Structural Dynamics of N-Propionyl-d-glucosamine Probed by Infrared

Article pubs.acs.org/JPCA

Structural Dynamics of N‑Propionyl‑D‑glucosamine Probed by Infrared Spectroscopies and Ab Initio Computations Chen Han, Juan Zhao, Fan Yang,* and Jianping Wang* Beijing National Laboratory for Molecular Sciences, Molecular Reaction Dynamics Laboratory, Institute of Chemistry, Chinese Academy of Science, Beijing 100190, People’s Republic of China S Supporting Information *

ABSTRACT: N-Acylglucosamine is an important component in many oligosaccharides in eukaryotes, where it plays a very important biological role. Located between a glucose ring and an alkyl group of such species is an amide unit (−CONH−), which exhibits an infrared absorption band, mainly due to the CO stretching, in the region of 1600−1700 cm−1, similar to the amide-I band found in polypeptides. In this work, vibrational properties of such an “amide-I mode” in Npropionyl-D-glucosamine (GlcNPr) are examined in three typical solvents (water, methanol, and dimethylsulfoxide) by using steady-state infrared and femtosecond infrared dispersed pump−probe spectroscopies. As a result of solute−solvent interactions, multiple structured GlcNPr−solvent clusters are formed in water and methanol but are unlikely in dimethylsulfoxide. The vibrational relaxation rate of the amide-I mode is slightly frequency-dependent, supporting the presence of multiple solvated structures. Further, the amide-I lifetime is significantly shorter in GlcNPr than that in a well-known monopeptide, N-methylacetamide, which can be attributed to the presence of additional downstream vibrational modes caused by the sugar unit. Ab initio molecular dynamics simulations are used to reveal microscopic details of the first solvation shell of GlcNPr. Our results demonstrate that the amide-I mode in glucosamine exhibits both structural and solvent sensitivities that can be used to characterize the three-dimensional arrangement of sugar residues and their structural dynamics in glycopeptides.

1. INTRODUCTION Glycoproteins are known to play crucial roles in biological processes such as protein folding, signal transuding, and molecular recognition via the interplay between peptidic and saccharidic units.1−7 Because structural changes can occur on various time scales during these processes, it is of great importance to monitor the structural dynamics of glycoproteins in real time. Unfortunately, due to structural diversity that is far more than that for proteins and nucleic acids, it remains a challenge to effectively characterize the structures and structural dynamics of glycoproteins. A common strategy is to examine partial structures of glycoproteins containing key amino acid and sugar residues. This simplification is justified because it has become clear that only a portion of glycoproteins plays a key role in relevant biological functions.2,3 Novel glycopeptides have been synthesized to show an ordered structure having sugar residues periodically arranged along the peptide backbone,8 while well-defined conformations in the N-glycoprotein linkage region have also been observed.9−13 In naturally occurring and synthetic glycopeptides, Nacetylglucosamine is a very common sugar residue, which also often exists in gluxolipids, lipopolysaccharides, and mureins of cell walls.1,9,14 Such a glucose derivative can be generally termed as N-acylglucosamine. A remarkable chemical group presented in glucosamine residues is a trans-amide unit © 2013 American Chemical Society

(−CONH−), which can be distributed in certain fashion in glycopeptides. Thus, this provides a structural probe to characterize the three-dimensional arrangement of sugar residues and their structural dynamics and distributions in glycopeptides. Analogous to what is often seen in peptides, the trans-amide unit in glucosamine also shows a linear infrared (i.e., FTIR) absorption band in the frequency region of 1600−1700 cm−1, which is an amide-I band that mainly involves vibrational excitation of the CO bond stretching. Even though infrared spectroscopy has been recognized as a useful tool to examine glycoprotein structures for quite some time,15−17 the infrared investigation specifically on such an amide-I chromophore of glucosamine is still missing. In other words, the information of vibrational properties of such an amide-I mode in glucosamine still remains unknown. The very first evidence that the amide-I mode could work as a structural marker would be the observation of its frequency sensitivity to the nearby chemical environment, solvent surroundings, as well as hydrogen bonding interactions. Structural sensitivities of such have Special Issue: Prof. John C. Wright Festschrift Received: January 3, 2013 Revised: April 11, 2013 Published: April 30, 2013 6105

