Article pubs.acs.org/JPCA
Noncatalytic Hydrothermal Elimination of the Terminal D‑Glucose Unit from Malto- and Cello-Oligosaccharides through Transformation to D‑Fructose Hiroshi Kimura,† Masaru Nakahara,† and Nobuyuki Matubayasi*,†,‡ †
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan Japan Science and Technology Agency (JST), CREST, Kawaguchi, Saitama 332-0012, Japan
‡
S Supporting Information *
ABSTRACT: Noncatalytic hydrothermolyses of malto- and cello-oligosaccharides (di-, tri-, tetraose), linked by α- and β-1, 4-glycosidic bonds, respectively, were investigated at 100−140 °C. In situ 13C NMR spectroscopy was applied to elucidate the position and pathways of the glycosidic bond breakage and the dependence of the hydrolysis rate on the bond type. Spectral analysis was carried out quantitatively as a function of time with the mass balance confirmed, and it was shown for both the malto- and the cello-oligosaccharides that the terminal D-glucose unit with a free anomeric carbon is selectively eliminated after transformation to D-fructose. Site-selective breakage of the glycosidic bonds proceeded on the order of hours. The initial apparent rates for terminal hydrolysis were found to be independent of the degree of oligomerization but dependent on the type of glycosidic bond. Rate constants were larger for the α-1,4-linked malto-oligosaccharides by a factor of 3−4 than for the β-1, 4-linked cello-oligosaccharides. The pathways and mechanisms for the malto- and cello-oligosaccharide hydrothermolyses are common and can be understood in terms of the elementary reactions of the di- and monosaccharides. tetrasaccharide, D-maltotetraose, the α-1,4-glycosidic bond at the rightmost terminal D-glucose unit with a free anomeric carbon (denoted as 1/4G) is special; only this terminal unit can be converted into the unstable open-chain form with ring opening.24,25 The other internal and leftmost units, 2/4G, 3/4G, and 4/4G, are not allowed to take the open-chain form as they are blocked by the glycosidic bond. Therefore, monosaccharide elimination is expected not to occur randomly. This idea is tested in the present work. The regularity in reaction pathways has been hard to find, even when it is present, for high-temperature hydrothermolyses of carbohydrates.6,8−12,26−28 This is due to the existence of a large number of product species including isomers. Further complication may arise when a catalyst is added to supercritical or subcritical aqueous medium. To resolve the complicated reaction systems of starch and cellulose, spectroscopic analysis needs to be conducted at atomic resolution with carefully designed experimental conditions. In this study, a low-temperature range
1. INTRODUCTION Progress of the physical chemistry of the structures1−5 and reactions6,7 of biomass is crucial to meet the increasing demand for green chemistry. In particular, hydrothermal treatment of biomass in supercritical and subcritical conditions is promising since the reaction medium is water. It is thus of great importance to establish the pathways and mechanisms for hydrothermal conversion of such biomass molecules as starch and cellulose into simple synthetic fuels (biofuels) and starting materials (feedstock) for drug, polymer, and device-component preparation.6,8−23 To resolve the complicated pathways of carbohydrate reactions, comprehensive studies on model systems are needed as the first step using oligosaccharides and controlling the number and geometry of glycosidic bonds. In the present work, we focus on starch-related malto-oligosaccharides with α-1,4-glycosidic bonds and cellulose-related cello-oligosaccharides with β-1,4-glycosidic bonds and systematically carry out detailed structural and mechanistic studies on the hydrothermolyses of the oligosaccharides. The questions addressed are whether the α- or β-1,4-glycosidic bond is hydrolyzed site selectively or randomly and whether the pathways and mechanisms are common to the malto- and cello-oligosaccharides. As seen in Figure 1a for the © 2012 American Chemical Society
Received: April 10, 2012 Revised: August 6, 2012 Published: October 5, 2012 10039
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was also examined. The sample was loaded into a Pyrex NMR tube (SHIGEMI, 10.0 mm o.d.). The sample tube was flame sealed after the tube inside was purged with argon. D-Cellotriose and D-cellotetraose were weighed to form nearly saturated solutions (0.1 and 0.02 M, respectively) and loaded into an NMR microtube (SHIGEMI, 10.0 mm o.d. with a 12 mm thickness spacer at the bottom), which was used to reduce the amount of expensive substrate. To prepare a nearly saturated solution, the sealed tube was put into a hot water bath and sonicated for 1 h at 100 °C. For full detection and determination of the openchain (unstable) and ring forms, nearly saturated solutions of D-maltose (∼3 M) and D-cellobiose (∼1 M) were additionally prepared using a hot water bath at 100 °C.29 The apparatus and experimental procedures were the same as before.7 The reaction temperature was set to 100, 110, 120, 130, and 140 °C and controlled within ±1 °C. In the in situ 13C measurements, proton irradiation was turned off to keep the spectral intensity proportional to the number of carbon atoms. One in situ measurement took 0.5−1 h, which is short enough compared to the time scale of the reaction discussed below. For the ring and open-chain forms of the disaccharides, D-maltose and D-cellobiose, we also performed ab initio MO calculations in vacuum and PCM (continuum) water using the GAUSSIAN 09 program.30 The geometry was optimized using the hybrid density functional B3LYP with the correlation-consistent polarized valence triple-ζ (cc-pVDZ) basis set. At the optimized geometry, a single-point energy calculation was carried out at the B3LYP level of theory with the augmented cc-pVDZ (augcc-pVDZ) basis set.
