Thioamide, a Hydrogen Bond Acceptor in Proteins and Nucleic Acids

Subscriber access provided by University of Colorado Boulder

Letter

Thioamide, a Hydrogen Bond Acceptor in Proteins and Nucleic Acids V. Rao Mundlapati, Sanjeev Gautam, Dipak Kumar Sahoo, Arindam Ghosh, and Himansu S. Biswal J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b01810 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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.

The Journal of Physical Chemistry Letters 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 26

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

The Journal of Physical Chemistry Letters

Thioamide, a Hydrogen Bond Acceptor in Proteins and Nucleic Acids V. Rao Mundlapati†,‡, Sanjeev Gautam †,‡, Dipak Kumar Sahoo†,‡, Arindam Ghosh †,‡ and Himansu S. Biswal*,†,‡ †

School of Chemical Sciences, National Institute of Science Education and Research

(NISER), PO- Bhimpur-Padanpur, Via-Jatni, District- Khurda, PIN - 752050, Bhubaneswar, India ‡

Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai

400094, India *Corresponding Author: Himansu S. Biswal E-mail: [email protected], Phone No: +91-674-2494 185/186

ACS Paragon Plus Environment


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

ABSTRACT: Thioamides are used as potential surrogates of amides to study the structure and dynamics of proteins and nucleic acids. However, incorporation of thioamides in biomolecules leads to changes in their structures and conformations mostly attributed to the strength of the amide-N-H•••S=C hydrogen bond. In most cases it is considered weak owing to small electronegativity of sulfur and in some as strong as conventional H-bonds. Herein, adopting PDB structure analysis, NMR spectroscopy and quantum chemistry calculations we have shown that thioamides in a geometrical and structural constraint free environment are capable of forming strong H-bonds like their amide counterparts. These studies also enabled us to determine the amide-N-H•••S=C H-bond enthalpy (ΔH) very precisely. The estimated ΔH for amide-N-H•••S=C H-bond is ~ -30 kJ/mol which suggests that the amide-N-H•••S=C H-bond is a strong H-bond and merits its inclusion in computational force fields for biomolecular structure simulations to explore the role of amide-N-H•••S=C H-bonds in nucleobase pairing and protein folding.

TOC GRAPHICS


Page 2 of 26

Page 3 of 26

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


The concept of hydrogen bond (H-bond) dates back a century1, but only less than a decade ago the concept was extended to third and fourth row elements of the periodic table.212

It is now established that sulfur of methionine and cysteine side chain can form strong H-

bonds in proteins.8-9 The formation of noticeably strong amide-N-H•••Se H-bonds with the selenium of selenomethionine in proteins has recently been reported.11 Contrary to the general consensus and given the electronegativity of sulfur is smaller than oxygen, sulfur forms surprisingly strong H-bonds. These cited few examples explicate the exigency of the understating of the H-bond. The aforementioned work deployed the structural data of proteins and gas phase infra red (IR) spectroscopy to ascertain the strength of amide-N-H•••S H-bonds in proteins. These high precision gas phase experimental data on sulfur centered H-bonds (SCHB) have been exploited by chemists and biochemists in recent times,13-17 e.g. F. Biedermann and H. J. Schneider in a review titled "Experimental Binding Energies in Supramolecular Complexes"16 enlisted solution phase H-bond energies of almost all type of H-bonds except SCHBs. This suggests that till today SCHBs have not been studied meticulously in solution. One of the striking examples of SCHBs that is yet to be addressed at the molecular level is the hydrogen bond abilities of thioamides in proteins and nucleic acids. Some of the experimental and theoretical reports on model molecular systems suggest that the thioamide-N-H is a stronger H-bond donor and the thioamide-C=S is a weaker H-bond acceptor than the corresponding amide counterparts.18-19 The opposite trend has also been noticed in many cases.20-21 These discrepancies become more apparent in biological molecules such as proteins and nucleic acids.22-28 It goes without saying that thioamide incorporation in biomolecules in place of amide is an active field of research in chemistry because of several differing properties that results due to this substitution. The H-bond ability of thioamides has been used extensively in solution phase to probe the structural aberration in proteins22-28 and nucleic acids29-35. For instance, Raines and co-workers observed that