dx.doi.org/10.1021/jp400096a | J. Phys. Chem. A 2013, 117, 6105−6115

The Journal of Physical Chemistry A

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been demonstrated in peptides and proteins.18−20 Presumably, the amide linkage in sugar side chains may also serve as a vibrational probe for the local structure and local environment of glycoproteins. The linear IR absorption band of the amide-I mode in condensed phases is often inhomogeneously broadened even in a single amide group containing a peptidal compound, Nmethylacetamide (NMA).21 Such a broadened line shape contains very important information about equilibrium structural distributions and solute−solvent interactions. However, such information is not directly extractable from a linear IR spectrum. Moreover, multiple solute−solvent complexes are often formed through intermolecular hydrogen bonds so as to cause complicated structural dynamics even in the single amideI chromophore case.21−24 For glycopeptides, multiple structural configurations in both peptidic and saccharidic regions are highly possible. Furthermore, because of multiple solventaccessible sites, multiple solvation structures are also possible. These structures may have their amide-I bands overlapped substantially in the spectral window of less than a 100 cm−1, thus complicating the IR spectra structure assignment. Femtosecond (fs) mid-IR pump−probe spectroscopy, on the other hand, is able to provide information about ultrafast structures and dynamics of molecules in condensed phases because of its time- and frequency-resolving powers. By bringing molecules to vibrational excited states, one can follow vibrational energy flows and obtain relevant population relaxation dynamics. Because molecular vibration is sensitive to both intra- and intermolecular interactions, the IR pump− probe method is naturally an effective tool for examining ultrafast structural dynamics. In a typical IR pump−probe experiment, a set of vibrational states is perturbed by an intense broad-band pump pulse so as to cause an excitation from the vibrational ground state (ν = 0) to its first excited state (ν = 1). This yields a fundamental transition (bleaching of the ground state plus the stimulated emission of the first excited state) and an induced absorption at the frequency of the excited state (ν = 1 → 2). The absorption frequency is usually red-shifted with respect to the fundamental transition frequency due to vibrational anharmonicity. The broad-band pump can efficiently excite molecules with a set of vibrational transition dipoles preferentially aligned along the polarization axis of the pump pulse, which leads to an anisotropic distribution of the excited molecules. After a certain delay time, a weak probe pulse is introduced to detect the perturbed vibrational states. The polarization of the probe pulse can be parallel or perpendicular or have a magic angle (54.7°) to that of the pump pulse. These polarized pump−probe signals can be used to reveal the relaxation pathways of vibrationally excited solute molecules. Further, by probing at selected frequencies, it is also possible to dynamically (and structurally) discern different molecular species. Employing the amide-I mode as an intrinsic conformational probe, recent nonlinear IR experiments have gained new insight into the structures and their fluctuations and conformational transitions of peptides in condensed phases.25 As mentioned above, a chemically structurally simple but dynamically quite intriguing example of such is a model monopeptide compound containing a single trans-amide unit, that is, NMA. The structural and vibrational dynamics of NMA and its NH deuterated form (dNMA) in different solvents have been examined recently by using experimental and simulated nonlinear IR spectroscopic methods.21−23,26−31 These inves-

tigations showed that the amide-I vibrational population of NMA exhibited a solvent-dependent biphasic relaxation, with time constants ranging from hundreds of fs to a few picoseconds (ps). In the present work, we examine the amide-I vibrational dynamics of N-propionyl-D- glucosamine (GlcNPr or 2propionylamino-2-deoxy-D-glucose, Scheme 1) in deuterated Scheme 1. Structure of GlcNPr

water (D2O), deuterated methanol (CH3OD), and dimethylsulfoxide (DMSO). GlcNPr is a very interesting molecule because it contains the trans-amide unit that resembles that in NMA, but it also contains larger chemical groups (glucose and ethyl) that differ from NMA and it thus structurally more complicated. A thorough study of the amide-I vibrational dynamics in GlcNPr would help us to understand the structural and environmental sensitivities of the amide group of the glucosamine side chain in glycopeptides, which is the major motivation of the present study. Steady-state IR and transient IR pump−probe spectroscopic methods in combination with quantum chemical computations are used to examine the influences of solvent molecules on the amide-I vibrational relaxations, as well as those on the structural dynamics of GlcNPr. Anharmonic computations are carried out to understand the amide-I vibrational relaxation channels. Ab initio molecular dynamics (MD) simulations are performed to gain more insights into solvation dynamics and to elucidate local hydrogen bonding structures of GlcNPr in explicit solvent on the ultrafast time scale.

2. MATERIALS AND METHODS 2.1. Material and Steady-State Infrared Spectroscopy. N-propionyl-D-glucosamine (purity ≥95%, TCI) was lyophilized three times in D2O for NH/ND exchange and then dissolved in D2O, CH3OD, and DMSO, with a final concentration of ∼30 mM in each case. Note that there are five exchangeable hydrogen atoms in GlcNPr (Scheme 1); however, for simplification, the NH/ND exchanging product is still referred to as GlcNPr throughout the text. IR spectra were measured using a Nicolet 6700 spectrometer equipped with a -nitrogen-cooled mercury−cadmium−telluride (MCT) detector. The IR measurement was carried out at room temperature with a spectral resolution of 1 cm−1. Samples were placed in homemade CaF2 IR cell with a 50 μm thick Teflon spacer. Dry air was used to purge the FTIR spectrometer during the entire IR experiments. 2.2. Infrared Pump−Probe Spectroscopy. An ultrafast 800 nm, 3 mJ, 1 kHz laser system (Spitfire Pro, SpectraPhysics) pumps an optical parametric amplifier (OPA800C, Spectra-Physics) and produces mid-IR pulses with an energy of ∼4 μJ and a pulse width of ∼60 fs. The OPA was tuned to be 6106