Figure 1. Notations for the D-glucose (G) and D-fructose (F) units in the malto- and cello-oligosaccharides. (a) For the G-type D-maltotetraose, the position of the G unit is denoted by the superscripts, 1/4, 2/4, 3/4, and 4/4, respectively, for 1/4G, 2/4G, 3/4G, and 4/4G. Numbering starts from the rightmost terminal unit that can take the open-chain form (1/4Gopen‑chain). Isomerization and transformation are not allowed for the n/4 G (n ≥ 2) units due to the presence of glycosidic bonds. (b) For the F-type D-cellobiose, the same numbering and notation systems are adopted. Carbon sites within the G and F units are numbered as shown in the figure.
3. RESULTS AND DISCUSSION We will show that the terminal D-glucose residue with a free anomeric carbon is selectively eliminated from oligosaccharides after transformation to D-fructose, irrespective of the type of 1,4glycosidic bond and the degree of oligomerization. To help one understand the complicated structures of the oligosaccharides, hereafter we express the open-chain form, the pyranoses (6-membered ring), and the furanoses (5-membered ring) of α- and β-types for D-glucose (G) and D-fructose (F) as Gopen‑chain, G6‑α, G6‑β, G5‑α, G5‑β, Fopen‑chain, F6‑α, F6‑β, F5‑α, and F5‑β. The isomeric species are thus indicated as the subscript. The rightmost terminal is denoted as unit 1 in the superscript. The unit connected to unit 1 is termed unit 2, and the other units are further called units 3 and 4. The locations of the units in the di-, tri-, and tetrasaccharides are expressed as l/2 (l = 1, 2), m/3 (m = 1, 2, 3), and n/4 (n = 1, 2, 3, 4), respectively. For example, the location of the G unit is specified as l/2G, m/3G, and n/4G. With our notation, the open-chain form of the G-type D-maltotetraose and the β-pyranose of the F-type D-cellobiose are expressed as 4/4 G6‑α−3/4G6‑α−2/4G6‑α−1/4Gopen‑chain and 2/2G6‑β−1/2F6‑β, respectively, as shown in Figure 1. The position of the carbon atom is further numbered as in Figure 1. The number 1 carbon atom (i.e., anomeric carbon atom) of the G unit is denoted as C1, the number 2 carbon atom is C2, and so on. Furthermore, the units of the hydrolyzed oligosaccharides are not renumbered from the state before the reaction to show clearly where glycosidic bond cleavage occurs. Usually the monosaccharides of D-glucose and D-fructose are expressed as Glc and Frc, respectively, and moreover, the pyranose and furanose forms are indicated by p and f, respectively; by this, for example, α-pyranose of D-glucose is denoted as α-Glcp, which corresponds to G6‑α in our nomenclature. A simplified set of notations is adopted in this paper since we have to distinguish
of 100−140 °C is taken in the absence of catalysts so that early products due to glycosidic bond breakage can be identified in real time within NMR time resolution.7 Site-selective information is required for determining the position of the monosaccharide elimination. We employ the in situ 13C NMR spectroscopy which enables us to noninvasively distinguish, identify, and quantify the chemical species involved in hydrothermolyses of the malto- and cello-oligosaccharides as a function of reaction time. 13C chemical shifts are sensitive enough to distinguish the location of the D-glucose unit. The in situ NMR method can provide real-time information not only on the reactants but also on the intermediate and final products and is especially useful to examine the total mass balance; mass balance analysis is crucial to explore the reaction pathways and mechanisms. The experimental procedures are described in section 2. Results and discussion are described in section 3; assignment of 13 C spectra before and during hydrothermolysis is shown in sections 3.1 and 3.2, respectively, and the pathways and mechanisms for hydrothermolysis are established in section 3.3. Conclusions are summarized in section 4.