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

incorporation of thioproline (Pros) in collagen triple helix stabilizes the triple helix whereas incorporation of Glys significantly destabilizes it.27 In an extensive work, Petersson and coworkers used stronger H-bond donor ability of thioamide-N-H and weaker H-bond acceptor ability of the thioamide-C=S than the amide-C=O to explain the stabilizing and destabilizing effects of thioamide backbone substitution in α-helix, β-sheet and polyproline type II helix secondary structure motif.28 It is hard to accept a similar reasoning in proteins where thousands of H-bonds are at play to optimize the protein structure. From the above few examples alone it is not straight forward to conclude that the thioamide-C=S is indeed a weaker H-bond acceptor in biomolecules. On the other hand, our earlier reports which suggested that divalent sulfur in methionine and cysteine are capable of forming very strong amide-N-H•••S H-bonds,8-11 encouraged us to further investigate the (thio)amide-N-H•••S=Cthio(amide) H-bonds in proteins and nucleic acids in a systematic manner. We first collected the atomic coordinates of proteins and nucleic acids from protein data bank (PDB)36, then used dispersion corrected DFT to fix H-atom positions by carrying out geometry optimization of 10-15 residues around the amide-N-H•••S=C H-bonds, while freezing all other atomic positions as in the PDB structure. We also performed natural bond orbital analysis (NBO) to find out donor (lone pair of S/O/N) and acceptor (σ*N-H) interaction energies (EDA) responsible for H-bonding. Figure -1 depicts few examples of proteins, nucleic acids and the interacting orbitals where thioamide-C=S is involved in H-bonding. The EDA values along with the H-bonded residues are provided in Table-S1 of supporting information (SI). The EDA values vary from 21 kJ/mol to 77 kJ/mol, suggesting that in some cases conformational constraints imposed by other residues restrict the N-H and C=S groups to come close enough to have substantial overlap between lone pair of S and σ*N-H to form strong H-bonds. However, the N-H•••S=C H-bond strengths are comparable to those of the N-H•••O=C and N-H•••N H-bonds in proteins and nucleic acids (see Table S1of SI)). Since no experimental


Page 4 of 26

Page 5 of 26

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


data of the N-H•••S=C H-bond energies are available, it is very difficult to arrive at any conclusion based on the NBO analysis alone. Hence, it is a pertinent question whether any conventional and standard spectroscopic methods can be employed to unveil the strength of N-H•••S=C H-bonds in solution. An obvious choice is NMR spectroscopy as this technique is capaciously utilized in solution to quantify several noncovalent interactions including Hbonds.37-40 Herein with the help of NMR spectroscopy we have reported for the first time accurate experimental N-H•••S=C H-bond energies in solution for the model systems without any conformational or structural restrictions.

Figure 1. Representative examples of amide-N-H•••S=C hydrogen bonds observed in (a) apoCalmodulin thioamide variant (PDB: 1qx5, Glus1007 substitution)41, H-bond between trans-amide-N-H (Met1011) and trans-thioamide C=S (Glus1007), (b) complex of 6thioguanosine monophosphate (6-thio-GMP) and NUDT15 ((PDB: 5lpg)42, H-bond between and trans-amide-N-H (Leu138) and cis-thioamide C=S (6-thio-GMP) (c) 11-mer DNA duplex containing 6-thioguanine (PDB:1kbm)29, H-bond between and cis-amide-N-H and cis-



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

thioamide C=S. The plots of donor-acceptor interacting natural bond orbitals (NBO) i.e. overlap of p-type sulfur/oxygen/nitrogen lone pair and N-H σ* for (d) 1qx5, (e) 5lpg and (f) 1kbm with the donor-acceptor interaction energies for N-H•••S=C H-bonds. As it is very difficult to determine the N-H•••S=C H-bond strength in biomolecules without interference from the environment and conformational constraints, we first screened 23 dimers consisting of cis(trans)-(thio)amide-N-H•••S(O) H-bonds keeping their relevance in biomolecules in mind . Figure 2 displays B97D-SMD/aug-cc-pVDZ optimized H-bond complexes of amides and thioamides with their binding enthalpies. The other complexes are provided in Figure S1 of SI. The H-bond enthalpies (ΔH) were calculated at the coupledcluster singles, doubles, and triples (CCSD (T)) level for the B97D optimized structures by using CCSD(T)/aug-cc-pVDZ electronic energies and B97D-SMD/aug-cc-pVDZ thermochemistry and solvation energies (taking CHCl3 as solvent, the detailed computational method is provided in the SI). The same solvent (CDCl3) was also chosen for the NMR experiments. Geometry optimization at lower level and single point energy calculation at the CCSD(T) level have been regularly employed to get accurate energetics of non-covalent interactions in biomolecules.43-44 The H-bond distances (dH•••X, X=O or S) and angles (∠NH•••X) of the optimized structures are provided in table S2. In most of the cases the NH•••O=C and N-H•••S=C H-bonds are found to be linear with average H-bond angles of 171° and 166°, respectively. The computed N-H•••S H-bond enthalpies are in the range of ~ -25 to -30 kJ/mol and are close to those of the N-H•••O H-bonds. The average H-bond enthalpy (ΔHavg.) for the N-H•••S H-bond is -25.2 kJ/mol and that of the N-H•••O H-bond is -27.6 kJ/mol. The N-H•••O=C H-bonds are considered as strong H-bonds45 and their H-bond enthalpies in non-polar solvents fall in the range of ~ -20 to -35 kJ/mol.46 Hence, the NH•••S=C H-bond can also be considered as a strong H-bond. The ΔH of homo and mixed dimers of 2-pyridone (2-PY) and 2-thipyridone (2-TPY) are ~ -60 kJ/mol as they account for