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resonant with the amide-I transition of GlcNPr (∼6 μm). The spectral bandwidth of the IR laser pulse (∼230 cm−1) spanned the fundamental (ν = 0 → 1) and overtone (ν = 1 → 2) transitions of the amide-I mode. Polarization-controlled pump−probe measurements were carried out using wire-grid IR polarizers. The pump and probe beam energies were set to 1 μJ and 50 nJ, respectively, using a combination of an IR polarizer and λ/2 wave plate made of MgF2. The pump pulses were chopped at 500 Hz. The pump−probe pulse polarization angle was set to 54.7 (magic angle) or 0° (parallel). The transmitted probe pulse was dispersed in an IR monochromator equipped with a liquid-nitrogen-cooled 64-element array MCT detector (Infrared Associates). Data acquisition and analysis were carried out using a home-written LabView program. 2.3. Ab Initio Molecular Dynamics Simulations. Ab initio MD simulations using Car−Parrinello simulation protorol32 were performed on GlcNPr systems solvated in H2O, DMSO, and CH3OH. Density functional theory (DFT) with the Becke, Lee, Yang, and Parr (BLYP) gradient-corrected functional33,34 for the exchange and correlation terms was used. The plane wave basis set was used with a kinetic energy cutoff of 70 Ry restricted to the Γ point of the Brillouin zone, and Martins−Trouillier pseudopotentials35 was also used. The core−valence interaction of C, N, and O was treated by s and p potentials with pseudization radii of 1.23, 1.12, and 1.05 au, respectively (taking the same radius for s and p), while H atoms were treated as an s potential with a 0.5 au radius. Energy expectations were calculated in reciprocal space using the Kleinman−Bylander transformation.36 The simulation systems were composed of a single GlcNPr molecule surrounded by 23 H2O, 4 DMSO, and 7 CH3OH molecules. The side lengths of the simulation boxes were set to 11.6, 10.9, and 9.6 Å of H2O, 13.6, 13.6, and 8.4 Å of DMSO, and 10.7, 11.4, and 9.4 Å of CH3OH. No H/D exchange in GlcNPr or solvents was considered. The MD simulations were performed at a constant pressure of 1.01325 bar (under an atmosphere) using a fictitious electron mass of 500 au, a time step of 5 au (∼0.12 fs), and periodic boundary conditions. The MD simulations consisted of three steps, wave function optimization, equilibration, and MD production. The equilibration phase was set to be 240 fs with a control of temperature through velocity rescaling. The MD simulations were performed with separate Nosé− Hoover chain thermostats37−39 for the ionic and electronic degrees of freedom used for better temperature control. The average ionic temperatures were 300 K. In the present study, due to computational expense, only a 6 ps MD trajectory was obtained for the three selected solute−solvent systems. This provided a very good starting point to evaluate the dynamics of the first solvation layer for GlcNPr at the quantum mechanical level. Structural dynamics including a local hydrogen bonding interaction between the solute and solvent could be examined. On the other hand, one may use the classical MD simulations for a longer-time sampling of the structural dynamics at the level of molecular mechanics; however, this requires a proper set of empirical force fields to be developed and tested for GlcNPr. 2.4. Quantum Chemical Computations. Molecular structural optimization and anharmonic normal-mode frequency calculations for GlcNPr in various solvents were carried out for each GlcNPr−solvent (D2O, DMSO, or CH3OD) cluster for explicit solvation, additionally using the standard integral equation formalism version of the polarizable continuum (IEF-PCM)40 for implicit solvation. Here, all of

the polar H atoms in GlcNPr and in NMA were replaced by the D atoms in order to compare with the experiments. Calculations on a single GlcNPr molecule and a NMA molecule in the gas phase were also performed in comparison. The computations were carried out at the level of B3LYP/631+G** using Gaussian 09.41 The potential energy distribution (PED) analysis, which can be used to characterize the degree of vibrational delocalization, was carried out for the amide-I mode on the basis of internal coordinates using the vibrational energy distribution analysis (VEDA) program.42

3. RESULTS AND DISCUSSIONS 3.1. Steady-State IR Absorption Spectra. The IR absorption spectra of GlcNPr in D2O, CH3OD, and DMSO in the amide-I region are shown in Figure 1. In each solvent, the

Figure 1. (A) IR spectra of GlcNPr in the amide-I region in D2O, CH3OD, and DMSO. (B−D) Fitting with Gaussian functions (red lines for sum and blue lines for its components) is also shown in each case.

IR spectrum has its characteristic peak position, line shape, and full width at half-maximum (fwhm). In D2O and DMSO, the amide-I band shows apparently a single peak, with the central frequency found to be at 1626.2 cm−1 (in D2O) and 1656.0 cm−1 (in DMSO) and the fwhm determined to be 39.0 cm−1 for the former and 25.3 cm−1 for the latter. Here, a large red shift from DMSO to D2O indicates a stronger hydrogen bonding interaction between the amide unit and water. However, in D2O, the line shape of the amide-I band is broad and slightly asymmetric, with a shoulder showing on the low-frequency side, suggesting that there may be two or more GlcNPr structures. The amide-I spectrum of GlcNPr in CH3OD, sitting between those in D2O and DMSO, on the other hand, shows a clear doublet feature in which a weaker band appears on the lower-frequency side, also suggesting the presence of more than one structured GlcNPr species. To characterize possible structured species in each case, curve fitting the IR spectra of GlcNPr in these solvents was 6107



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Table 1. Fitting Parameters of the Amide-I Spectra of GlcNPr in Three Solvents, including the Peak Position (νmax, cm−1), fwhm (Γ, cm−1), and Relative Weight (w) of the Integrated Peak Area D2 O

CH3OD

DMSO

component

νmax

Γ

w

νmax

Γ

w

νmax

Γ

w

a b c

1609.6 1629.0 1652.9

30.0 28.8 26.1

0.30 0.63 0.07

1622.6 1647.2

28.7 26.7

0.37 0.63

1656.2

25.0

1.00

Figure 2. Magic-angle dispersed pump−probe spectra of GlcNPr as a function of delay time (upper) in D2O (A), CH3OD (B), and DMSO (C). The negative and positive peak positions and those at roughly half height are marked in the zero-time spectra (lower part of each panel).

carried out, and results are given in Figure 1B−D, with fitting parameters listed in Table 1. In D2O and CH3OD, the spectra can be fitted with three and two significant amide-I components, respectively (labeled as component a, b, and c), while in DMSO, one Gaussian seems to fit the IR spectrum reasonably. Here, D2O and CH3OD can serve as either a hydrogen-bond donor or acceptor, but DMSO can only act as a hydrogen-bond acceptor. Because only when the hydrogen bonding interaction occurs on the CO group can it significantly red shift the amide-I frequency, component c in D2O and component a in DMSO can be tentatively assigned as weakly bonded GlcNPr species, while component b in D2O and in CH3OD can be assigned as GlcNPr species with CO group hydrogen-bonded to one solvent molecule, and component a in D2O and in CH3OD can be assigned to a more completely solvated species. In the case of CH3OD, there is also a very weak high-frequency component that can be assigned to “free” GlcNPr. This picture of multiple solvated GlcNPr structures seems to be consistent with computational results shown in later sections of this work. However, it should be pointed out that even though fitting by Gaussian functions is

a well-accepted way to decompose the IR spectra, recent study also suggested that non-Gaussian dynamics could be true for the amide-I mode in certain cases.43 Further, the difference in these IR absorption line shapes suggests that the size and chemical properties of solvent molecule play very important roles in solvation. In addition, these IR line shape do not show concentration dependence between 5 to 30 mM of GlcNPr (data not shown), suggesting insignificant intermolecular aggregations. 3.2. Pump−Probe Spectra. Magic-angle dispersed pump− probe spectra of the GlcNPr amide-I band are shown in Figure 2. The transient spectra at zero delay time are also given in each case. Negative peaks (blue) are due to the fundamental transitions (ν = 0 → 1) that contain the ground-state bleaching plus the stimulated emission of the first excited state, while positive peaks (red) are due to the overtone transitions (ν = 1 → 2) that contain only the first excited-state absorption. The overtone transition peak shifts toward the low-frequency side due to anharmonicity. The dispersed pump−probe signal decays as a function of delay time, which decays almost completely on the time scale of ∼3 ps, and the ν = 1 → 2 signal 6108



