Sigal A 2015

Subscriber access provided by West Virginia University | Libraries
Article
Determination of Hydrogen Bond Structure in Water Versus Aprotic
Environments to Test the Relationship Between Length and Stability
Paul A. Sigala, Eliza A. Ruben, Corey W Liu, Paula M. B. Piccoli, Edward
G. Hohenstein, Todd J. Martínez, Arthur J. Schultz, and Daniel Herschlag
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja512980h • Publication Date (Web): 14 Apr 2015
Downloaded from http://pubs.acs.org on April 16, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society 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.
Page 1 of 34 Journal of the American Chemical Society
1
2
3
Determination of Hydrogen Bond Structure in Water Versus Aprotic Environments to Test
4
5 the Relationship Between Length and Stability
6
7
8
9
10
11
12
13
14
15 Paul A. Sigala†,∆, Eliza A. Ruben†,Corey W. Liu‡, Paula M. B. Piccoli§, Edward G. Hohenstein#,
16
Todd J. Martínez#, Arthur J. Schultz§,≠, and Daniel Herschlag†,#,*
17
18
19
20
21 †
Dept. of Biochemistry, Stanford University, Stanford, California 94305
22 ‡
Stanford Magnetic Resonance Laboratory, Stanford University, Stanford, California 94305
23 §
24
Intense Pulsed Neutron Source, Argonne National Laboratory, Argonne, Illinois 60439
#
25 Dept. of Chemistry, Stanford University, Stanford, California 94305
∆
26 Present address: Dept. of Molecular Microbiology, Washington University, St. Louis, Missouri
27 63110
28 ≠
Present address: X-ray Science Division, Argonne National Laboratory, Argonne, Illinois
29
60439
30 *
31 e-mail: herschla@stanford.edu
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
1
ACS Paragon Plus Environment
Journal of the American Chemical Society Page 2 of 34
1
2
3
ABSTRACT
4
5
6 Hydrogen bonds profoundly influence the architecture and activity of biological
7
8 macromolecules. Deep appreciation of hydrogen bond contributions to biomolecular
9
10
11
function thus requires a detailed understanding of hydrogen bond structure and energetics
12
13 and the relationship between these properties.Hydrogen bond formation energies (∆Gf)are
14
15 enormously more favorable in aprotic solvents than in water, andtwo classes of
16
17
18 contributing factors have been proposed to explain this energetic difference, focusing
19
20 respectively on the isolated and hydrogen-bonded species: I)water stabilizes the dissociated
21
22 donor and acceptor groups much better than aprotic solvents, thereby reducing the driving
23
24
25 force for hydrogen bond formation; and II) water lengthens hydrogen bonds compared to
26
27 aprotic environments, thereby decreasing the potential energy within the hydrogen bond.
28
29
30
Each model has been proposed to provide a dominant contribution to ∆Gf, but incisive tests
31
32 that distinguish the importance of these contributionsare lacking. Here wedirectlytest the
33
34 structural basis of Model II.Neutron crystallography, NMR spectroscopy, and quantum
35
36
37 mechanical calculations demonstrate that O-H•••O hydrogen bonds in crystals, chloroform,
38
39 acetone, and water have nearly identical lengths and very similar potential energy surfaces
40
41 despite ∆Gfdifferences greater than 8 kcal/mol across these solvents. These results rule out
42
43
44 a substantial contribution fromsolvent-dependent differences in hydrogen bond structure
45
46 and potential energy after association (Model II) and thus support the conclusionthat
47
48 differences in hydrogen bond ∆Gfare predominantly determined by solvent interactions
49
50
51 with the dissociated groups (Model I).These findings advance our understanding of
52
53 universal hydrogen-bonding interactions and have important implications for biology and
54
55
56
engineering.
57
58
59
60
2
1
2
3
INTRODUCTION
4
5
6 Hydrogen bonds are ubiquitous chemical interactions whose formation and
7
8 propertiescontribute enormously toprotein and nucleic acid folding, molecular recognition,
9
10
11
enzymatic catalysis, and drug-receptor interactions. A thorough understanding of hydrogen bond
12
13 structure and energetics is thus required to deeply understand hydrogen bond contributions to
14
15 biomolecular functionand to optimize the engineering ofnew molecular systems with novel
16
17
18 functional properties. In this work, we test the relationship between hydrogen bond length and
19
20 the free energy change (∆Gf) upon hydrogen bond formation.∆Gf reflects the overall stability of a
21
22 hydrogen-bonded complex relative to the dissociated donor and acceptor groups (Fig. 1A) and is
23
24
25 the most common thermodynamic measure for assessing hydrogen bond contributions to protein
26
27 folding, ligand binding, and enzyme catalysis.1
28
29
Substantial prior work has indicated that the energetic properties of hydrogen bonds are
30
31
32 strongly influenced by the nature of the surrounding environment. Extensive studies of small-
33
34 molecule complexes have provided overwhelming evidence that hydrogen bonds formed in
35
36
37 aprotic environments are enormously more stable than in water. For example, ∆Gf for hydrogen
38
39 bond formation between 4-nitrophenol and 4-nitrophenolate is 7–8 kcal/mol more favorable in
40
41 acetonitrile and tetrahydrofuran than in water, and the hydrogen bond between fluoride and
42
43
44 hydrogen fluoride is nearly 40 kcal/mol more stable in the gas phase than in water.2-5
45
46 Particular attention has been focused in the literature on the structural and energetic role
47
48 of hydrogen bonds with lengths of 2.4–2.6 Å, as these hydrogen bonds are significantly shorter
49
50
51 than the majority of 2.8–3.2 Å hydrogen bonds observed in protein and small-molecule
52
53 structures. Such short hydrogen bonds display distinguishing spectroscopic features (e.g. 1H
54
55
56
NMR chemical shifts of 14–18 ppm) in aprotic organic solvents, have large formation free
57
58
59
60
3
1
2
3
energies in aprotic environments, and are frequently referred to as “short, strong hydrogen
4
5
6 bonds”.6-10 In contrast, spectroscopic signals indicative of short hydrogen bonds have not
7
8 typically been observed for hydrogen-bonded complexes in water,8,11 where ∆Gf values are
9
10
11
small.
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 Fig. 1. Models for the effect of solvent on the equilibrium formation of O-H•••O– hydrogen
48
bonds.(A) Schematic depiction of hydrogen bond lengths and formation free energy (∆Gf),
49
50 which reflects the stability of the hydrogen-bonded complex relative to the dissociated groups.
51
52 (B) Schematic depictionof Model I in which hydrogen bond stability is determined by the
53
54 differential interactions of water versus aprotic solvents with the dissociated donor and acceptor
55
56
groups, with identical hydrogen-bonded species in the differing solvents. (C) Schematic
57 depiction of Model II in which solvent-solute interactions lengthen a hydrogen bond in water
58
59
60
4
1
2
3
relative to an aprotic organic solvent, resulting in the longer hydrogen bond as the more stable
4
5 structure in water and the shorter hydrogen bond as the more stable structure in an aprotic
6
7 environment. Charges and partial charges depict possible effects that accompany hydrogen bond
8
9 shortening and interactions with solvent molecules. The larger dots represent the deeper potential
10
11
energy well posited in aprotic organic solvents according to Model II.
12
13
14
15 Based on these structural and energetic observations, two general models have been
16
17 proposed in the literature to account for the enhanced stabilities of hydrogen bonds in aprotic
18
19 environments compared to water.These modelscan provide independent and additive
20
21
22 contributions to ∆Gf and focus on either the dissociated donor and acceptor groups (Fig. 1A, left
23
24 side of equilibrium) or the hydrogen-bonded complex (Fig. 1A, right side of equilibrium) and the
25
26
27
differential effects of the surroundings on these species, as elaborated below (see additional
28
29 discussion in Text S1).
30
31 Model Iaddresses the differential ability of protic versus aprotic solvents to stabilize the
32
33
34 separatedhydrogen bond donor and acceptor groups.2,3,12-14 According to proposals that this
35
36 model provides a dominant contribution to hydrogen bond ∆Gf, the isolated charge of a
37
38 dissociated oxyanionic acceptor will be enormously more stable in water than in aprotic organic
39
40
41 solvents, resulting in a substantially reduced hydrogen bond formation energy in water compared
42
43 to aprotic environments (Fig. 1B).