2. EXPERIMENTAL SECTION A series of malto-oligosaccharides, D-maltose monohydrate (Aldrich, >98%), D-maltotriose (Wako, >97%), D-maltotetraose (Hayashibara, >96%), and cello-oligosaccharides, D-cellobiose (Wako, >98%), D-cellotriose (Funakoshi, >95%), and D-cellotetraose (Funakoshi, >95%), and deuterated water D2O (ISOTEC, 99.95 atom % D) were used without further purification. In the structural and kinetic studies, the malto-oligosaccharides and −3 D-cellobiose were prepared at 0.5 M (mol dm ) in D2O; for determining the reaction order of hydrolysis, a 0.2 M solution 10040
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Figure 2. 13C spectral evidence for the open-chain and ring forms of the F-type D-maltose in the nearly saturated solution of ∼3 M. These spectra were obtained at 100 °C over a long accumulation time of 80 h, until which the reaction proceeds slightly; hydrolyzed monosaccharides, G and F, are yielded to be ∼2%, together with a trace amount of anhydrosaccharides and/or their derivatives. Anomeric carbons of the G and F units are identified and quantified in the chemical-shift range shown: C1 of the G unit and C2 of the F unit (see Figure 1 for carbon numberings). For assignment of the monomeric G and F, see ref 7. By referencing the chemical shifts for the F monomer and considering the upfield shift due to glycosidic bond formation, the peaks derived from the 2/2G−1/2F dimeric forms were assigned.
Gopen‑chain ⇄ Fopen‑chain.7,35−39 The F monomer is more reactive than the G monomer as previously shown,7 and the same reactivity difference is expected also for the cases of the oligosaccharides.40 The key role is played by the anomeric site in controlling isomerization and transformation, so the 2/2G unit at the left-hand-side terminal is not allowed to be isomerized and transformed because the anomeric site is blocked by the α-1, 4-glycosidic bond. Now we are at a position to discuss the population fractions of the chemical species involved in the pre-equilibrium before the hydrolysis starts. The fractions of the cyclic forms for the G-type D-maltose and D-cellobiose are in the following orders
D-glucose
and D-fructose. A single-letter abbreviation is more convenient to see the carbohydrate configuration within the present context. In addition, the rightmost 1G (1/2G, 1/3G, and 1/4 G) and 1F (1/2F, 1/3F, and 1/4F) terminals with a free anomeric carbon are called a reducing unit, while the other units are called a nonreducing unit. 3.1. 13C Spectra before Hydrolysis. First, we discuss the isomer distribution before analyzing hydrolysis of oligosaccharides. In general, many carbohydrates exist in equilibrium between open-chain and cyclic forms.7,24,25 Of these isomeric forms, the open-chain form has been considered to play a key role in the isomerization and transformation because of the low stability and most likely resemblance to the transition state.24 Figure 2 illustrates the in situ 13C NMR spectrum of a concentrated (∼3 M) solution of α-1,4-linked D-maltose at 100 °C after a long accumulation time of 80 h, until which hydrolysis of the glycosidic bond can hardly occur; a trace amount of anhydrosaccharides and/or their derivatives was also seen as byproducts.27,31 What isomers are formed in the F (2/2G−1/2F) and G (2/2G−1/2G) types? For the F type, as seen in Figure 2, the isomeric forms detected at equilibrium are the open chain, 2/2 G6‑α−1/2Fopen‑chain, and the rings, 2/2G6‑α−1/2F6‑β, 2/2G6‑α−1/2F5‑α, and 2/2G6‑α−1/2F5‑β. Clearly, the terminal 1/2G unit is transformed into 1/2F before elimination, just as in the case of the monosaccharide,7 see Figure 3. To the best of our knowledge, this is the first success in detecting by NMR the open chain of the F-type Dmaltose (2/2G6‑α−1/2Fopen‑chain);32,33 full detection was possible only at a high concentration of ∼3 M and was not at a lower concentration of 0.5 M, showing the relative instability of the open-chain form. For the G type, the pyranoses of 2/2 G6‑α−1/2G6‑α and 2/2G6‑α−1/2G6‑β are observed. The open-chain form, 2/2G6‑α−1/2Gopen‑chain, was not detected because of the lower solubility and stability.34 The furanoses of 2/2G6‑α−1/2G5‑α and 2/2 G6‑α−1/2G5‑β are absent since the OH group attached to the C4 site in the 1/2G unit is blocked by the glycosidic bond; see Figure 3. It is of great importance that the 1/2G unit is transformed into the 1/2F before hydrolysis. Such reversible transformation is considered to take place via keto−enol tautomerization between the open-chain forms, as is typically so for the monosaccharide,