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the lower-/higher-frequency side), with fitting parameters and assignments summarized in Table 2

decays somewhat faster. In D2O, at short delay time, negative and positive signals are peaked at 1628.0 and 1594.3 cm−1, respectively, with a peak separation of 33.7 cm−1. Such a peak separation is caused by the anharmonicity but usually appears to be larger than the true anharmonicity because of the finite spectral bandwidth, while in this case, it can also be influenced by the presence of more than one structured species. In CH3OD, because of a doublet IR absorption feature, there are two negative peaks and two positive peaks overlapping in the 1550−1700 cm−1 region. This causes a partial signal cancellation between the ν = 1 → 2 transition of component b (the high-frequency component) and the ν = 0 → 1 transition of component a (the low-frequency component). Nevertheless, the negative and positive features are still clearly seen in the pump−probe signal, with one broad negative peak and one broad positive peak located at ∼1648.9 and 1602.1 cm−1, respectively. In DMSO, the negative and positive signals are peaked at 1661.0 and 1630.8 cm−1, respectively, with a frequency separation of 30.2 cm−1. Fitting zero-time pump− probe signal yields the anharmonicity of ∼19.9 cm−1 in this case. This value is typical for the CO stretching dominating the vibrational mode, however, being somewhat larger than that found previously for dNMA in DMSO (e.g., 1621 and 12 cm−126). Fixed-frequency dynamics from the magic-angle pump− probe signals of GlcNPr in three solvents detected at selected probe frequencies, shown in the lower part of Figure 2, are given in Figure 3. The data are averaged over 3 cm−1 and

ΔAbs(t ) = α1e−t / τ1 + α2e−t / τ2

(1)

Here, α1 and α2 are amplitudes of two vibrational population relaxation processes, while τ1 and τ2 are their time constants. The results show that the amide-I vibrational population relaxation in each case has a biphasic process with two characteristic time scales, a fast component with τ = 100−300 fs and a relatively slow component with τ ranging from 0.8 to 1.2 ps. This biphasic feature suggests that the vibrational relaxation is not a simple process, which will be discussed further in section 3.6. In D2O, the partially overlapped fundamental and overtone transitions from the three components hinder a clear measurement of single-component dynamics. However, by probing at 1577.2 cm−1 (Figure 3A, triangles), the vibrational dynamics of the ν = 1 → 2 transition for mainly component a can be obtained. It shows two time constants, 112 and 843 fs. Probing at the central positive peak position (1594.3 cm−1), shown in Figure 3A (circles), yields the vibrational population relaxation for both components a and b. The time constants are found to be 131 fs and 1.03 ps, respectively, with the latter being somewhat slower than that of component a, which could be due to the involvement of component b. To probe the ν = 0 → 1 vibrational dynamics, the central negative peak position (1628.0 cm−1), shown in Figure 3D (circles), is used. This frequency mainly contains contribution from component b. The relaxation time constants are found to be 212 fs and 1.03 ps. The relaxation time parameters at 1639.6 cm−1 that contain contributions from components b and c (Figure 3D, triangles) are found to be 209 fs and 1.21 ps, respectively. Therefore, the results demonstrate that the amide-I component at the lowfrequency side has a relatively faster vibrational relaxation than those at the high-frequency side, both for the overtone and fundamental transitions. As shown in Table 2, the situation of GlcNPr in CH3OD is quite similar to that in D2O. By probing at different frequencies, the time constant for slow relaxation is found to increase slightly from component a to component b. However, in the case of DMSO, the vibrational dynamics probed at several frequencies exhibit quite similar time constants, suggesting the presence of only one structural component. The vibrational relaxation pathways of the amide-I excitations in these systems will be discussed in section 3.6. In addition, experimental anisotropies obtained from the parallel and magic-angle pump− probe signals of GlcNPr at central negative peak positions in the three solvents show a relaxation time constant of roughly 3−4 ps in D2O and in CH3OD, and it is somewhat longer in DMSO (data not shown), suggesting solvent-dependent reorientational times. 3.3. Chemical Environment Effects on the Amide-I Mode. Comparing the structural aspects of GlcNPr and dNMA allows one to understand the chemical environment influence on the amide-I mode. The glucose ring and ethyl group in GlcNPr have stronger electron-donating ability and larger steric effect than the two methyl groups in dNMA. This increases the atomic partial charges for the C, O, N, and D atoms in the amide unit (−0.31 e in GlcNPr and −0.27 e in dNMA, general atomic polar tensor charges44) and changes the potential energy surface of the amide-I mode, thus red shifting the amide-I frequency. Indeed, anharmonic normal-mode calculations of gas-phase GlcNPr and dNMA yield the amide-I mode

Figure 3. Magic-angle pump−probe signals of the amide-I mode of GlcNPr in D2O (A,D), CH3OD (B,E), and DMSO (C,F). Probing frequencies (in cm−1) are marked. See the text for details.