44
45
Model IIaddresses the hydrogen-bonded complex and posits that hydrogen bonds adopt
46
47
48 shorter equilibrium distances in aprotic environments than in water, and that such
49
50 shorteningdeepens the potential energy well of the hydrogen-bonded groups and results in a
51
52
53
much more favorable formation free energy (Fig. 1C, additional discussion in Text S1).8,12,15-23
54
55 This model recognizes that hydrogen bonds are largely electrostatic in character but have a
56
57 charge-transfer component that increases as a hydrogen bond shortens due to enhanced orbital
58
59
60
5
1
2
3
overlap.24,25 Hydrogen bonds formed in water might be preferentially longer due to favorable
4
5
6 interactions between more localized charges and solvent water molecules (Fig. 1C). In the
7
8 absence of strong stabilizing interactions with solvent, hydrogen bonds formed in aprotic
9
10
11
solvents might instead adopt shorter distances to maximize stabilization from charge
12
13 delocalization (see additional discussion in Text S1 and S2).Indeed, a prior computational study
14
15 suggested that the O•••O distance of thewater-hydroxidehydrogen bond lengthens from 2.4 Å in
16
17
18 the gas phase to 2.8 Å in water.12
19
20 Both models have been suggested to be important for protein function. For example,
21
22 “short, strong hydrogen bonds” have been proposed to form within buried protein interiors, due
23
24
25 to unique properties of the solvent-sequestered protein matrix that differ from water, and to
26
27 contribute substantially to enzyme catalysis because of their short lengths.6,9,11,19 Other
28
29
researchers have invoked the general observation of increased hydrogen bond formation free
30
31
32 energies in the absence of water to suggest that hydrogen bond stability might be enhanced
33
34 within protein interiors due to loop or domain closures or the tight packing of active site groups
35
36
37 that largely exclude bulk water molecules.26,27
38
39 Despite the ubiquity of the two models described above, therelative importance of their
40
41 energetic contributions to hydrogen bond stability in solution has never been distinguished and
42
43
44 assessed. Indeed, the central tenet of the second model that hydrogen bonds are longer in water
45
46 than in non-aqueous environments has remained untested dueto technical challenges in assessing
47
48 hydrogen bond structure in water.There have been occasional spectroscopic reports9,28-
49
50 31
51 suggesting that short hydrogen bond distances can form in partially aqueous environments at
52
53 sub-zero temperatures (e.g., 10% water/90% acetone at -50˚C),and X-ray crystallographic studies
54
55
56
of the 4-nitrophenol•4-nitrophenolate complex have reported nearly identical O•••O distances in
57
58
59
60
6
1
2
3
the absence (2.475 Å) or presence (2.465 Å) of two hydrating water molecules per unit
4
5
6 cell.32Under these heterogenous and low-temperature conditions, however, the solvation
7
8 properties of water molecules can substantially differfrom bulk water.33
9
10
11
We have critically assessed the structural basis forModel II(Fig. 1C) bysystematically
12
13 testingwhether hydrogen bonds are longer and have significantly different potential energy
14
15 surfaces in water relative to aprotic organic solvents.Our results provide direct evidence that the
16
17
18 weaker formation free energies observed for hydrogen bonds in water compared to aprotic
19
20 organic solvents are not accompanied by increased hydrogen bond lengths or substantially
21
22 differing potential energy surfaces across these environments. These results rule out the central
23
24
25 underpinning of Model II and, by elimination, support Model I as the dominant determinant of
26
27 hydrogen bond ∆Gf.This finding has important implications for understanding the contributions
28
29
of hydrogen bonds to the fundamental energetic and structural properties of biomolecules, their
30
31
32 complexes, and their functions.
33
34
35
36
37 RESULTS AND DISCUSSION
38
39 We approached this question by studying changes across a series of compounds in
40
41 multiple environments, rather than examining a single hydrogen-bonded complex, as we
42
43
44 reasoned that systematic studies could provide more extensive and incisive information about the
45
46 relationship between solvation and hydrogen bond structure and potential energy surface. We
47
48 focused on O-H•••O hydrogen bonds, as these interactions are common in nature and have been
49
50
51 the subject of extensive prior investigation.8,25,34,35
52
53
54
55
56
The dependence of hydrogen bond length on ∆pKa
57
58
59
60
7
1
2
3
Hydrogen bond formation energies (∆Gf)are commonly observed to depend linearly on
4
5
6 ∆pKa, the proton affinity difference between the hydrogen bond donor and acceptor groups, with
7
8 the largest formation energies typically observed at or near a∆pKa value of zero.5,36As the
9
10
11
shortest hydrogen bond lengths are also typically observed between groups with similar pKa
12
13 values,37 we first asked whether hydrogen bond lengthsalso show a broad linear dependence on
14
15 ∆pKa.We analyzed the crystal structures inSteiner and Saenger’s 1994 compilation of O-H•••O
16
17
18 hydrogen bonds determined by low-temperature (<130 K) neutron diffraction,35experimental
19
20 conditions that most accurately determine proton positions.Wealso determined low-temperature
21
22 neutron structures of six additionalhydrogen-bonded complexes, resulting in a composite,
23
24
25 structurally diverse data set of 68inter- and intramolecular hydrogen bonds with ∆pKa values
26
27 spanning nearly 20 units (Table S1).
28
29
We compiled the O•••O, O-H, and H•••O distances observed in the neutron structures for
30
31
32 each hydrogen-bonded complex and plotted these distances as a function of the ∆pKa values
33
34 determined for each hydrogen bond based on the experimental or calculated pKa values of the
35
36
37 interacting donor and acceptor groups. These plotsrevealeda steady dependence on ∆pKa, with
38
39 the O•••Odistance increasing linearly with a slope of 0.02 Å/pKa unit from 2.4 Å to almost 2.9 Å
40
41 as ∆pKa increased from 0 to 20 (R2 = 0.86) (Fig. 2A). The position of the hydrogen-bonded
42
43
44 proton also varied steadily with ∆pKa, with both O-H and H•••O distances converging on 1.2 Å
45
46 as ∆pKa approached zero (Fig. 2B).
47
48 This structural dependence on ∆pKa observed directly by neutron diffraction is consistent
49
50
51 with prior NMR and IR studies of O-H•••O hydrogen bonds in aprotic organic solvents and in
52
53 the gas phase that have reported systematic changes as a function of ∆pKa in 1H chemical shift
54
55
56
and O-H stretching frequency, spectroscopic observables that provide incisive readouts of
57
58
59
60
8
1
2
3
hydrogen bond structure.38-41 Our direct assessment of this structural dependence on ∆pKa in
4
5
6 hydrogen-bonded crystals provides a quantitative baseline from which to test and compare the
7
8 effects of differing solvent environments.
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 Fig. 2. The dependence of hydrogen bond length on ∆pKa. Dependence of O•••O distance (R2
46 = 0.86) (A) and O-H (squares) and H•••O distances (circles) (B) on ∆pKa for O-H•••O hydrogen
47
48 bonds in low-temperature neutron structures. Data points for newly determined structures are in
49
50 red. Inset in (A) is the neutron structure for 4-nitrophenol•4-methoxypyridine-N-oxide
51
52 determined herein (∆pKa = 5.1). Aqueous pKa values are used herein as a proxy for proton
53
54
affinity and so that each compound can be denoted by a single ∆pKa value that allows
55 comparisons between properties of the same compound in different environments.
56
57
58
59
60
9
1
2
3
Direct tests of hydrogen bond structure in aprotic solvents versus water via1H NMR
4
5
6 In a non-aqueous environment, both hydrogen bond length and formation free energy
7
8 correlate with ∆pKa.Prior studies3,36,42 have provided clear evidence that hydrogen bond
9
10
11
formation energies display a substantially steeper dependence on ∆pKa in aprotic organic
12
13 solvents than in water. The critical unresolved question that we set out to test was whether
14
15 hydrogen bond lengthsalso display a steeper dependence on ∆pKain aprotic organic solvents than
16
17
18 in aqueous solution, as predictedbased on the central tenet ofModel II(Fig. 1C) that hydrogen
19
20 bond formation energies depend directly onhydrogen bond lengths.
21
22 We exploited the ability of 1H NMR spectroscopy to directly detect hydrogen-bonded
23
24
25 protons and its high sensitivity to changes in hydrogen bond structure as small as 0.01 Å.341H
26
27 chemical shifts from solid state NMR correlate strongly with hydrogen bond distances
28
29
determined by neutron diffraction, with the chemical shift of the hydrogen-bonded proton
30
31
32 increasing from 12 – 20 ppm as the O•••O distance decreases from 2.7 Å to 2.4 Å and the O•••H
33
34 distance simultaneously decreases from 1.7 Å to 1.2 Å.23,34,43-46 This correlation has been
35
36
37 ascribed to decreased shielding from sigma bond electron density around the proton as it
38
39 migrates away from its covalently associated oxygen toward the center of the hydrogen bond as
40
41 O•••O distances shorten,23,35,47 andit provides a powerful means to detect and quantify changes in
42
43
44 hydrogen bond structure within solution environments.44,48This approach has been used to
45
46 interrogate hydrogen bond structure in aprotic solvents,8,45,48 but aqueous environments remain
47
48 challenging to study due to proton exchange betweenwater molecules and hydrogen-bonded
49
50
51 solutes.