2/2
G6‐α−1/2G6‐β (59%) >
2/2
2/2
2/2
G6‐β −1/2G6‐β (59%) >
G6‐α−1/2G6‐β (41%)
(1a)
G6‐β −1/2G6‐α(41%)
(1b)
Here the numbers in parentheses are the mole fractions (%) of interest normalized by the overall 2/2G−1/2G types.41 The fractions for D-maltose in eq 1a are identical to those for D-cellobiose in eq 1b. In the F-type D-maltose and D-cellobiose, the open chain and rings isomerized at the anomeric site are populated in the following orders 2/2
G6‐α−1/2 F6‐β (55%) >
−1/2 F5‐α (9%) >
2/2
G6‐α−1/2 F5‐β (31%) >
2/2
G 6‐ α
2/2
G6‐α−1/2 Fopen‐chain(5%)
2/2
G6‐β −1/2 F6‐β (54%) >
−1/2 F5‐α (10%) >
2/2
2/2
G6‐β −1/2 F5‐β (31%) >
G6‐β −1/2 Fopen‐chain(5%)
(2a) 2/2
G 6‐ β (2b)
where the fractions are determined against the overall 2/2G−1/2F types. In the case of the 2/2G−1/2F types, the fractions for Dmaltose in eq 2a are identical to those for D-cellobiose in eq 2b, just as in the case of the 2/2G−1/2G types. Thus, the glycosidebond type does not affect the isomer fractions in the equilibrium. What factors determine the isomer fractions (or isomer stability order) for the disaccharides? Previously it was shown7 that for the monomeric G and F the fractions of the open-chain and ring forms are in the following orders 10041
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Figure 3. Reaction pathways of the hydrothermolysis of the disaccharide D-maltose. All isomeric forms of the disaccharides are shown. Curly braces denote a set of all isomers of each saccharide; for the monosaccharides only the representative isomers are shown here for brevity.
quantum-chemical calculation of each of the G- and F-type forms in PCM water. For the G-type D-maltose, the computed stability order in PCM water is given as
G6‐β (54%) > G6‐α(44%) ≫ G5‐β (1%) > G5‐α(0.5%) > Gopen‐chain(0.04%)
(3)
2/2
G6‐α−1/2G6‐α ≈
2/2
>
2/2
F6‐β (48%) > F5‐β (36%) > F5‐α(8%) > Fopen‐chain(5%) > F6‐α (4%)
(4)
G6‐α−1/2G6‐β G6‐α−1/2Gopen‐chain
(5)
This is in good agreement with the experimental result, eq 1a. For D-cellobiose, the computed stability order in PCM water is
Compare the isomer distributions for the dimer and monomer cases (eqs 1a, 1b, and 3 and eqs 2a, 2b, and 4, respectively) and one can find the following common tendencies: (i) the pyranoses (6-membered rings) of the G type predominantly exist with the sum kept nearly 100%, (ii) the β-anomer is more favored than the α-anomer in the pyranoses of the G type, (iii) the fraction of each G-type dimeric form corresponds well to that of the corresponding monomeric form, (iv) similarly, the fraction of each G−F form is in good agreement with the corresponding monomeric F form, and (v) the fraction of the open-chain form is larger in the F type than in the G type. Points i−v can be explained by the hydration effect as in the cases of the monosaccharides studied previously.7 The empirical rule previously found is that the hydration effect on the monosaccharides depends on the difference between the number of equatorial or pseudoequatorial OH groups and that of axial or pseudoaxial OH groups (orientation effect). Let us see to what extent the experimental findings described above are rationalized theoretically in terms of the electronic energy and hydration free energy. The hydration effect on the isomer distribution for the disaccharides can be understood from
2/2
G6‐β −1/2G6‐α ≈
2/2
G6‐β −1/2G6‐β
>
2/2
G6‐β −1/2Gopen‐chain
(6)
which also agrees with the experimental result, eq 1b. On the contrary, the stability orders for D-maltose and D-cellobiose calculated in vacuum disagree with those given by eqs 1a and 1b, respectively.42,43 This is essentially due to the hydration effect. The stability reversal is observed also for the F-type form. For D-maltose, the computed stability order in PCM water is 2/2
G6‐α−1/2 F5‐β ≈
>
2/2
G6‐α−1/2 F6‐β >