centered at the labeled frequency. These magic-angle traces reveal the amide-I vibrational population relaxation dynamics in the absence of molecular reorientations. The probe frequencies are selected either on the positive (ν = 1 → 2 transition) or negative (ν = 0 → 1 transition) peak position or on the lower-/ higher-frequency side of these peaks but still having reasonable optical density change. The choice of these frequencies is based on the fitting result shown in Figure 1B−D, with the aim of examining whether these sub-bands have different dynamics. Each of the decays can be fitted with a double exponential function (red lines for central peaks and green lines for those at 6109



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Table 2. Biexponential Fitting Parameters for the Pump−Probe Measurements of the Amide-I Vibrational Relaxations of GlcNPr in Three Solvents solvent D2 O

transition

frequency (cm−1)

α1

τ1 (fs)

α2

τ2 (fs)

assignment

ν=1→2

1577.2 1594.3 1628.0 1639.6

0.47 0.56 0.35 0.34

0.53 0.44 0.65 0.66

0.24 0.31 0.16 0.14

1610.3 1630.8 1661.0 1676.5

0.51 0.52 0.36 0.34

843 ± 80 1025 ± 33 1030 ± 31 1210 ± 164 1027 918 ± 32 1076 ± 19 1095 ± 15 1182 ± 42 1068 1174 ± 51 1217 ± 49 1217 ± 19 1213 ± 72 1205

a a+b b b+c

1587.5 1602.1 1648.9 1658.2

112 ± 15 131 ± 3 212 ± 9 209 ± 16 166 147 ± 19 125 ± 5 141 ± 18 156 ± 27 143 133 ± 17 123 ± 8 163 ± 14 208 ± 38 157

ν=0→1

CH3OD

averagea ν=1→2 ν=0→1

DMSO

averagea ν=1→2 ν=0→1 averagea

a

0.76 0.69 0.84 0.86 0.49 0.48 0.64 0.66

a a+b a+b b a a a a

Average lifetime at four probing frequency positions.

anharmonic frequency (and anharmonicity) of 1702.1 cm−1 (19.3 cm−1) for the former and 1716.4 cm−1 (18.5 cm−1) for the latter. Thus, in the gas phase, the amide-I frequency of GlcNPr is about 14.5 cm−1 lower than that of dNMA. However, in the solution phase, the glucose ring and ethyl group can affect the solvation of the amide unit. These factors all play a role in determining the amide-I spectral features of the solvated GlcNPr. Further, our results show that there is no intramolecular hydrogen bonding interaction between the hydroxyl group on C5 or C9 of the glucose ring and the amide CO group (Scheme 1). The reason is simple that the amide unit is unlikely to form a hydrogen bond with a nearby hydroxyl group because a seven-membered ring structure is sterically unfavorable. It is evidently shown that in DMSO, the amide-I frequency of GlcNPr (1656.0 cm−1) is very close to the case of dNMA (1659.0 cm−1);21,23,28 otherwise, the former would be much red-shifted from the latter. 3.4. Solvent Influences at the Chemical Bond Level. Ab initio MD simulations were carried out to investigate local structural and hydrogen bonding interactions of GlcNPr in H2O, DMSO, and CH3OH. Snapshots of three GlcNPr− solvent clusters at 0.5 ps are given in Figure 4A. The MD simulations results suggest that the O atom of the amide unit in GlcNPr can form two hydrogen bonds with surrounding H2O molecules but only one hydrogen bond with the surrounding CH3OH molecule. For DMSO, there is no observable hydrogen bonding interaction on the O site of the amide unit because the H atoms in DMSO are nonpolar. On the other hand, the N−H group of the amide unit in GlcNPr can form one O···H hydrogen bond with all three solvent molecules. Thus, in the first solvation layer of GlcNPr, the amide group can form possibly on average three hydrogen bonds with D2O, two with CH3OD, and only one with DMSO. The site-to-site pair radial distribution function (RDF), usually denoted as g(r), is computed from the MD trajectories. Figure 5 shows RDFs between specific atom pairs of GlcNPr and solvents. Even though in each case the g(r) curves are not smooth, they all converge to 1 at larger r, showing a typical feature of the radial distribution function. The information on hydrogen bonding structures can be obtained from the RDF plots. For the case of [O(C)···H] in Figure 5A, the first peak

Figure 4. (A) GlcNPr−solvent clusters in D2O, CH3OD, and DMSO taken from the ab initio MD snapshots at 0.5 ps. (B) Additional structures of possible GlcNPr-D2O GlcNPr−CH3OD clusters. The amide-I vibrational properties of these structures were computed and are listed in Table 3.

appears at ∼1.6 Å in H2O and 1.7 Å in CH3OH, reflecting average hydrogen bond distances in the first solvation layer in these two cases. For the [O(C)···H] curve of DMSO, there is no obvious peak in the region of the first solvation shell, suggesting a less organized and non-hydrogen-bonded configuration. On the other hand, each [H(−N)···O] curve in Figure 5B shows a peak at ∼1.7 Å of H2O, CH3OH, and DMSO, indicating a similar hydrogen bond distance for the three cases. Because of multiple hydrogen bonding sites, GlcNPr may have more than one solvated species coexist in water and in methanol as well under equilibrium condition. These solute−solvent hydrogen bonding interactions, including [O(C)···H] and [H(−N)···O], are the primary source of amide-I frequency shifts and fluctuations. 3.5. Anharmonic Vibrations. To further characterize the solvent effect on the amide-I mode of GlcNPr, several solute− solvent clusters were selected. The GlcNPr−(D2O)3, GlcNPr− (CH3OD)2, and GlcNPr−DMSO clusters were directly 6110