52
53 We focused on the intramolecular hydrogen bond within salicylate monoanions (Fig.
54
55
56
3A), as a prior 1H NMR study29 of these compounds in 10% water/acetone (-50 ˚C) hinted that it
57
58
59
60
10
1
2
3
might be possible to detect the hydrogen-bonded proton on a high-field instrument in fully
4
5
6 aqueous solution (i.e., 100% water) and thus enable the critical experimental test. Furthermore,
7
8 these compounds are available with differing substituents that vary the donor-acceptor ∆pKa over
9
10
11
a wide range, and equilibrium hydrogen bond formation free energies (∆Gf, defined in Fig. 3A)
12
13 measured for salicylates indicate stronger hydrogen bonds and a much steeper dependence on
14
15 ∆pKa in aprotic organic solvents than in water42 (see additional discussion in Text S3, Supporting
16
17
18 Information). In addition, as we describe below, multiple observations indicate that the hydrogen
19
20 bond behavior of these intramolecular complexes is very similar to that of intermolecular
21
22 complexes.
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
11
1
2
3 1
Fig 3.Solution H NMR studies of hydrogen bonding in substituted salicylate
4
5 monoanions.(A) Schematic depiction of intramolecular hydrogen bond formation in salicylates
6
7 and 1H NMR spectra of 5-methylsalicylate (∆pKa = 5.0), 4-methoxysalicylate (∆pKa = 4.2), 3,5-
8
9 dichlorosalicylate (∆pKa = 3.5), 5-formylsalicylate (∆pKa = 2.9), 5-nitrosalicylate (∆pKa = 2.8),
10
11
3-nitrosalicylate (∆pKa = 2.8), and 3,5-dinitrosalicylate (∆pKa = 0.5) in chloroform, acetone, and
12 water at 4 ˚C. The spectrum of 3,5-dinitrosalicylate in water which was acquired in 10%
13
14 DMSO/water at -3 ˚C. (B–D) Dependence of 1H NMR chemical shift in each solvent on ∆pKa
15
16 (slopes = 0.8–1.0 ppm/pKa unit, R2 = 0.82–0.92) (B), 1H NMR chemical shifts in chloroform
17
18 versus water (slope = 1.0, R2 = 0.98) (C), and 1H NMR chemical shifts in acetone versus water
19 (slope = 0.8, R2 = 0.89) (D).
20
21
22
23
24
We acquired 1H NMR spectra of a series of salicylates (aqueous ∆pKavaried over 5 units)
25
26 in chloroform (ε = 5), acetone (ε = 21), and water (ε = 80), solvents of widely differing dielectric
27
28
29
(ε) and hydrogen bonding ability. A far-downfield peak corresponding to the intramolecular
30
31 hydrogen-bonded proton was readily detected in all spectra, including those obtained in water,
32
33 where the downfield peak was mildly broadened due to slow exchange with bulk water (Fig.
34
35
36 3A).
37
38 There were modest (<1 ppm) differences in the absolute chemical shift measured for each
39
40
salicylate across the three solvents but no trend that distinguished the behavior between any of
41
42
43 the solvents. Indeed, the 1H NMR chemical shift measured for each salicylate’s hydrogen-
44
45 bonded proton and its change across the series of salicylates were strikingly similar in all three
46
47
48
solvent environments, with increases of 1.0 ± 0.1 ppm per unit decrease in ∆pKa (Fig. 3B). A
49
50 plot of the observed 1H NMR chemical shift in chloroform versus water is accurately fit (R2 =
51
52 0.98) by a linear function with a slope of 1.0, further indicating a nearly identical dependence of
53
54
55 hydrogen bond proton position on ∆pKa in these two solvent environments despite their nearly
56
57 17-fold difference in dielectric constant and their disparate capacities to hydrogen bond with
58
59
60
12
1
2
3
solutes (Fig. 3C).Similar trends are seenfor the observed 1H NMR chemical shifts in acetone
4
5
6 versus water (R2 = 0.89) (Fig. 3D),acetone versus chloroform (R2 = 0.89) (Fig. S1A), and water
7
8 versus the previously reported29 chemical shifts in 10% water/acetone (R2 = 0.92) (Fig. S1B). By
9
10
11
plotting the NMR data in each solvent versus the NMR data in the other solvents, any
12
13 uncertainty in the accuracy of the intrinsic ∆pKa values for salicylates is eliminated from the
14
15 analysis.
16
17
18 As noted above, 1H NMR chemical shifts can be used toestimate hydrogen bond
19
20 lengths34,43,44and thus to quantifydifferences in hydrogen bond structure for the same salicylate or
21
22 across the series of salicylates in discrete solvents (correlation functions given in Supporting
23
24
25 Information, Materials and Methods). The hydrogen bond O-O distancesestimated for 3,5-
26
27 dinitrosalicylate in chloroform, acetone, and water were all within 0.01 Å of oneanother and
28
29
within 0.03 Å of the O-O distance observed in a low-temperature neutron crystal structure of this
30
31
32 compound (Table S2), providing additional evidence against substantial environmental effects on
33
34 hydrogen bond length.
35
36
37 Across the series, the observed 1H chemical shifts suggest that salicylate hydrogen bonds
38
39 shorten by 0.02 – 0.03 Å (O•••O distance) and 0.04 – 0.05 Å (H•••O distance) per unit decrease
40
41 in ∆pKain all three solvents.49These distance changesare very similar to the corresponding
42
43
44 changes in O•••O(0.02 Å/pKa unit) and H•••O(0.03 Å/pKa unit) distances determined by neutron
45
46 diffraction for hydrogen bonds in diverse small-molecule crystals (Fig. 2A&2B). Analysis of X-
47
48 ray diffraction data in the Cambridge Structural Database forcrystalline salicylates also indicate
49
50
51 a0.017 Å/pKa unit change in O•••O distance that is nearly identical to that estimated by
52
53 solution1H NMR for the salicylates and very similar to the 0.021 Å/pKa unit change in O•••O
54
55
56
distance observed by neutron diffraction for intermolecular hydrogen bonds (Fig. S2).Thus, the
57
58
59
60
13
1
2
3
physical properties of salicylate hydrogen bonds are not unique but are broadly similar to the
4
5
6 observed behavior of the diverse inter- and intramolecular O-H•••O hydrogen bonds included in
7
8 the crystallographic datasets (Fig.S2 and S3, and additional discussion in Text S7).The 1.0 ± 0.1
9
10
11
ppm/pKa unit change in chemical shift of the hydrogen-bonded proton across the series of
12
13 salicylates in the three solvents is also identical, within error, to the 0.9 ± 0.2 ppm/pKa unit
14
15 chemical shift change reported for the intermolecular O-H•••O hydrogen bond between
16
17
18 substituted phenols and trimethylamine-N-oxide in chloroform,38 further supporting the
19
20 conclusions that salicylate hydrogen bonds have similar properties to other hydrogen-bonded
21
22 groups, including intermolecular complexes, and that hydrogen bond structure is not strongly
23
24
25 influenced by solvent environment.
26
27 We also studied the effect of solvent on the hydrogen bond in 2-hydroxyphenylacetate,
28
29
which, while still intramolecular, is less structurally constrained than the salicylates. Despite this
30
31
32 additional flexibility, we observed nearly identical 1H NMR chemical shifts for the hydrogen-
33
34 bonded proton in chloroform (13.4 ppm) and 10% water/acetone (13.2 ppm) (Fig. S9), providing
35
36
37 additional evidence that supports our conclusion that solvent effects do not substantially alter the
38
39 structures of intra- and intermolecular hydrogen bonds.
40
41
42
43
44 Probing hydrogen bond potentials in aprotic solvents versus water with 2H isotope effects
45
46 To further test the conclusionthat hydrogen bonds formed in water are not substantially
47
48 lengthened relative to aprotic solvents and to evaluate possible differences in the potential energy
49
50
51 surface of hydrogen bonds formed in different environments, we measured the deuterium (2H)
52
53 isotope effect on the NMR chemical shifts of the hydrogen-bonded protons. For hydrogen bonds
54
55
56
with anharmonic potentials, substitution of the bridging proton for deuterium leads to a
57
58
59
60
14
1
2
3
shortening of the O-D bond relative to the original O-H bond50,51(most frequently observed in
4
5
6 hydrogen bonds less than ~2.7 Å) that can be detected by NMR as an upfield shift in the position
7
8 of the 2H peak relative to the 1H peak.8The magnitude of this 1H - 2H chemical shift has been
9
10
11
observed to increase with decreasing hydrogen bond length, an effect ascribed to increased
12
13 anharmonicity of the bond potential as the central barrier to proton transfer lowers with
14
15 decreasing hydrogen bond length.52 Thus, measurement of the1H - 2H chemical shift for a
16
17
18 hydrogen bond provides an incisive readout of the general shape and anharmonicity of the
19
20 potential energy surface.