2/2
G6‐α−1/2 F6‐α >
2/2
G6‐α−1/2 F5‐α
2/2
G6‐α−1/2 Fopen‐chain
(7)
which is comparable to the experimental result in eq 2a. For Dcellobiose, the computed stability order in PCM water is 2/2
G6‐β −1/2 F5‐β >
≈ 10042
2/2
2/2
G6‐β −1/2 F6‐β ≈
G6‐β −1/2 F6‐α >
2/2
2/2
G6‐β −1/2 F5‐α
G6‐β −1/2 Fopen‐chain
(8)
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Similarly, this is in agreement with the experimental result expressed as eq 2b.44 3.2. 13C Spectra during Hydrolysis. Now we discuss the hydrolysis reaction with treating isomers distinctly. To reveal the pathways and mechanisms for the hydrothermolyses of maltoand cello-oligosaccharides, it is essential to clarify which form of G or F is observed as the dominant monomer fragment and see how the number and type of 1,4-glycosidic bonds affect the product distribution. We spectroscopically distinguish and quantify the reactant and hydrolyzed products including the G- and F-type forms as functions of reaction time during hydrolysis. 3.2.1. D-Maltose. Let us first show what species are observed during the hydrothermolysis of the disaccharide, D-maltose. Figures 4a and 4b illustrate the expanded in situ 13C spectra after reactions for 0.5 and 40 h, respectively, at 120 °C in dilute 0.5 M solution in the chemical-shift range corresponding to the anomeric carbons of the G (C1) and the F (C2) units. At the initial reaction stage (0.5 h), there are detected 4 peaks that come from the C1 of the 1/2G and 2/2G units in the G-type D-maltose; see Figure 4a. The hydrothermal transformation from the 1/2G unit into 1/2F was not directly observed at 0.5 h. The situation gradually varies with time. At 40 h reaction time, as seen in Figure 4b, the total number of the newly observed peaks except for those derived from the initial 2/2G−1/2G type is 5; 2 medium peaks (pyranoses of α- and β-types) come from the C1 of the 2/2G monomer generated,45 2 weak peaks (furanose and pyranose of β-type) from the C2 of the 1/2F monomer are generated, and 1 weak one from the C2 of the 1/2F unit in the 2/2G−1/2F type is generated. Noticeably, only the residual 2/2G and the newly born 1/2 F monomers are produced with the mass balance exactly kept. In view of these spectral observations before and during hydrolysis, the scheme for the D-maltose hydrolysis can be established as in Figure 3. After the transformation of the terminal 1/2G unit into 1/2F, the glycosidic bond scission is exclusively initiated. The unit transformation from 1/2G to 1/2F can be understood according to the hydrothermal keto−enol tautomerization between the open-chain forms that are observed by the in situ 13C NMR at the high concentration mentioned in section 3.1. The open-chain and ring forms of the 2/2G−1/2G type are in rapid equilibrium with each other (thick arrows), and the situation is the same as that for the case of the 2/2G−1/2F type. The time dependence of the 13C spectrum will be discussed later in a quantitative manner, and the reaction scheme in Figure 3 will be validated on the basis of the time evolution explored (see section 3.3.1). 3.2.2. D-Maltotriose and D-Maltotetraose. How do the product and isomer distributions depend on the degree of oligomerization? Application of the in situ 13C NMR spectroscopy enabled us to distinguishably assign the location of the G unit in the higher-oligomerized D-maltotriose and D-maltotetraose, as seen in Figures 4c and 4d, respectively. When the 13C spectra in Figures 4b, 4c, and 4d are compared, the following features of the product and isomer distributions are the same as in the cases of the disaccharides: (i) the populations of the G-type isomers and (ii) generation of the F monomer and carbohydrate counterpart. The F types, 3/3G−2/3G−1/3F for D-maltotriose and 4/4 G−3/4G−2/4G−1/4F for D-maltotetraose, are not detected as a result of the drastic population and/or solubility drop due to the presence of a series of glycosidic bonds: cf., 2/2G−1/2F for D-maltose. The similar product distribution among the maltooligosaccharides (di-, tri-, tetraose) shows that the pathways and mechanisms for hydrolysis are independent of the degree of oligomerization, as shown below in detail.