Page 6 of 26

Page 7 of 26

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


two H-bonds. The 2-PY and 2-TPY dimers (the strongest H-bond dimers) were chosen as model systems for experiments. The reasons of selecting 2-PY and 2-TPY are two folds: (a) they have very high ΔH values and hence, an appreciable amount of their dimers can be formed in solution enabling an easy detection, (b) the 1H NMR signal of NH (δNH > 10.5 ppm) are observed in the far lower field than aromatic CH (δCH > 6-8 ppm) and hence δNH can be monitored as a function of concentration or temperature without any interference of other 1

H NMR signals. The association constant or equilibrium constants (K) of 2 − PY ⇆

ሾ2 − PYሿଶ and 2 − TPY ⇆ ሾ2 − TPYሿଶ can be estimated by monitoring the equilibrium process as function of concentration or temperature since at high concentrations and low temperatures the formation of dimers is favorable.

Figure 2. Model molecular complexes used to study amide-N-H•••S and N-H•••O H-bonds in biomolecules. The structures were optimized at B97D-SMD/aug-cc-pvDZ level. The negative values correspond to the H-bond enthalpies in kJ/mol obtained at CCSD(T)-SMD/aug-cc-



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

pVDZ//B97D-SMD/aug-cc-pVDZ level of theory. The H-bond distances (dNH•••O and dNH•••S) and C=O and C=S bond lengths in Å are also shown in the figure. t: trans (thio)amide, c: cis (thio)amide, oo: amide-N-H•••O=C H-bond, so: thioamide-N-H•••O=C H-bond, os: amideN-H•••S=C H-bond, ss: thioamide-N-H•••S=C H-bond. 2-PY: 2-Pyridone, 2-TPY: 2Thiopyridone, NNDMA: N, N-dimethylacetamide, NNDMTA: N, N-dimethylthioacetamide, NMA: N-methylacetamide, NMTA: N-methylthioacetamide. As depicted in Figures 3(a) and 3(b), increase in concentration of 2-PY and 2-TPY caused downfield shifts of the NH peaks (1H NMR spectra are provided in Figure S2 of SI). This confirms the formation of dimers through N-H•••O and N-H•••S H-bonds at higher concentrations of 2-PY and 2-TPY, respectively. The variation of chemical shift (δNH) as a function of concentrations of 2-PY and 2-TPY could be fitted to a 1:1 binding isotherm, yielding association free energies (ΔGC, subscript “C” : concentration dependent studies) of 16.7 and -14.6 kJ/mol for 2-PY and 2-TPY, respectively. The ΔGC values are in reasonably good agreements with those computed at the CCSD(T) level (see Table-S3 of SI). It should be noted that ΔGC for N-H•••O and N-H•••S are very similar. Using the computed entropy (ΔS), the N-H•••O and N-H•••S H-bond enthalpies are estimated (ΔHC) to be -62.6 and -59.8 kJ/mol, respectively. Similar values of N-H•••O H-bond enthalpies are reported by Frey and Leutwyler for 2-pyridone⋅uracil and 2-pyridone⋅thymine Watson-Crick and Wobble isomers.47 This suggests that each N-H•••O and N-H•••S H-bond contribute ~ -30 kJ/mol to the total enthalpy and the N-H•••O and N-H•••S H-bond strengths are of similar magnitude. To ensure the existences of monomers at lower concentrations and dimers at higher concentrations, we measured translational diffusion coefficients at two extreme concentrations by fitting the data of the conventional diffusion-ordered spectroscopy (1H DOSY) experiment to the Stejskal–Tanner equation. In many recent works, DOSY has been used in investigating ionic-liquids48-50, supramolecular assembly and several noncovalent


Page 8 of 26

Page 9 of 26

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


interactions such as hydrogen and halogen bonds.51-52 Figures 3(c) and 3(d) show Stejskal– Tanner plots of logarithmic normalized signal strength (loge (I/I0)) versus squared gradient pulse power (G2), the slope is proportional to the diffusion coefficient.