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 Fig 4. Deuterium isotope effects of hydrogen bond structure in water and organic
56 solvents.(A) Dependence of the deuterium isotope shift (1H – 2H NMR chemical shift) on ∆pKa
57
58
59
60
15
1
2
3
in chloroform (red, R2 = 1.0), acetone (black, R2 = 0.99), and water (blue, R2 = 0.95) for 5-
4
5 methylsalicylate (∆pKa = 5.0, spectra in inset), 4-methoxysalicylate (∆pKa = 4.2), 5-
6
7 formylsalicylate (∆pKa = 2.9), and 3,5-dinitrosalicylate (∆pKa = 0.5). (B) Plot of the deuterium
8
9 isotope shift versus the 1H NMR chemical shift for each salicylate in chloroform, acetone, and
10
11
water (colors as in (A)) and for previously published O-H•••O– hydrogen-bonded complexes in
12 aprotic organic solvents (gray).8(C) The HH and HD isotopologues of 1,8,9-
13
14 trihydroxyanthracene monoanion formed by partial deuterium exchange. (D)1H NMR spectra of
15
16 the 1,8,9-trihydroxyanthracene monoanion in DMSO or 90% water/DMSO without or with
17
18 partial deuterium exchange of labile hydrogen atoms. XD = mole fraction of deuterium.
19
20
21
22 We acquired 2H NMR spectra for the same salicylates in chloroform, acetone, and water.
23
24 The 2H NMR peak in each case was readily detected for all compounds except for 3,5-
25
26
27
dinitrosalicylate in water (Fig. S4). The magnitudes of the 1H – 2H shifts observed for individual
28
29 salicylates in distinct solvents were within 0.1 ppm of one another, and the change in the 1H – 2H
30
31 shift as a function of ∆pKa was nearly identical in the three environments (Fig. 4A). In all cases
32
33
34 the 1H – 2H shift increased similarly with increasing 1H NMR chemical shift, as previously
35
36 observed for diverse O-H•••O hydrogen bonds in non-aqueous solvents (Fig. 4B) and as
37
38 expected for progressively shorter hydrogen bonds whose individual anharmonicities and
39
40
41 potential energy surfaces are very similar in both water and aprotic solvent environments.
42
43
44
45
Physical coupling of adjacent hydrogen bonds in aprotic solvents versus water
46
47
48 Prior studies have observed that when two hydrogen bonds are donated to a common
49
50 acceptor atom, substituting proton for deuterium in one of the hydrogen bondsresults in a
51
52
53
shortening of the neighboring hydrogen bond. This shortening can be detected as an increase in
54
55 the 1H NMR chemical shift of the hydrogen bond that retains its proton, and it indicates that the
56
57 structures of the two hydrogen bonds are coupled.45,53
58
59
60
16
1
2
3
To test whether water attenuates this coupling, as might occur if there were strong
4
5
6 interactions with water molecules that changed the electronic properties of the hydrogen-bonded
7
8 groups and altered the potential energy surface, we acquired 1H NMR spectra of 1,8,9-
9
10
11
trihydroxyanthracene monoanion (Fig. S5) dissolved in DMSO or in 90% water/DMSO (this
12
13 compound is insoluble in 100% water) without and with partial substitution of labile hydrogen
14
15 atoms with deuterium (Fig. 4C).Without deuterium exchange, we observed a single downfield 1H
16
17
18 NMR peak at 15.1 ppm in both solvents, corresponding to the two magnetically equivalent
19
20 hydrogen-bonded protons of the symmetric O-H•••–O•••H-O isotopologue and reflecting nearly
21
22 identical hydrogen bond structures in both solvent environments (Fig. 4D). Upon partial
23
24
25 deuteration, a second downfield peak was detected in both solvents, signaling formation of the
26
27 O-H•••–O•••D-O isotopologue and indicating that these neighboring hydrogen bonds are coupled
28
29
in both solvents. The observation that the downfield chemical shiftof the O-H•••–O•••D-O
30
31
32 isotopologue is the same (15.3 ppm), within error, intheaprotic and aqueous solvation
33
34 environments, provides further evidence that hydrogen bonds havevery similar structures and
35
36
37 potential energy surfaces in both non-aqueous solvents and in water.
38
39
40
41 Theoretical calculations of hydrogen bond structure in aprotic solvents versus water
42
43
44 Quantum mechanical (QM) calculations can, in principle, provide an independent and
45
46 rigorous test of the effect of solvation on hydrogen bond structure, allowing discrete solvent
47
48 properties to be separated and independently tested. However, computational complexities
49
50
51 prevent modeling of both the hydrogen-bonded solutes and a full atomistic solvation
52
53 environment at the highest levels of QM theory, and the development of accurate explicit
54
55
56
solvation models remains an ongoing challenge.54,55
57
58
59
60
17
1
2
3
To approach this question computationally, we first asked the most simple and
4
5
6 computationally straightforward question of whether broadly varying the general solvent
7
8 property of dielectric, in the absence of specific solvent-solute interactions, substantially alters
9
10
11
hydrogen bond structure. We performed QM calculations on the water-hydroxide ion and formic
12
13 acid-formate ion hydrogen bonds embedded in a polarizable continuum56 whose dielectric was
14
15 varied between 5 and 80. This dielectric range mimics the dielectric difference between
16
17
18 chloroform and water, two of the solvents tested experimentally and between which hydrogen
19
20 bond ∆Gf values vary enormously.For both hydrogen bonds, ab initio calculations at multiple
21
22 levels of theory and basis sets returned energy-minimized O•••O and H•••O distances near 2.5 Å
23
24
25 and 1.5 Å, respectively, that increased by only 0.04 ± 0.01 Å as the continuum dielectric
26
27 increased from 5 to 80 (Fig. 5A, Fig. S6, Table S3, and additional discussion in Text S4,
28
29
Supporting Information). This small change is nearly identical to the 0.03 Å difference
30
31
32 previously reported for the N-H•••O=C hydrogen bond (N•••O and H•••O distances) in the acetic
33
34 acid-acetamide dimer at solvent dielectrics corresponding to water and chloroform.13These
35
36
37 results indicate that increasing solvent dielectric alone from the value of a low-polarity organic
38
39 solvent such as chloroform to the value of water does not substantially perturb hydrogen bond
40
41 structure.
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18
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 Fig. 5 Theoretical calculations of the effect of solvent environment on hydrogen bond
29 structure. Calculated structure of the water-hydroxide hydrogen bond in (A) a polarizable
30
31 continuum of variable dielectric without explicit solvent or (B) a polarizable continuum with a
32
33 dielectric value of 5 with two explicit water molecules. Distances are in angstroms (Å).
34
35 Calculations were performed at the B3LYP level using the 6-311++G(d,p) basis set.
36
37
38
39 To test whether intramolecular hydrogen bonds display a similar or different sensitivity to
40
41 solvent dielectric compared to intermolecular hydrogen bonds, we performed analogous QM
42
43
44 calculations for the intramolecular hydrogen bond in 5-methylsalicylate, one of the compounds
45
46 studied experimentally herein (Table S3). Our calculations suggest that the O•••O and H•••O
47
48 distances within this hydrogen bond increase by ≤0.02 Å as the dielectric increases from 5 to 80,
49
50
51 supporting our conclusion that the structures of inter- and intramolecular hydrogen bonds display
52
53 a similarlyverylow sensitivity to solvent dielectric.
54
55
Calculations using a continuum solvation model also afforded a straightforward means to
56
57
58 test the effect of increasing solvent dielectric from 5 to 80 on the one-dimensional potential
59
60
19
1
2
3
energy surface of the water-hydroxide ion hydrogen bond. These computations, summarized in
4
5
6 Fig. S7 and discussed in more detail in Text S5, suggest that the shape of the potential energy
7
8 well for O-H•••O- hydrogen bonds is very similar at dielectric constants of 5 to 80, in agreement
9
10
11
with our conclusion based on the 2H NMR studies above.