Figure 4. 13C spectral evidence for the hydrolyzed products in the hydrothermolyses of the malto-oligosaccharides, (a and b) D-maltose, (c) D-maltotriose, and (d) D-maltotetraose at a concentration of 0.5 M. Spectrum (a) was obtained at the initial condition with no reactions proceeding, and the other spectra (b, c, and d) were obtained after reacting for 40 h at 120 °C when all of the hydrolyzed products are identified. Peaks derived from the 1G (1/2G, 1/3G, and 1/4G), 2G (2/2G, 2/3G, and 2/4G), 3G (3/3G and 3/4G), and 4G (4/4G) units were assigned by considering the upfield shift caused by glycosidic bond formation. Anomers of α and β types are distinguishable by their populations in view of the population difference in the case of the monosaccharide, G; see ref 7.
3.2.3. D-Cellobiose, D-Cellotriose, and D-Cellotetraose. The reactivity of carbohydrates depends on the type of glycosidic bonds (α and β).46 Here let us examine how the product distribution involved in hydrolysis varies with the type of glycosidic bonds. Figure 5 shows in situ 13C spectra for the β-1,4-linked cello-oligosaccharides after reaction for 72 h at 120 °C. When the 13 C spectra in Figures 5a, 5b, and 5c are compared to the corresponding spectra for the malto-oligosaccharides in Figures 4b, 4c, and 4d, the spectral characteristics are essentially common except that the anomeric carbon peaks of the cello-oligosaccharides emerge at a lower magnetic field than of the 10043
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the 1,4-glycosidic bonds occur. For the monosaccharide G, we have shown that the isomer fractions were constant through the reaction as well and treated the isomers as a set in the kinetic analysis.7 Indeed, according to the polarographic method applied for the monosaccharide G, the rate constants for isomerization between the open-chain and the cyclic pyranoses were found to be ∼10−3 s−1 at 25 °C,25,47 which are much larger (by 106−107) than those for the forward and backward hydrothermal transformations, G ⇄ F. This demonstrates the validity of the collective treatment of the isomers for each monosaccharide in the kinetic analysis of the slower hydrothermolysis. In view of these results, for the oligosaccharides, the rates for isomerization between the open-chain and the ring forms are expected to be much faster than those for the transformation from the G-type form to the F-type and for hydrolyses of both the α- and the β-1,4-glycosidic bonds. Collective treatment of the isomers can be conducted also in the kinetic analysis of the oligosaccharide hydrolysis described below. As for the temperature effect, the equilibrium fractions of the isomers for each oligosaccharide vary slightly with increasing temperature (see Figure SI-1, Supporting Information). Thus, the temperature effect on the isomer fractions is considerably small. The same tendency was observed for the G monomer.7 As noted in section 3.2.1, in the following we adopt a convention that the unit of the hydrolyzed oligosaccharides is not renumbered. 3.3.1. Time Evolution. We show the time evolution of the concentrations of the products involved in the malto- and cellooligosaccharide hydrolyses in Figure 8 and the time dependence of the consumption of the reactant oligosaccharides in Figure 9. Let us first see how the hydrolyzed products are evolved with time at 120 °C.48 For the disaccharides, D-maltose and 2/2 1/2 D-cellobiose ( G− G, seen in Figure 8a), it is found that the F-type form, the 2/2G−1/2F dimer, is generated in addition to the hydrolyzed 2/2G and 1/2F monomers at the early reaction stage (∼1 and ∼3 h, respectively). After that, the concentration of the 2/2G−1/2F dimer reaches a stationary state with the carbon mass balance kept. The same aspects of the time dependence of the F-type form have been observed for the hydrothermal transformation from the monosaccharide G to F.7 The stationary concentration of 2/2G−1/2F demonstrates the reversibility of the transformation between the G- and the F-type forms, 2/2G−1/2G ⇄ 2/2 G−1/2F. This is parallel to the reversibility of the transformation between the monosaccharides, G ⇄ F.7,36−39 The concentration ratio of the F type (2/2G−1/2F) to the G type (2/2G−1/2G) is, however, found for both the α- and the β-1, 4-linked disaccharides to be an order of magnitude smaller than that of the monomeric F to G. This can arise from the steric hindrance due to the presence of glycosidic bond in the oligosaccharides. Hydrolyses of the disaccharides of D-maltose and 2/2 D-cellobiose give rise to the monomeric G and 1/2F in equal amounts until ∼3 and ∼12 h, respectively. These findings clearly indicate that the scission of the glycosidic bond takes place after the transformation from 2/2G−1/2G to 2/2G−1/2F. After that (>∼3 and >∼12 h, respectively, for the α- and β-bond), the concentration ratio of G to F becomes larger than unity. This is due to the transformation of F to G as shown in a previous paper.7 The hydrolysis rate of the glycosidic bond is faster than the reversible transformation of the monomeric G ⇄ F, which leads to the additional increment of the G monomer; correspondingly, the concentration of the F monomer approaches the nearly stationary state (∼0.1) at a longer time; see Supporting Information (Figure SI-2a).
