilibration of the carbohydrate solution for at


29


Page 30 of 45

least 30 min, by mixing the three solutions. For uncatalysed reactions, the nucleophile and electrophile solutions were diluted with a 50 mM buffer solution prior to mixing. Citrate was used as buffer in experiments carried out at pH 3.0-6.0, while phosphate was used as buffer in experiments carried out at pH 7.0. In general experiments, 1 mM nucleophile was mixed with 10 mM carbohydrate and 10 mM aniline catalyst. In slower reactions (e.g., formation of GlcNAc oximes), the concentrations were increased to 5 mM nucleophile, 50 mM carbohydrate and 50 mM catalyst, maintaining the ratio of nucleophile/electrophile/catalyst at 1:10:10. Peak identities were determined as follows: The peak of the nucleophile was identified by retention time by comparison with an injection of a solution containing only the nucleophile. The peak of the product was identified by peak collection and subsequent HR-MS analysis. Equation 1 was fitted to the kinetic HPLC data using GraphPad Prism 6.07 to provide the intrinsic second-order forward rate constants, k2, for each of the uncatalyzed reactions. For fast reactions, C୞ୣ values were obtained by fitting directly from the data (two-parameter fit), whereas for slower reactions,

C୞ୣ values were obtained from separate experiments where the reactions were left to reach equilibrium. Apparent rate constants, k2,app, for catalyzed reactions performed with a fixed nucleophile/carbohydrate/catalyst ratio of 1:10:10, were obtained using the same equation as for the uncatalyzed reactions. The apparent rate constants, k2,app, are dependent on the applied catalyst ratio, but they are directly comparable to the intrinsic rate constants, k2, of the uncatalyzed reactions. The ratio, k2,app/k2, is a measure of the efficiency of the individual catalysts, i.e, the rate enhancement observed in the presence of the catalyst.

C୞ ሺtሻ =

‫ ۇ‬ష େ౛ౖ ∙‫ۈ‬ୣ ‫ۉ‬

బ ౛మ ిబ ఽ ిా షిౖ ౡ ౪ మ ౛ ిౖ

‫ۊ‬ ିଵ‫ۋ‬

మ ిబ ిబ షి౛ ష ఽ ా౛ ౖ ౡమ ౪ ిౖ

మ ి౛ ౖ ∙ୣ బ ిఽ ిబ ా

‫ی‬

ିଵ

(Equation 1)


30

Page 31 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Determination of equilibrium concentrations for glucosylamine formation by NMR analyses. The reactions of D-[1-13C]glucose with different aniline catalysts, in the absence of an α-nucleophile, were studied by NMR spectroscopy. Briefly, two solutions containing D-[113

C]glucose (100 mM), and catalyst (100 mM aniline, 5-methoxyanthranilic acid, or 1,4-

diaminobenzene), both in 50 mM aqueous (H2O) citrate buffer (both solutions adjusted to pH 3.0, 4.0, 5.0, or 6.0, separately), or 50 mM aqueous (H2O) phosphate buffer (both solutions adjusted to pH 7.0, separately), were rapidly mixed in a 1:1 ratio and transferred to a 5 mm NMR tube. A closed capillary containing DMSO-d6 was added to the NMR tube, and

13

C-NMR

spectra were acquired at different time points. The samples were left to reach equilibrium, which typically occurred after 4-8 hours. Spectra were acquired using a 7.8 s relaxation delay (D1) and 512 scans to allow for peak integration of the different 13

C]glupyranose

and

13

N-aryl-D-[1-13C]glucopyranosylamines.28

C-labeled signals of D-[1Peak

identification

was

performed by reference to literature values.28, 38-39, 7 Tables S3-S5 list the equilibrium percentages for the different compounds containing the 13C-label. Kinetic measurements of glucose oxime formation from pre-equilibrated glucosylamine solutions by NMR analyses. Solid O-benzylhydroxylamine hydrochloride (final concentration of 5 mM) was added to pre-equilibrated solutions of D-[1-13C]glucose (50 mM), and catalyst (50 mM aniline, 5-methoxyanthranilic acid, or 1,4-diaminobenzene) in 50 mM aqueous (H2O) citrate buffer, pH 6.0. Following rapid mixing, the samples were transferred to a 5 mm NMR tube. A closed capillary containing DMSO-d6 was added to the NMR tube, and 13C-NMR spectra were acquired continuously for 3.5 h. Spectra were acquired using a 7.8 s recycle time (D1) and 64 scans to allow for peak integration of the different

13

C-labeled signals of D-[1-13C]glucose, N-


31


Page 32 of 45

aryl-D-[1-13C]glucosylamines, and D-[1-13C]glucose O-benzyloximes. Peak identification was performed by reference to literature values, as described above.