12
13 To address the computationally more complex question of whether specific hydrogen
14
15 bonding interactions between solutes and explicit solvating water molecules substantially
16
17
18 lengthen hydrogen bonds compared to aprotic environments, we performed QM calculations on
19
20 the water-hydroxide ion dimer at a dielectric of 5 that included two additional explicit hydrating
21
22 water molecules (Fig. 5B), and we compared the calculated distances to those obtained above in
23
24
25 the absence of explicit hydration. These calculations gave a nearly identical O•••O distance in
26
27 the absence (2.535 Å) or presence (2.517 Å) of explicit hydrating water molecules (Fig. 5B),
28
29
consistent with the prior crystallographic observation that the presence or absence of two
30
31
32 hydrating water molecules had nearly no effect on the length of the 4-nitrophenol:4-
33
34 nitrophenolate hydrogen bond.32Prior QM studies that involved more extensive solvation shells
35
36
37 of explicit water molecules have also reported O•••O distances of 2.5-2.6 Å for the water-
38
39 hydroxide ion complex,57-59suggesting that the very small length difference observed in our
40
41 calculations plus or minus two hydrating water molecules is not substantially altered by inclusion
42
43
44 of a larger network of solvating water molecules. Overall, these calculations suggest that the
45
46 inductive effects on hydrogen bond structure from specific solvation by water molecules are
47
48 small and on the order of~0.02 Å, in contrast to prior suggestions in the literature that O-H•••O
49
50
51 hydrogen bonds are substantially lengthened in water relative to low polarity environments by as
52
53 much as several tenths of an angstrom.12,16
54
55
56
57
58
59
60
20
1
2
3
CONCLUSIONS AND IMPLICATIONS
4
5
6 Since Pauling’s seminal description in The Nature of the Chemical Bond,24 hydrogen
7
8 bonds have been recognized as ubiquitous and critical interactions whose structural and energetic
9
10
11
properties make dominant contributions to aqueous solvation and enormously influence the
12
13 fundamental architectures and activities of all biological macromolecules.Hydrogen bonds
14
15 formed within the complex interiors of folded biopolymers link together structural elements,
16
17
18 tether bound ligands, and stabilize charge localization during enzymatic catalysis.
19
20 Although hydrogen bonds are present in bulk solution and in protein interiors, the nature
21
22 of thesetwo environments differs greatly. In aqueous solution, dissolved solutes are surrounded
23
24
25 by extensive solvation shells of abundant water molecules, which can form hydrogen bonds with
26
27 solutes and aggressively compete for hydrogen bond formation between solutes. Protein
28
29
interiors, however, largely exclude water molecules and instead surround hydrogen-bonded
30
31
32 groups with highly anisotropic solvation environments composed of structured arrays of charged,
33
34 polar, and hydrophobic groups.
35
36
37 The effect of these solvation differences between the interior protein matrix and water on
38
39 the structural and energetic properties of hydrogen bonds has been the subject of considerable
40
41 debate and speculation in the literature and has remained unresolved.6,12,17,26,42,60In this work, we
42
43
44 have overcome longstanding technical limitationsto determinethe structural properties of
45
46 hydrogen-bonded solutes in water.These direct and systematic measurements, in combination
47
48 with prior studies of hydrogen bond formation free energies in water versus aprotic solvents,
49
50
51 elucidate the fundamental relationship between hydrogen bond length and stability in solution
52
53 and have importantimplications for understanding hydrogen bond contributions to biomolecular
54
55
56
function.
57
58
59
60
21
1
2
3
4
5
6 The structural properties of hydrogen bonds show little sensitivity to solvent environment.
7
8 We observed very similar structures for individual hydrogen bonds in widely differing solvent
9
10
11
and crystalline environments (Fig. S2B and S2C). Furthermore, the sensitivity of hydrogen bond
12
13 length to changes in the proton affinity of the hydrogen bond donor and/or acceptor (∆pKa) was
14
15 also nearly identical for diverse small molecules in water, aprotic solvents, and in crystals, with a
16
17
18 0.02 – 0.03 Å change in O•••O distance per unit change in ∆pKa (Fig. 2A, Fig. 6B, and Fig. S2).
19
20 This sensitivity is also nearly identical to that previously reported for a series of phenolates
21
22 bound to ketosteroid isomerase61,62(0.02 Å/pKaunit) or photoactive yellow protein53 (0.03
23
24
25 Å/pKaunit), providing additional evidence for similar hydrogen bond structural properties across
26
27 widely differing solvation environments.
28
29
Equilibrium distance is the most common read-out of hydrogen bond structure, but we
30
31
32 have also probed the shape of the potential energy well for O-H•••O–hydrogen bonds. High-level
33
34 QM calculations (Fig. S7) and indirect experimental measurements, including the 2H isotope
35
36
37 effect on the NMR chemical shift of the hydrogen-bonded proton (Fig. 4), suggest minimal
38
39 changes in the hydrogen bond potential energy surface acrosssolvents with widely varying
40
41 dielectrics (5–80)and hydrogen-bonding ability (water versus aprotic).
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22
1
2
3
Fig. 6. Hydrogen bond formation free energy but not hydrogen bond length is exquisitely
4
5 sensitive to solvent.Comparison of the dependence of hydrogen bond formation energy (∆Gf)
6
7 (A) versus hydrogen bond O•••O distance (B) on ∆pKa for substituted salicylates in an aprotic
8
9 organic solvent (red) versus water (blue). The ∆Gfvalues were previously published42 and were
10
11
determined in DMSO and water. The O•••O distances were determined from the 1H NMR
12 chemical shifts measured in chloroform and water as reported herein.
13
14
15
16
Hydrogen bonds are shorter than the sum of the van der Waals radii of the interacting
17
18
19 atoms, indicating a degree of orbital mixing and covalent character.24,63-65Some degree of partial
20
21 covalent character of hydrogen bonds could contribute to the minimal change in O-H•••O–
22
23
24
hydrogen bond structure across environments. Indeed, analogous covalent bond lengths in
25
26 organic molecules vary little (e.g., 0.01-0.02 Å)in different solvent environments.66,67The
27
28 constancy of hydrogen bond lengths between protic and aprotic solvents may also arise because
29
30
31 interaction energies of solvating water molecules with the oxygen atoms in an O-H•••O–
32
33 hydrogen bond are not sufficiently large, relative to the interaction energies within the O-H•••O–
34
35 complex, to perturb the electronic properties of the hydrogen-bonded groups and therebyreshape
36
37
38 the potential energy well (see additional discussion in Text S6).Consistent with this view, our
39
40 QM calculations (Fig. 5B) and prior theoretical57-59,68and crystallographic32studies suggest that
41
42
43
the electronic perturbations due to the formation of additional hydrogen bonds to hydrating water
44
45 molecules are small and result in only 0.01-0.02 Å changes in the length of a solute-solute
46
47 hydrogen bond over the conditions investigated herein.69 Indeed, even the presence of a second
48
49
50 short (pKa-matched) hydrogen bond only results in a minor <0.1 Å perturbation to a hydrogen
51
52 bond formed to a common acceptor atom (Fig. 5B),53,57,58 suggesting that even short hydrogen
53
54 bonds do not greatly perturb the accessibility of the valence electron cloud for making an
55
56
57 additional interaction.
58
59
60
23
1
2
3
4
5
6 Environment rather than hydrogen bond length is the primary driving force for hydrogen
7
8 bond stability. Our findings clarify the physical basis for the widely differing hydrogen bond
9
10
11
formation free energies for a given hydrogen bond pair in water versus aprotic environments.
12
13 Large energetic differences (>8 kcal/mol) in the stability of individual hydrogen bonds in these
14
15 discrete environments (Figs. 1A & 6A) are not accompanied by large differences in hydrogen
16
17
18 bond length (Fig. 6B) or potential energy surface, disproving the central tenet of Model II (Fig.
19
20 1C) that hydrogen bond length and stability are directly coupled for a given complex. Hydrogen
21
22 bond lengths for each compound studied changed only slightly (i.e., ~0.01-0.02 Å) in widely
23
24
25 differing environments, indicating that hydrogen bond length is not the primary driving force for
26
27 the more stable hydrogen bonding in low-dielectric and poorer solvating environments (see
28
29
additional discussion in Text S6). Prior investigators have questioned the general connection
30
31
32 between hydrogen bond structure and stability,2,12,68,70 and our work provides a direct and
33
34 systematic test of this relationship.
35
36
37 By ruling out a significant contribution from Model II, our resultsstrongly favor Model
38
39 I,2,13,14 that the differences in hydrogen bond formation free energy for a given complex in protic
40
41 versus aprotic solvents is dominated by the differential ability of these solvents to stabilize the
42
43
44 dissociated donor and acceptor groups (Fig. 1B, left side of equilibrium) (see additional
45
46 discussion in Text S6). Solvation energies for the hydrogen-bonded groups (Fig. 1B, right side of
47
48 equilibrium) are also expected to differ in water versus aprotic environments.However, as is
49
50
51 critical for the overall energetics of hydrogen bond formation, this difference is expected to be
52
53 smaller than the corresponding difference for the isolated charge of the dissociated acceptor
54
55
56
group. Our findings emphasize the need to understand the nature of the solvation environments
57
58
59
60
24
1
2
3
in which hydrogen bonds form and to consider interactions with both sides of the equilibrium
4
5
6 when analyzing hydrogen bond stability (∆Gf), or any other equilibrium process.