Figure 5. 13C Spectral evidence for the products involved in hydrolyses of the cello-oligosaccharides: (a) D-cellobiose (0.5 M), (b) D-cellotriose (0.1 M), and (c) D-cellotetraose (0.02 M). These were all examined at 120 °C and obtained after reacting for 72 h. Only in the case of D-cellotetraose, the anomeric carbon peaks derived from the G units in the oligomers (di-, tri-, and tetraose) involved are degenerated at 103−104 ppm; in contrast, for D-maltotetraose shown in Figure 4d, the peaks derived from the hydrolyzed oligosaccharides are well resolved at higher magnetic field (100−101 ppm). This is because for the β-1,4-linked cello-oligosaccharides the configuration is more or less linearly stretched and the steric environments of the G units are similar to each other; on the contrary, the α-1,4-linked malto-oligosaccharides can introduce some helical structure with the site-specificity possessed.
malto-oligosaccharides; the α-1,4-linked malto-oligosaccharides have larger steric and crowding effects than do the β-1,4-linked cello-oligosaccharides. 3.3. Pathways and Mechanisms. Here we establish the pathways and mechanisms for oligosaccharide hydrolysis by comparing the hydrolyses of malto- and cello-oligosaccharides. The reaction scheme depicted in Figure 6 will be confirmed from the time evolution of the concentrations of the reactive species involved. It will turn out that the terminal 1G (1/2G, 1/3G, and 1/4 G) unit is selectively eliminated after transformation to the 1F 1/2 ( F, 1/3F, and 1/4F) unit, irrespective of the degree of oligomerization and type of 1,4-glycosidic bond. Before going to the kinetic analysis, we need to confirm how the equilibrium fractions of the isomers for each oligosaccharide depend on the reaction time and temperature. As depicted in Figure 7, the isomer fractions for the malto- and cellooligosaccharides are found to be identical to each other and constant all over the reaction time even when the hydrolyses of 10044
dx.doi.org/10.1021/jp3034165 | J. Phys. Chem. A 2012, 116, 10039−10049
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Figure 6. Reaction pathways of hydrothermolyses of the malto- and cello-oligosaccharides. k’s are the first-order rate constants for the corresponding reaction pathways shown by the arrows; for the notation of the symbols see the text (section 3.3.2).
To validate the selective scission of the 1,4-glycosidic bond through the transformation to the F-type mentioned just above, hydrolyses of trisaccharides and tetrasaccharides are of essence because the G and F monomers eliminated can be distinguished from the residual fragment, in contrast to the case of the disaccharides. By exploring the hydrolyses of the trisaccharides, 3/3 2/3 1/3 D-maltotriose and D-cellotriose ( G− G− G), we can clearly show which is dominant as the first monomer fragment, G or F, and which terminal glycosidic bond is to be hydrolyzed. As seen in Figure 8b, only the 1/3F monomer and the 3/3G−2/3G dimer are generated in equal amounts at the early stage of reaction (≤2 and ≤6 h, respectively), until which the 1/3G monomer is absent despite the larger population of the 3/3G−2/3G−1/3G trimer before hydrolysis. The absence of the 1/3G monomer is crucial to validate the selective glycosidic bond scission after the transformation to the F type. If the terminal 1/3G unit were hydrolyzed without the transformation from 3/3G−2/3G−1/3G to 3/3 G−2/3G−1/3F, equal amounts of the 1/3G monomer and the 3/3 G−2/3G dimer must be observed. However, this possibility is negated in view of the absence of the G monomer at the early stage.