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The PED values are also calculated and listed in the same table. For comparison, a similar calculation was also carried out for isolated GlcNPr molecule in the gas phase. As expected, upon solvation, the amide-I vibrational frequency in these solute−solvent clusters is red-shifted from the gas-phase value, and the amount of frequency shift is mainly determined by the hydrogen bonding interactions on the carbonyl oxygen. Accompanying the frequency red shift, the amide-I transition intensity is enhanced significantly. This is because the hydrogen bonding interactions can soften the anharmonic potential surface of the amide-I mode in GlcNPr, change its anharmonicity, and increase the transition dipole strength. The anharmonic frequency difference between GlcNPr− D···D2O and GlcNPr−(D2O)3 is predicted to be significant (41.6 cm−1), explaining the observed broad IR band feature in Figure 1. Here, the calculated frequencies of each GlcNPr− D2O cluster show an apparent one-to-one correspondence to the fitted IR peak components given in Figure 1B, suggesting different hydration situations for different GlcNPr−D2O clusters. For example, component a located at 1609.6 cm−1 can be assigned to the GlcNPr−(D2O)3 structure, with a calculated amide-I frequency at 1608.6 cm−1. Component b located at 1629.0 cm−1 can be assigned to the GlcNPr−(D2O)2 and GlcNPr−O···D2O structures, whose calculated amide-I frequencies are 1630.3 and 1631.4 cm−1, respectively. Component c, located at 1652.9 cm−1, can be assigned to the GlcNPr−D···D2O structure, whose calculated amide-I frequency is 1650.2 cm−1. However, the amide-I frequency may also be affected by the solvated glucose groups; thus, the solvation in D2O could be more complicated than expected. Nevertheless, these results clearly show that both the hydrogen bonding site and number of formed hydrogen bonds play a role in determining the amide-I frequency. Similarly, for the case of GlcNPr in CH3OD, the IR components can be assigned roughly to different solute−solvent complexes. In addition, these results seem to support the assignment of differently solvated GlcNPr species in Figure 1, and different electrostatic interactions in the three solvents are responsible for the subtle differences in the observed frequencies (Table 1). The computations also predict that the amide-I diagonal anharmonicity in GlcNPr−solvent clusters decreases somewhat from the gas-phase value. The largest decrease is found for the GlcNPr−(D2O)3 system, whose anharmonicity is 15.7 cm−1. For the GlcNPr−DMSO cluster, the computed value is 17.9

Figure 5. Radial distribution functions for selective pairs of atoms of GlcNPr in H2O, DMSO, and CH3OH. (A) [O(C)···H)]; (B) [H(−N)···O].

extracted from the snapshot of the ab initio MD simulations (Figure 4A). One GlcNPr−(D2O)2, two GlcNPr−D2O, one GlcNPr−(CH3OD)3, and two GlcNPr−CH3OD clusters were also constructed, which are shown in Figure 4B. For the GlcNPr−(D2O)2 complex, two D2O molecules are hydrogen bonded to the carbonyl oxygen and the ND group. For the two GlcNPr−D2O clusters, one has a hydrogen bonding site on the carbonyl oxygen (GlcNPr−O···D2O), while the other has a hydrogen bonding site on the ND group (GlcNPr−D···D2O). Here, the O or D atom in GlcNPr forms a hydrogen bond with the solvent molecule. This is also the case for the two GlcNPr− CH3OD clusters. Thus, a few representative hydrogen-bonded structures of GlcNPr in the three solvents are taken into account in Figure 4. A hydrogen bond with the hydroxyl groups on the glucose ring is not considered. These systems are embedded in a dielectric continuum matching the static dielectric constant for each solvent.40 The dielectric constants of the three solvents are 80.4 for water, 33.6 for methanol, and 42.7 for dimethylsulfoxide. The anharmonic normal-mode frequency, anharmonicity, intensity, and the magnitude of the transition dipole moment of the amide-I mode in these GlcNPr−solvent complexes are calculated and listed in Table 3.

Table 3. GlcNPr−Solvent Systems and Anharmonic Transition Frequency (ν, cm−1), Anharmonicity (Δi, cm−1), Transition Intensity (I, km/mol), Transition Dipole Moment (μ, Debye), and PED (%) of the Amide-I Modea system

ν

Δi

I

μ

GlcNPr GlcNPr−Dc···D2O GlcNPr−Oc···D2O GlcNPr−(D2O)2 GlcNPr−(D2O)3 GlcNPr−Dc···CH3OD GlcNPr−Oc···CH3OD GlcNPr−(CH3OD)2 GlcNPr−(CH3OD)3 GlcNPr−DMSO

1702.1 1650.2 1631.4 1630.3 1608.6 1649.5 1631.3 1629.7 1610.1 1642.0

19.3 18.5 17.6 17.6 15.7 18.2 17.9 17.1 16.0 17.9

190.7 495.8 643.5 665.0 682.3 501.3 630.4 667.8 760.7 535.3

0.211 0.346 0.397 0.403 0.407 0.348 0.393 0.404 0.434 0.361

PEDb CO CO CO CO CO CO CO CO CO CO

s s s s s s s s s s

(83), (80), (77), (76), (66), (80), (78), (75), (70), (79),

CN s (4), CCN d (3) CN s (5), CCN d (4), ND ib (3) CN s (6) CNs (7) CNs (16), CCN d (5), HCN d (3) CN s (6), CCN d (4) CN s (6), CC s (3) CN s (8) CN s (14) CN s (6), CCN d (4)

The PCM solvent model is applied in all cases. bOnly the contributions ≥ 3% are listed. Modes are defined by s = stretch, d = deformation, and ib = in-plane bend. cAtom with which the hydrogen bond is formed with a solvent molecule. a