7
8
9
10
11
The origins of changes in hydrogen bond length and formation free energies (∆Gf) across a
12
13 series of hydrogen bond donors and acceptors.Although the length of a hydrogen bond for a
14
15 particular complex is largely insensitive to solvent environment, hydrogen bond lengths across a
16
17
18 series of compounds show a regular dependence on ∆pKa, the proton affinity difference between
19
20 the donor and acceptor groups (Fig. 2 and Fig. 6B).37,68This dependence on ∆pKa holds whether
21
22 the donor or acceptor is varied, and as noted above it displays a common dependence of 0.02-
23
24
25 0.03 Å (O•••O distance) per unit change in ∆pKa71regardless of solution, crystalline, or protein
26
27 environment(Fig. S2).53,61,62
28
29
Increased charge density of a hydrogen bond donor or acceptor group, which correlate
30
31
32 strongly with pKa,72,73presumably results in larger electrostatic attractive forces and greater
33
34 orbital mixing and covalent character between the hydrogen-bonded groups and thus shorter
35
36
37 hydrogen bond distances with decreasing ∆pKa. The formation free energy (∆Gf) for hydrogen-
38
39 bonded complexes also tends to vary linearly with ∆pKa for a homologous series of compounds
40
41 within a constant solvent environment.3,5,36,42 Overall, differences in hydrogen bond ∆Gf values
42
43
44 across a series of compounds reflect changes in the hydrogen bond potential energy, from
45
46 contributions classically ascribed to ionic and covalent factors, as well as differential solvation
47
48 effectsfor the free and hydrogen-bonded species for each complex in the series for each given
49
50
51 solvent (see additional discussion in Text S6).
52
53
54
55
56
57
58
59
60
25
1
2
3
Short hydrogen bonds inside proteins do not provide a read-out of hydrogen bond stability
4
5
6 or the nature of the local electrostatic environment. Structural studies have identified short
7
8 hydrogen bond distances (e.g. 2.4 – 2.6 Å) in the interior of numerous proteins, where they have
9
10
11
been suggested to form due to unique properties of the solvent-sequestered protein matrix that
12
13 differ from water and to make substantial energetic contributions to protein stability and function
14
15 (e.g. catalysis).6,11,19 Our results demonstrate that formation of a short hydrogen bond within a
16
17
18 protein or in other environments does not provide a read-out of the local solvation environment
19
20 or hydrogen bond stability, as short lengths appear to be independent of solvent properties and
21
22 are not tightly coupled to formation energy (Fig. 6).
23
24
25 Several observations suggest the presence of slightly shorter hydrogen bonds within
26
27 protein interiors than in other protein environments.17,74 Our results demonstrate that such
28
29
differences are not expected from and do not reflect the local protein solvation environment and
30
31
32 underscore the importance of exploring multiple potential origins. We speculate that packing and
33
34 binding interactions within the anisotropic protein interior may constrain hydrogen bonds from
35
36
37 relaxing to a preferred geometry in some instances, and such constraints might at times favor
38
39 shorter17,74 or longer53,75 hydrogen bonds depending on the idiosyncrasies of the local structural
40
41 environment. These complexities emphasize the need for direct, systematic tests of the structural
42
43
44 and energetic contributions from hydrogen bonds in enzyme interiors76,77, rather than inferring
45
46 such contributions from distance observations alone.
47
48
49
50
51 Why do enzymes exclude water? Recognition that hydrogen bond formation is generally weak
52
53 in water compared to aprotic environments has led to proposals that hydrogen bonds are stronger
54
55
56
(i.e.,more stable) within the non-aqueous protein matrix because water molecules are largely
57
58
59
60
26
1
2
3
excluded.11,17 Indeed, it is often stated that enzymes have evolved flexible surface loops or
4
5
6 domains that close over active sites upon substrate binding in order to exclude water and thus to
7
8 strengthen hydrogen bond formation.6,11,26,27,78-80
9
10
11
Researchers have suggested that hydrogen bonds adopt shorter distances within
12
13 enzymesupon better matchingof proton affinities (pKavalues) between protein hydrogen bond
14
15 donor groups and substrate acceptor groups in the reaction transition state due to water exclusion
16
17
18 and that such shortening provides a mechanism to enhance hydrogen bond strength (i.e. potential
19
20 energy) and thus stability.6,11,19However, as noted above, our results provide direct evidence that
21
22 the sensitivity of hydrogen bond distance to ∆pKa is not strongly influenced by solvation
23
24
25 environment or directly coupled to stability (Fig. 6A & 6B), suggesting that modulation of
26
27 hydrogen bond structure via water exclusion is not a general mechanism for proteins to enhance
28
29
hydrogen bond formation free energies.
30
31
32 Others have taken note of the general increase in hydrogen bond stability within non-
33
34 aqueous environments and proposed that enzymes might exclude water in order to decrease the
35
36
37 local polarity of active site as a mechanism to strengthen hydrogen bond formation and increase
38
39 catalysis.17,26,27To provide catalysis, however, enzymesmust stabilize transition states relative to
40
41 the stability of such species in aqueous solution. Enhanced stability of a hydrogen-bonded
42
43
44 complex, which occursfor the case when both the dissociated groups and the complex arealready
45
46 in a non-aqueous environment (Fig. 1B, bottom equilibrium),does not indicate that the hydrogen-
47
48 bonded complex in a low polarity environment, in the absence of other active site features, is
49
50
51 intrinsically more stable than that same complex in water and can thereby provide
52
53 catalysis.Indeed, a destabilizing rather than stabilizing effect for a charged or polar hydrogen-
54
55
56
bonded complex is expected upon lowering the environment polarity (Fig. S8). In other words,
57
58
59
60
27
1
2
3
all else being equal, the polar or charged complex would prefer to remain in the solvent of higher
4
5
6 polarity or dielectric.
7
8 Wesuggest that enzymes have evolved domain closures and tightly packed active sites
9
10
11
that largely exclude water molecules to optimally position and interact with bound substrates and
12
13 thus maximize favorable binding interactions withactive site groups whose electrostatic nature
14
15 and geometric positioningare complementary to the shape and charge distribution of reaction
16
17
18 transition state.It remains an ongoing experimental and computational challenge to understand
19
20 the electrostatic nature of enzyme active sites, to fully decipher the physical consequences of
21
22 excluding water, and to test the relative contributions to catalysis from positioning and charge
23
24
25 stabilization and indeed to determine whether such factors are formally separable.60,75,81-85
26
27
28
29
Implications for computational models. The strong correlation of hydrogen bond length with
30
31
32 ∆pKa suggests that expected values from such correlations might be used as benchmarks for
33
34 computation and as simple checks of X-ray structural models. As there is evidence in certain
35
36
37 situations for perturbations of hydrogen bond lengths by local structural features within
38
39 proteins,53,75 a substantial mismatch to an expected value could indicate model error or
40
41 interesting local interactions. In addition, as force fields often include empirical corrections,
42
43
44 including terms to constrain hydrogen bond lengths could improve model accuracy. On an
45
46 additional practical level, our observations suggest that molecular dynamics force fields will not
47
48 require terms that mimic quantum mechanical coupling between solvent molecules and
49
50
51 hydrogen-bonded complexes to adjust the shape and depth of potential energy wells for
52
53 hydrogen bonds in different environments. In addition, the near constancy of hydrogen bond
54
55
56
distances across the range of solvation conditions and dielectric constants of 5-80 studied herein
57
58
59
60
28
1
2
3
raises the possibility that benchmarking computational force fields to gas phase data may, in
4
5
6 some instances, create unnecessary complications through the need for additional terms to adjust
7
8 the hydrogen bond properties from those at very low dielectrics (i.e., 1-4) to those observed
9
10
11
across a broad range of solvation conditions at higher solvent dielectrics.