Computational Methods. DFT calculations were carried out in Gaussian 0940 using the B3LYP41-44 functional with the D3 empirical dispersion correction30 and the 6-311+G(d,p)45-46 basis set. Bulk solvent effects were modeled with the conductor-like polarizable continuum model (CPCM)47-48 for water. Geometry optimizations to local minima and transition states (TS) were carried out with the Berny algorithm49 with tight convergence criteria and quantum mechanical evaluation of the Hessian at each optimization step. Two-electron integrals were evaluated using an ultrafine grid. Initial estimates for product and reactant structures were drawn in Maestro34 and optimized to local minima. Initial estimates for transition state (TS) structures were obtained using a relaxed coordinate scan with a step size of 0.1 Å for transforming reactants into a product or vice versa. The geometry with the highest energy in the relaxed coordinate scan was optimized to a transition state. Vibrational harmonic frequencies were calculated for all stationary points to verify that transition states and local minima (reactants, products) had exactly one and zero imaginary frequencies, respectively. Intrinsic reaction coordinate (IRC) calculations were carried out for all transition states to validate that the TS connected the expected reactants and products. Thermodynamic quantities were calculated for the stationary points at 298.15 K and 1 atm with the default procedure in Gaussian. We used acetaldehyde as a model for a generic open-chain carbohydrate. This truncated carbohydrate model is based on the assumption that the remainder of the carbohydrate does not contribute to the difference between reactions with different catalysts. This assumption need not hold in the actual systems, but it removes conformational variability in the calculations, and thus allows


32

Page 33 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


isolation of the non-conformational (inductive) effects of the three catalysts. The aminooxy nucleophile was truncated to methoxyamine and the catalysts were included in their unmodified form. Relative reaction rates were estimated from the Eyring equation (Equation 2)31: ݇rel = exp ቀ

∆∆G‡ ோ்

ቁ

(Equation 2)

Hemiaminal dehydration. The rate limiting step in the nucleophilic catalysis of oxime formation is widely accepted to be the dehydration of the catalyst-hemiaminal resulting from the condensation of (protonated) aldehyde and catalyst.50 We selected the protonated catalysthemiaminal (structures Ia, Ib, and Ic in Fig. 5a and Fig. S55) as the starting point for our calculations, and thus assume that this species has been formed in a previous nucleophilic addition step. DFT calculated free energies for dehydration of the protonated catalysthemiaminal are shown in Fig. S55a, b, c for aniline, PDA, and distally protonated PDA, respectively. Fig. 5b in addition to these energies shows the free energy barrier for dehydration with a distal glucosylamino group on PDA. We assume that the dehydration occurs through a direct internal proton transfer from N to O. The possibility of water mediating the proton transfer (i.e. acting as a proton shuttle) at this step is discussed below. Imine to oxime conversion. The next stage in the nucleophilic catalysis of oxime formation is the conversion of the condensation product between the catalyst imine and aminooxy nucleophile into the target oxime and free catalyst. The calculated structures and Gibbs free energies for this process are shown in Fig. S56 for (a) aniline, (b) PDA and (c) distally protonated PDA, with the key barriers shown separately in Fig. 6b. The largest barrier was found for the direct N to N proton transfer step, and is for each catalyst lower than the largest barrier calculated for the


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preceding hemiaminal dehydration (Fig. 5 and Fig. S55), supporting that dehydration of the catalyst-hemiaminal is rate-determining. Water assisted proton transfers. Protic solvents may facilitate proton transfer reactions by acting as proton shuttles, leading to substantially reduced reaction barriers.23,4,7 Inspired by previous DFT studies,51 we examined if the principal barriers calculated for hemiaminal dehydration (TS_I, Fig. 5a and Fig. S55) and for imine-to-oxime conversion (TS_II, Fig. 6b and Fig. S56) could be lowered by the participation of water proton shuttles for mediating the proton transfer between N and O or N and N, respectively. We selected the PDA catalyzed reaction for examining these effects, and successively tried to incorporate a single water molecule or a water dimer in the transition states (i.e. TS_Ib and TS_IIb). Geometrical complementarity between the transition state structure in question and the two types of water bridge determined whether a single water molecule or two water molecules produced a converged transition state structure. In case of TS_Ib, only a single water molecule efficiently bridged the proton-donating/accepting N and O atoms, while only the water dimer could bridge the germinal nitrogens in TS_IIb without structural distortion. For the hemiaminal dehydration mechanism, the single water molecule acting as a proton shuttle between N and O lowered the barrier for proton transfer by 12.5 kcal/mol (from 32.4 kcal/mol to 20.0 kcal/mol), see Fig. S57. For the proton transfer in imine-tooxime conversion, the proton shuttle consisting of two water molecules lowered the barrier by 14.7 kcal/mol (from 22.8 to 8.1 kcal/mol), see Fig. S58. These results indicate that the participation of water proton shuttles is plausible in the actual mechanism. However, the effect is assumed to be additive across the three catalyzed reactions studied herein, and the catalyst differences between aniline, PDA, and distally protonated PDA discussed above should remain in the presence of proton shuttles. Also, it is seen that the water proton shuttles do not change the


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rank-ordering of the barriers associated with TS_Ib and TS_IIb, and thus hemiaminal dehydration is still predicted to be rate-limiting.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publication website. Details on the equation for determination of second order rate constants, Supplementary tables (complete data sets for second order rate constants including standard deviations and equilibrium constants, and measured equilibrium percentages for glucosylamine formation by NMR), and supplementary figures (HPLC chromatograms, data fitting, additional data from NMR and DFT studies). Copies of NMR spectra, and DFT optimized structures and energies (PDF).

AUTHOR INFORMATION Corresponding Authors *For K.J.J.: [email protected] *For M.B.T.: [email protected] ORCID Knud J. Jensen: 0000-0003-3525-5452 Mikkel B. Thygesen: 0000-0002-0158-2802 Funding Sources Generous support by the Danish National Research Foundation (grant no. DNRF79) and by the Villum Foundation (grant no. VKR022710) is gratefully acknowledged. Notes


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The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank Dr. John Sullivan and Dr. Artur Muszyński for production and purification of the EPS octasaccharide unit from Mesorhizobium loti.

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Table-of-Content Graphic


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Glycoconjugate Oxime Formation Catalyzed at ... - ACS Publications

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