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cm−1, being in reasonable agreement with the determined value from the pump−probe trace at zero delay time (17.0 cm−1). The change of the amide-I vibrational property, from the gas to solution phases, is due to solvation. Our analysis above yields a picture of the first solvation shell at the chemical bond level. The amide-I mode undergoes a mode delocalization process upon solvation, and such a delocalization effect can be reflected by PED. The distributed potential energies listed in Table 3 show that the amide-I mode is dominated by the CO stretching motion, similar to what is known for peptides. However, upon solvation, the amide-I mode becomes delocalized, picking up more backbone stretches (e.g., the C−N stretching) and bends at the cost of lowering the CO stretching contributions. This is clearly seen in the cases of water and methanol. In addition, we find that upon solvation, the amide-I mode would pick up solvent motions, for example, the DOD bending; even their contribution is below the PED threshold (3%) set in Table 3 (thus, data are not given). These intra- and intermolecular couplings can facilitate the vibrational relaxations and can explain, for example, why the population relaxation dynamics of the amide-I mode of GlcNPr in D2O is faster than that in other solvents. 3.6. Accelerated Vibrational Dynamics. The twocomponent feature of the amide-I vibrational dynamics observed in these solvated GlcNPr systems (Figure 3) indicates a fast and a slow population relaxation processes. In condensed phases, the increased density of states usually makes it difficult to assign a definite relaxation pathway for a given vibrational excitation. Because of this, even in simple peptide systems, the amide-I mode vibrational population relaxation mechanism is still not fully understood.23,26,45 It is useful to compare the results of GlcNPr and dNMA in order to get insight into the physical origins of the observed amide-I vibrational dynamics of GlcNPr. In solvated dNMA, a biphasic population relaxation of the amide-I mode has also been reported, with typical time constants of 0.38 and 2.1 ps26 and 0.45 and 4 ps in D2O.21 These time constants become 0.43 and 2.1 ps, respectively, in DMSO26 and 0.26 and 2.3 ps, respectively, in fully deuterated DMSO.23 For dNMA, the fast process may be due to vibrational energy dissipation to the strongly coupled modes via the intramolecular vibrational energy redistribution (IVR), while the slow process may be associated with the vibrational energy transfer to solvent through solute−solvent interactions.26 Clearly, the time constants for both fast and slow components in solvated GlcNPr are somewhat shorter than those observed in dNMA. In particular, the slow relaxation process is about twice faster. This can be seen more easily by comparing the average values shown in Table 2 with the results of dNMA. For the amide-I mode in GlcNPr, the IVR processes can occur through exchange of vibrational excitation between the amide-I and other intramolecular modes with which it is anharmonically coupled. To understand the acceleration, we perform ab initio computations on both GlcNPr and dNMA under various conditions. The energy-accepting candidates for the amide-I vibrational relaxation can be the downstream modes of the amide-I mode. Figure 6 shows a comparison of experimental and ab initio computed IR spectra of deuterated GlcNPr (panel A) and dNMA (panel B) in the spectral region of 1300−1700 cm−1. Anharmonic frequencies are computed and listed in Tables S1 and S3, respectively, for the GlcNPr and dNMA systems in the Supporting Information. The diagonal

Figure 6. FTIR spectra of GlcNPr (A) and NMA (B) in D2O in the region of 1300−1700 cm−1, showing the amide-I mode and its downstream modes, including the well-known amide-II. Calculated stick IR spectra of the two species forming a hydrogen bond with three D2O molecules were also shown for comparison.

anharmonicities of these modes and pairwise off-diagonal anharmonicities are listed in Tables S2 and S4 (Supporting Information), respectively. Here, because only intramolecular modes are concerned, the GlcNPr−(D 2 O) 3 cluster is considered without applying the PCM solvent model, which results in slight differences in frequencies from the case listed in Table 3. The same treatment is used for dNMA. Clearly, there are more downstream modes available in GlcNPr than in dNMA. Instead of simple band assignment, we listed the PED values of these modes in GlcNPr and dNMA in Tables S1 and S3 (Supporting Information), respectively. Tables S2 and S4 (Supporting Information) revealed nonzero diagonal and offdiagonal anharmonicities of the listed modes. Because the offdiagonal anharmonicity is proportional to the bilinear vibrational anharmonic couplings, the results show that these downstream modes are weakly coupled to each other and to the amide-I mode as well. In Figure 6A, the computed bar located at ν = 1496.0 cm−1 is the amide-II mode in GlcNPr. The PED analysis reveals that the next two low-frequency modes (Table S1, Supporting Information) are mainly methyl umbrella distortions with sizable transition strength. This picture is in agreement with the experimentally observed doublet in this region (Figure 6A). A similar doublet is also seen in dNMA (Figure 6B). In previous works,26,46,47 the doublet was also believed to be due to the Fermi resonance. However, on the basis of the results presented here, we believe that the main cause is methyl umbrella distortion. The higher density of modes below the amide-I mode presented in GlcNPr suggests more relaxation channels for the vibrational excitation, thus leading to the accelerated amide-I vibrational relaxations in GlcNPr. This also suggests that the amide-II is not the only possible energyaccepting mode. To further investigate this, we compare the vibrational properties of the amide-I and -II modes in water. First, we find that the vibrational coupling strength between the amide-I and -II modes is similar in the two cases. The off-diagonal anharmonicity of the amide-I and amide-II for GlcNPr− (D2O)3 and dNMA−(D2O)3 systems are found to be 1.5 cm−1 (Table S2, Supporting Information) and 2.4 cm−1 (Table S4, Supporting Information), respectively, at the level of B3LYP/631+G**, suggesting similarly weak coupling in both cases. The predicted result for dNMA is in reasonable agreement with 6112