12
13
14
15 MATERIALS AND METHODS
16
17
18
A full description of all experimental and computational methods is given in Supporting
19
20 Information. Small-molecule neutron structures were determined experimentally at the Intense
21
22 Pulsed Neutron Source (Argonne National Laboratory) or retrieved from the Cambridge
23
24
25 Structural Database and analyzed for hydrogen bond distances. ∆pKa values were assigned based
26
27 on experimental values or computational prediction. 1H and 2H NMR spectra were acquired on
28
29 UNITY
800 or 600 MHz Varian INOVA spectrometers equipped with a 5-mm, triple-resonance,
30
31
32 gradient 1H(13C/15N) probe or a 10 mm broadband probe. NMR samples consisted of 25 mM
33
34 substituted salicylic acid and triethylamine (TEA) in 100% water (containing 5% D2O), acetone-
35
36 d6 (with 0.05% tetramethylsilane, TMS), or CDCl3 (with 0.05% TMS). Spectra for samples
37
38
39 dissolved in acetone-d6, chloroform-d6, and DMSO-d6 were acquired using the s2pul pulse
40
41 sequence, and chemical shifts were referenced internally to TMS (0 ppm). Spectra for aqueous
42
43
44
samples were acquired using the 1331 binomial pulse sequence for water suppression, a 30 ppm
45
46 spectral width (carrier frequency set on the water resonance), an excitation maximum of 14–17
47
48 ppm, and chemical shift referencing to the water resonance (5.0 ppm at 4 ˚C). Hydrogen bond
49
50
51 distances were estimated from the measured 1H NMR chemical shifts for the detected salicylate
52
53 hydrogen-bonded protons using published correlation functions.34,44 QM calculations were
54
55 performed at the HF, B3LYP, and MP2 levels using multiple basis sets.
56
57
58
59
60
29
1
2
3
ASSOCIATED CONTENT
4
5
6 Supporting Information.
7
8
9 Additional discussion of hydrogen bond structure and energetics, additional analysis and
10
11 comparison of structural properties of intra- and intermolecular hydrogen bonds, crystallographic
12
13 coordinates and analysis of neutron structures, crystallographic data and refinement statistics,
14
15 and QM calculated hydrogen bond distances. This material is available free of charge via the
16
17
Internet at http://pubs.acs.org.
18
19
20
21 AUTHOR INFORMATION
22
23 Corresponding Author
24 *
25 herschla@stanford.edu
26
27
28
29
30 Notes
31
32 The authors declare no competing financial interest.
33
34
35
36 ACKNOWLEDGEMENTS
37
38 We thank J. Schwans, M. Miller, and P. Almond for assistance with NMR sample preparation,
39
40 neutron crystallography, and X-ray crystallography, respectively. We thank J. Bowie, S.
41 Benkovic, B. Cravatt, P. Kim, and C. Perrin for critical feedback on the manuscript. Funding was
42
43 provided by NSF grant MCB-1121778 to D.H. P.A.S. is a recipient of a Burroughs Wellcome
44
45 Fund Career Award at the Scientific Interface. Research at Argonne National Laboratory was
46
47 supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences,
48
Materials Sciences and Engineering Division (Argonne Contract DE-AC02-06CH11357).
49
50
51
52
53 REFERENCES
54
55 (1) We intentionally refrain from using the term “hydrogen bond strength”. This term, although
56 widely adopted in the literature, lacks a precise or universally agreed definition but has typically referred
57
58
59
60
30
1
2
3 to either a formation enthalpy (∆Hf) or free energy (∆Gf) change that reflects the energetic difference
4
between the hydrogen-bonded complex and the dissociated donor and acceptor groups.
5
6
(2) Guthrie, J. P. Chem. Biol.1996, 3, 163-170.
7 (3) Stahl, N.; Jencks, W. P. J. Am. Chem. Soc.1986, 108, 4196-4205.
8 (4) Shan, S. O.; Loh, S.; Herschlag, D. Science1996, 272, 97-101.
9 (5) Magonski, J.; Pawlak, Z.; Jasinski, T. J. Chem. Soc. Faraday Trans.1993, 89, 119-122.
10 (6) Cleland, W. W.; Frey, P. A.; Gerlt, J. A. J. Biol. Chem.1998, 273, 25529-25532.
11 (7) Cleland, W. W. Arch. Biochem. Biophys.2000, 382, 1-5.
12 (8) Hibbert, F.; Emsley, J. Adv. Phys. Org. Chem.1990, 26, 255-379.
13 (9) Zhao, Q. J.; Abeygunawardana, C.; Talalay, P.; Mildvan, A. S. Proc. Natl. Acad. Sci. U.S.A.1996,
14 93, 8220-8224.
15 (10) Perrin, C. L.; Nielson, J. B. Annu Rev Phys Chem1997, 48, 511-544.
16 (11) Frey, P. A.; Whitt, S. A.; Tobin, J. B. Science1994, 264, 1927-1930.
17
(12) Warshel, A.; Papazyan, A. Proc. Natl. Acad. Sci. U.S.A.1996, 93, 13665-13670.
18
19
(13) Pasalic, H.; Aquino, A. J.; Tunega, D.; Haberhauer, G.; Gerzabek, M. H.; Georg, H. C.; Moraes,
20 T. F.; Coutinho, K.; Canuto, S.; Lischka, H. J. Comput. Chem.2010, 31, 2046-2055.
21 (14) Pasalic, H.; Tunega, D.; Aquino, A. J.; Haberhauer, G.; Gerzabek, M. H.; Lischka, H. Phys.
22 Chem. Chem. Phys.2012, 14, 4162-4170.
23 (15) Kreevoy, M. M.; Liang, T. M. J. Am. Chem. Soc.1980, 102, 3315-3322.
24 (16) Gerlt, J. A.; Kreevoy, M. M.; Cleland, W. W.; Frey, P. A. Chem. Biol.1997, 4, 259-267.
25 (17) Gao, J.; Bosco, D. A.; Powers, E. T.; Kelly, J. W. Nat. Struct. Mol. Biol.2009, 16, 684-690.
26 (18) Bowie, J. U. Curr. Opin. Struct. Biol.2011, 21, 42-49.
27 (19) Cleland, W. W.; Kreevoy, M. M. Science1994, 264, 1887-1890.
28 (20) Lin, I. J.; Gebel, E. B.; Machonkin, T. E.; Westler, W. M.; Markley, J. L. Proc. Natl. Acad. Sci.
29 U.S.A.2005, 102, 14581-14586.
30
(21) Feierberg, I.; Aqvist, J. Biochemistry2002, 41, 15728-15735.
31
32 (22) Perrin, C. L. Science1994, 266, 1665-1668.
33 (23) Mildvan, A. S.; Harris, T. K.; Abeygunawardana, C. Methods Enzymol.1999, 308, 219-245.
34 (24) Pauling, L. The Nature of the Chemical Bond; Cornell University Press: Ithaca, 1960.
35 (25) Jeffrey, G. A. An Introduction to Hydrogen Bonding; Oxford University Press: New York, 1997.
36 (26) Richard, J. P.; Amyes, T. L.; Goryanova, B.; Zhai, X. Curr. Opin. Chem. Biol.2014, 21C, 1-10.
37 (27) Dewar, M. J.; Storch, D. M. Proc. Natl. Acad. Sci. U. S. A.1985, 82, 2225-2229.
38 (28) Lin, J.; Frey, P. A. J. Am. Chem. Soc.2000, 122, 11258-11259.
39 (29) Mock, W. L.; Morsch, L. A. Tetrahedron2001, 57, 2957-2964.
40 (30) Hess, R. A.; Reinhardt, L. A. J. Am. Chem. Soc.1999, 121, 9867-9870.
41 (31) Frey, P. A.; Cleland, W. W. Bioorg. Chem.1998, 26, 175-192.
42 (32) Marimanikkuppam, S. S.; Lee, I. S. H.; Binder, D. A.; Young, V. G.; Kreevoy, M. M. Croat.
43
Chem. Acta1996, 69, 1661-1674.
44
45 (33) Venables, D. S.; Schmuttenmaer, C. A. J. Chem. Phys.2000, 113, 11222-11236.
46 (34) Jeffrey, G. A.; Yeon, Y. Acta Crystallogr., Sect. B: Struct. Sci.1986, 42, 410-413.
47 (35) Steiner, T.; Saenger, W. Acta Crystallogr., Sect. B: Struct. Sci.1994, 50, 348-357.
48 (36) Shan, S. O.; Herschlag, D. Methods Enzymol.1999, 308, 246-276.
49 (37) Gilli, P.; Pretto, L.; Bertolasi, V.; Gilli, G. Acc. Chem. Res.2009, 42, 33-44.
50 (38) Brycki, B.; Brzezinski, B.; Zundel, G.; Keil, T. Magn. Reson. Chem.1992, 30, 507-510.
51 (39) Zundel, G. Adv. Chem. Phys.2000, 111, 1-217.
52 (40) Roscioli, J. R.; McCunn, L. R.; Johnson, M. A. Science2007, 316, 249-254.