Figure 7. Time dependence of the isomer populations of the maltoand cello-oligosaccharides. Populations of the G- and F-type forms are given along the left- and right-hand axes, respectively. Nomenclature and reaction times shown in parentheses are of the cello-oligosaccharides. Since there are small differences ( 2/2G6‑α−1/2Gopen‑chain (34 kJ mol−1), where the parenthesized numbers are the free energy difference from the most stable form, the symbol ≈ means a smaller difference than the thermal energy (RT, 3.3 kJ mol−1 at 120 °C), and the symbol > means a larger difference than RT. For the G-type D-cellobiose, the computed stability orders in vacuum and PCM water are, respectively, 2/2G6‑β−1/2G6‑α > 2/2G6‑β−1/2G6‑β (7 kJ mol−1) > 2/2 G 6‑β − 1/2 G open‑chain (55 kJ mol −1 ), and 2/2 G 6‑β − 1/2 G 6‑α ≈ 2/2 G6‑β−1/2G6‑β (2 kJ mol−1) > 2/2G6‑β−1/2Gopen‑chain (43 kJ mol−1). The difference between 2/2G6‑α−1/2G6‑α and 2/2G6‑α−1/2G6‑β or between 2/2 G6‑β−1/2G6‑α and 2/2G6‑β−1/2G6‑β is smaller in PCM water than in vacuum, but the reverse relationship shown in eqs 1a and 1b cannot be reproduced. The computed values show that the species stability for the disaccharides depends on hydration, as in the cases of the monosaccharides.7 Although the improved force field is developed, all-atom free energy calculation is not yet done for these species found here; see ref 43. (43) Hansen, H. S; Hünenberger, P. H. J. Comput. Chem. 2011, 32, 998−1032. (44) The stability orders of the F-type isomers of D-maltose in vacuum and PCM water are, respectively, computed as 2/2G6‑α−1/2F5‑β ≈ 2/2 G6‑α−1/2F5‑α (3 kJ mol−1) > 2/2G6‑α−1/2F6‑β (16 kJ mol−1) ≈ 2/2 G6‑α−1/2F6‑α (18 kJ mol−1) > 2/2G6‑α−1/2Fopen‑chain (42 kJ mol−1), and 2/2G6‑α−1/2F5‑β ≈ 2/2G6‑α−1/2F6‑β (1 kJ mol−1) > 2/2G6‑α−1/2F5‑α (11 kJ mol−1) > 2/2G6‑α−1/2F6‑α (15 kJ mol−1) > 2/2G6‑α−1/2Fopen‑chain (21 kJ mol−1), where the numbers in parentheses are the free energy differences from the most stable form. For the F-type D-cellobiose, the stability orders in vacuum and PCM water are, respectively, computed as 2/2 G6‑β−1/2F5‑β ≈ 2/2G6‑β−1/2F6‑β (3 kJ mol−1) > 2/2G6‑β−1/2F5‑α (8 kJ mol−1) ≈ 2/2G6‑β−1/2F6‑α (19 kJ mol−1) > 2/2G6‑β−1/2Fopen‑chain (32 kJ mol−1), and 2/2G6‑β−1/2F5‑β > 2/2G6‑β−1/2F6‑β (6 kJ mol−1) ≈ 2/2 G6‑β−1/2F5‑α (7 kJ mol−1) ≈ 2/2G6‑β−1/2F6‑α (8 kJ mol−1) > 2/2 G6‑β−1/2Fopen‑chain (15 kJ mol−1). The stability orders in PCM water are reversed by hydration and correspond to the experimental results expressed as eqs 2a and 2b. (45) The two medium peaks are derived not only from the 2/2G monomer but also from the 1/2G monomer except at the early stage of reaction. This is because the hydrolyzed 1/2F monomer is hydrothermally transformed into the 1/2G monomer with increasing time. However, the key to understanding the mechanisms for glycosidic bond scission is essentially expressed as 2/2G−1/2G ⇄ 2/2G−1/2F → 2/2G + 1/2 F. In view of this, therefore, these peaks were expressed as 2/2G only, not both 2/2G and 1/2G. (46) Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry, 5th ed.; W. H. Freeman and Co.: New York, 2002; Chapter 11. (47) For the oligosaccharides, there is no information on the isomerization rates between the open-chain and the ring forms. (48) In the reaction of D -cellotetraose at 120 °C, the 4/4 G−3/4G−2/4G−1/4G tetramer and the 4/4G−3/4G−2/4G trimer can 10049
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