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previous two-dimensional infrared (2D IR) measurements26,48 and computational results.49 Further, computations suggest that the amide-I mode delocalizes similarly in D2O-solvated GlcNPr and NMA, as shown in Table S5 for the former and Table S6 for the latter in the Supporting Information. The amide-I modes in GlcNPr and in NMA are similarly dominated by the CO stretching and delocalize similarly onto the backbone motion, as shown by PED. Also, as shown in Tables S5 and S6 (Supporting Information), similar delocalizations are also seen in the amide-II modes in the two species. Further, the acceleration in the slow component of GlcNPr may also be associated with the glucose vibrational modes, for example, the C−O stretching modes that are located at ∼1100 cm−1. The transition frequency, transition intensity, and PED of six strong C−O stretching modes are listed in Table S6 (Supporting Information). The presence of these hydrophilic groups, even though being at least two chemical bonds away from the amide unit in space, may provide additional vibrational energy dissipation pathways through Fermi resonance. Our pump−probe experiment shows that the time constant of the fast component is of slight frequency dependence. This is particularly the case in water (Table 2) where components b and c show relatively larger time constants than component a, suggesting that the IVR process can be affected by solute− solvent interactions.50 On the other hand, the slow relaxation component decays even faster, suggesting a relatively slow energy dissipation process via more weakly coupled solvent modes for less solvated GlcNPr species. This picture is consistent with the assignment of component a in the IR spectral region to less solvated GlcNPr. Finally, it should be pointed out that to further understand the vibrational relaxation dynamics, a narrow-band pump− broad-band probe method would be more helpful. The broadband pump−probe IR method used in this work has the advantage of simultaneously probing multiple frequency dynamics; however, it only covers the amide-I band and cannot resolve dynamical processes of other modes. Another promising method is the 2D IR method that possesses inherent doublefrequency resolution and can obtain information on frequency−frequency correlation functions, vibrational energy transfer, as well as equilibrium chemical exchange dynamics.51−60 In comparison with the 2D IR method, the broad-band pump−probe IR method only probes a projection of various relaxation pathways.

serve as either a hydrogen-bond donor or acceptor, its relatively larger size hindered a complete solvation of the amide unit. The amide-I band structure in the linear IR spectra, on the other hand, also exhibited clearly solvent sensitivity. The results suggested that there is more than one structured GlcNPr− solvent cluster in D2O as well as in CH3OD. Ab initio MD simulations showed that the amide unit in GlcNPr forms hydrogen bonds with D2O, CH3OD, and DMSO molecules quite differently in the first solvation shell. Quantum chemical computations of GlcNPr−solvent clusters showed that the hydrogen bonding interaction in the first solvation layer could account for a significant amount of the red shift observed in the linear IR spectra. These results revealed the structural and environmental sensitivities of the amide-I mode in solvated GlcNPr. Further, as a result of different solvent environments, these GlcNPr−solvent clusters exhibited different amide-I vibrational dynamics, which were clearly shown in kinetic traces extracted from the dispersed pump−probe spectra. Our results suggested that the GlcNPr−solvent structure with multiple hydrogen bonds has the amide-I vibration at the low-frequency side and a relatively fast vibrational dynamics, while that with fewer hydrogen bonds has the amide-I vibration at the high-frequency side and a relatively slow vibrational dynamics. This was supported by the computational results, which showed that the amide-I mode became more delocalized as the number of hydrogen bonds increased, in which case, the vibrational excitation energy may quickly relax to the bath modes. Furthermore, by comparing the dynamical behavior of the amide-I modes in GlcNPr and NMA, one sees that they have different vibrational relaxation times. In particular, the slow process is almost twice faster in GlcNPr. The frequency dependence discussed above cannot explain the acceleration. Rather, the acceleration is likely due to higher density of downstream modes below the amide-I mode, as well as the glucose ring modes that may provide more vibrational relaxation pathways than seen in NMA. Further investigations using narrow-band pump−broad-band probe and/or 2D IR methods shall provide additional useful information to better understand the vibrational relaxation pathways of the amide-I mode in various CONH-containing glycopeptide systems. The results presented here demonstrate that the amide-I mode in glucosamine can be a general and useful probe for the local structure and solvent environment for sugar residues in glycopeptides and glycoproteins. Potentially, the three-dimensional arrangement of multiple sugar residues and their structural distributions can be investigated in terms of the vibrational couplings between different amide-I modes. However, because the sugar amide-I mode falls into the protein amide-I frequency region, care should be taken when studying the relationship between the amide-I absorption band and the structure of glycopeptides. Even though previous IR studies of glycopeptides in the amide-I region did not assign the contribution from the sugar side chains, isotopic substitution (e.g., −13CONH−) could be used to effectively separate the amide-I modes from different units.

4. CONCLUDING REMARKS In this work, the amide-I vibrational dynamics of GlcNPr in different solvents were examined by using the steady-state and transient infrared spectroscopic methods, aided with quantum chemical computations and ab initio MD simulations. Three solvents, D2O, DMSO, and CH3OD, which have different dielectric constants, were used. The results revealed influences of the solute−solvent hydrogen bonding interaction as well as the glucose and ethyl groups on the amide-I vibrational properties in GlcNPr. The peak position of the amide-I mode of GlcNPr exhibited sensitivity to solvent. In comparison with the computed gasphase result, the largest red shift was seen in the case of D2O, and the smallest red shift was seen in DMSO. Here, D2O could serve as either a hydrogen-bond donor or acceptor. DMSO, being a less polar solvent, however, could only act as a hydrogen-bond acceptor. Even though CH3OD could also

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ASSOCIATED CONTENT

* Supporting Information S

Additional computational results of amide-I and its downstream vibrations (in particular, the amide-II mode) for deuterated GlcNPr as well as NMA for comparison and that of the glucose C−O stretches. The anharmonic frequency, transition intensity, 6113



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potential energy distribution, and diagonal and off-diagonal anharmonicities of selected modes are given. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (F.Y.); [email protected] (J.W.). Tel: (+86)-010-62656806. Fax: (+86)-010-62563167. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.H. thanks Dr. Kaicong Cai for helpful discussions and Ms. Jipei Shi for assistance in IR spectral measurement. This work was supported by the Knowledge Innovation Program (Grant No. KJCX2-EW-H01) and the Hundred Talent Fund from the Chinese Academy of Sciences. The support from the National Natural Science Foundation of China (91121020 and 21173231) is also acknowledged. The work was also supported by the NSFC instrumentation fund (20727001).

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