53 (41) Brycki, B.; Szafran, M. J. Chem. Soc. Perkin. Trans. II1984, 223-226.
54 (42) Shan, S. O.; Herschlag, D. Proc. Natl. Acad. Sci. U.S.A.1996, 93, 14474-14479.
55 (43) McDermott, A.; Rydenour, C. F. In Encyl. NMR; Grant, D. M., Harris, R. K., Eds.; John Wiley
56
and Sons (Sussex, England): 1996, p 3820-3825.
57
58 (44) Harris, T. K.; Mildvan, A. S. Proteins1999, 35, 275-282.
59
60
31
1
2
3 (45) Tolstoy, P. M.; Schah-Mohammedi, P.; Smirnov, S. N.; Golubev, N. S.; Denisov, G. S.; Limbach,
4
H. H. J. Am. Chem. Soc.2004, 126, 5621-5634.
5
6
(46) Harris, R. K.; Jackson, P.; Merwin, L. H.; Say, B. J.; Hagele, G. J. Chem. Soc. Faraday Trans.
7 I1988, 84, 3649-3672.
8 (47) Rohling, C. M.; Allen, L. C.; Ditchfield, R. J. Chem. Phys.1983, 79, 4958-4966.
9 (48) Koeppe, B.; Guo, J.; Tolstoy, P. M.; Denisov, G. S.; Limbach, H. H. J. Am. Chem. Soc.2013, 135,
10 7553-7566.
11 (49) The change in O•••O distance between 3,5-dinitrosalicylate and 5-nitrosalicylate estimated by
12 solution 1H NMR (0.055 Å) is very similar to the distance difference (0.046 Å) observed between the
13 neutron structure of 3,5-dinitrosalicylate (O•••O distance = 2.516 Å, Table S1) and an unpublished X-ray
14 structure of 5-nitrosalicylate (O•••O distance 2.562 Å), providing additional support for our use of NMR
15 to estimate changes in salicylate hydrogen bond lengths.
16 (50) Ichikawa, M. Acta Crystallogr., Sect. B: Struct. Sci.1978, 34, 2074-2080.
17
(51) Ichikawa, M. J Mol Struct2000, 552, 63-70.
18
19
(52) Altman, L. J.; Laungani, D.; Gunnarsson, G.; Wennerstrom, H.; Forsen, S. J. Am. Chem.
20 Soc.1978, 100, 8264-8266.
21 (53) Sigala, P. A.; Tsuchida, M. A.; Herschlag, D. Proc. Natl. Acad. Sci. U.S.A.2009, 106, 9232-9237.
22 (54) Guillot, B. J. Mol. Liq.2002, 101, 219-260.
23 (55) Jorgensen, W. L.; Tirado-Rives, J. Proc. Natl. Acad. Sci. U.S.A.2005, 102, 6665-6670.
24 (56) Tomasi, J.; Mennucci, B.; Cammi, R. Chem. Rev.2005, 105, 2999-3093.
25 (57) Pliego, J. R.; Riveros, J. M. J. Chem. Phys.2000, 112, 4045-4052.
26 (58) Lee, H. M.; Tarkeshwar, P.; Kim, K. S. J. Chem. Phys.2004, 121, 4657-4664.
27 (59) Chaudhuri, C.; Wang, Y. S.; Jiang, J. C.; Lee, Y. T.; Chang, H. C.; Niedner-Schatteburg, G. Mol.
28 Phys.2001, 99, 1161-1173.
29 (60) Warshel, A.; Aqvist, J.; Creighton, S. Proc. Natl. Acad. Sci. U. S. A.1989, 86, 5820-5824.
30
(61) Kraut, D. A.; Sigala, P. A.; Pybus, B.; Liu, C. W.; Ringe, D.; Petsko, G. A.; Herschlag, D. PLoS
31
32 Biol.2006, 4, e99.
33 (62) Hanoian, P.; Sigala, P. A.; Herschlag, D.; Hammes-Schiffer, S. Biochemistry2010, 49, 10339-
34 10348.
35 (63) Isaacs, E. D.; Shukla, A.; Platzman, P. M.; Hamann, D. R.; Barbiellini, B.; Tulk, C. A. Phys. Rev.
36 Lett.1999, 83, 4445-4445.
37 (64) Stevens, E. D.; Lehmann, M. S.; Coppens, P. J. Am. Chem. Soc.1977, 99, 2829-2831.
38 (65) Grzesiek, S.; Cordier, F.; Dingley, A. J. Methods Enzymol.2001, 338, 111-133.
39 (66) Torii, H.; Tatsumi, T.; Tasumi, M. J. Raman Spectrosc.1998, 29, 537-546.
40 (67) Andzelm, J.; Kolmel, C.; Klamt, A. J. Chem. Phys.1995, 103, 9312-9320.
41 (68) Chen, J. G.; McAllister, M. A.; Lee, J. K.; Houk, K. N. J. Org. Chem.1998, 63, 4611-4619.
42 (69) Our computations and prior results suggest that larger differences in hydrogen bond O•••O or
43
H•••O distance on the scale of 0.03 – 0.1 Å or larger may occur when comparing distances calculated at
44
45 dielectric values ≥5 to calculations at dielectric values of 1.0 (vacuum) or 1.4 (argon gas) (Table S3). As
46 the physical transition from a vacuum to solvent (including polarizable continuum models) is complex,
47 additional theoretical work will be needed to understand structural changes in this very low dielectric
48 region.
49 (70) Perrin, C. L. Acc. Chem. Res.2011, 43, 1550-1557.
50 (71) We emphasize that we have used a common pKa scale for all compounds in water, regardless of
51 the specific solvent environment studied, and that the value of ∆pKa itself is not the cause of the varying
52 hydrogen bond length but rather shares a common sensitivity with hydrogen bond length to other
53 molecular properties, especially atomic charge density.
54 (72) Gross, K. C.; Seybold, P. G. Int. J. Quantum Chem.2001, 85, 569-579.
55 (73) Gross, K. C.; Seybold, P. G.; Hadad, C. M. Int. J. Quantum Chem.2002, 90, 445-458.
56
(74) Joh, N. H.; Min, A.; Faham, S.; Whitelegge, J. P.; Yang, D.; Woods, V. L.; Bowie, J. U.
57
58 Nature2008, 453, 1266-U1273.
59
60
32
1
2
3 (75) Sigala, P. A.; Kraut, D. A.; Caaveiro, J. M.; Pybus, B.; Ruben, E. A.; Ringe, D.; Petsko, G. A.;
4
Herschlag, D. J. Am. Chem. Soc.2008, 130, 13696-13708.
5
6
(76) Kraut, D. A.; Sigala, P. A.; Fenn, T. D.; Herschlag, D. Proc. Natl. Acad. Sci. U.S.A.2010, 107,
7 1960-1965.
8 (77) Schwans, J. P.; Sunden, F.; Gonzalez, A.; Tsai, Y. S.; Herschlag, D. J. Am. Chem. Soc.2011, 133,
9 20052-20055.
10 (78) Malabanan, M. M.; Amyes, T. L.; Richard, J. P. Curr. Opin. Struct. Biol.2010, 20, 702-710.
11 (79) Perutz, M. Proc. Roy. Soc. London. Series B, Biol. Sci.1967, 167, 448.
12 (80) Wierenga, R. K.; Kapetaniou, E. G.; Venkatesan, R. Cell. Mol. Life Sci.2010, 67, 3961-3982.
13 (81) Fafarman, A. T.; Sigala, P. A.; Schwans, J. P.; Fenn, T. D.; Herschlag, D.; Boxer, S. G. Proc.
14 Natl. Acad. Sci. U. S. A.2012, 109, E299-308.
15 (82) Natarajan, A.; Schwans, J. P.; Herschlag, D. J. Am. Chem. Soc.2014, 136, 7643-7654.
16 (83) Sigala, P. A.; Fafarman, A. T.; Bogard, P. E.; Boxer, S. G.; Herschlag, D. J. Am. Chem. Soc.2007,
17
129, 12104-12105.
18
19
(84) Jha, S. K.; Ji, M.; Gaffney, K. J.; Boxer, S. G. Proc. Natl. Acad. Sci. U. S. A.2011, 108, 16612-
20 16617.
21 (85) Chimenti, M. S.; Castaneda, C. A.; Majumdar, A.; Garcia-Moreno, E. B. J. Mol. Biol.2011, 405,
22 361-377.
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
33
1
2
3
4
5
6
7
8 TABLE OF CONTENT GRAPHIC
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
34

Sigal A 2015

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Sigal A 2015

Uploaded by

Copyright:

Available Formats

Subscriber access provided by West Virginia University | Libraries

Journal of the American Chemical Society is published by the American Chemical

You might also like