Figure 3. (a,b) Concentration dependent NMR experiments for 2-PY and 2-TPY (solvent:CDCl3); (c,d) Fitting of Stejskal–Tanner equation to the decay of a spectral peak in a DOSY experiment; (e,f) Temperature dependent shifts of δNH

The proportionality constant depends on few experimental parameters and can be calculated readily. The ratios of diffusion coefficient of the monomer (Dm) to that of dimer (Dd) in both cases were found close to 1.2. The diffusion coefficient of a molecule (solute) in a solution is inversely proportional to the cube root of its molecular mass, if one assumes that the molecule is spherical, and temperature, density and viscosity of the solvent do not change in presence of the solute (here, 2-PY, 2-TPY and their dimers). The temperature dependent NMR studies were also carried out to estimate the H-bond enthalpies. Figures 3(e) and 3(f)



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

represent the chemical shift (δNH) variation with temperature. The temperature dependence of δNH is due to the equilibrium between monomer and dimer as 2 − PY ⇆ ሾ2 − PYሿଶ and 2 − TPY ⇆ ሾ2 − TPYሿଶ . Hence the plots 3(e) and 3(f) were fitted to a 1:1 binding isotherm yielding ΔHT (subscript “T”: temperature dependent studies) equals to -31.9 and -25.9 kJ/mol for 2-PY and 2-TPY, respectively. Huge discrepancies were observed between ΔHC and ΔHT values as obtained from concentration and temperature dependent NMR studies. ΔHT of ~ -15 kJ/mol for a strong amide-N-H•••O H-bond is too less. It is even less than the H-bond energy of Indole-Benzene dimer (-21.8 kJ/mol for an N-H•••π H-bond)53 and NH3-H2O dimers (-18.4 kJ/mol for an O-H•••N H-bond)54 as determined very precisely by gas phase laser spectroscopy experiments. In our opinion, the primary reason behind the discrepancy is the lack of enough data points in the temperature dependent studies. Due to infrastructure constraints we could not perform experiments below 250 K, whereas experiments above 350 K were not possible because of the limitation of the boiling point of the solvent (b. pt. of CHCl3 ~ 335 K). It is noteworthy to mention here that the concentration dependent NMR experiments provided better and precise information about the association free energies (ΔGC) and H-bond enthalpies (ΔHC). If one considers ΔHC, the H-bond enthalpy of an (thio)amide-N-H•••O and (thio)amide-N-H•••S H-bond could be ~ -25 to -30 kJ/mol. Finally we carried out deuterium exchange studies to show that N-H•••O and N-H•••S Hbond strengths are very similar. Figure 4 shows the area under the curve of the amide-NH peak in reference to different aromatic CH peaks in CDCl3 over time following the addition of D2O. The experiments were performed at high concentration of 2-PY and 2-TPY (75 mM) to ensure maximum presence of dimers in the solution. NMR-based amide hydrogen– deuterium exchange (H/D exchange) measurement has recently been used by T. Raines and coworkers to provide evidences of the intraresidue C5 hydrogen bond in proteins.55 Here we have used H/D exchange rates (kex) to compare the N-H•••O and N-H•••S H-bonds. The kex


Page 10 of 26

Page 11 of 26

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


(3-4 × 10-2 min-1) for 2-PY and 2-TPY dimers are of equal order of magnitude; corroborating the CCSD(T) energetics and concentration dependent NMR studies.

Figure 4. Integration of amide 1H NMR peaks of (a, c) 2-PY and (b, d) 2-TPY in CDCl3 over time following the addition of D2O.

In conclusion, although the amide-N-H•••S H-bonds have been widely studied in gas phase jet cooled condition as relevant contributors to biomolecular structures, attesting their existences and strengths in solution have been arduous owing to solvent and temperature effects and underestimated H-bond energies. The 2-thiopyridone dimer bestows an appropriate opportunity to detect and measure the (thio)amide-N-H•••S H-bond enthalpy in solution. Several NMR experiments viz. concentration and temperature dependent studies, DOSY and deuterium exchange studies confirm that the (thio)amide-N-H•••S H-bonds are as strong as the classical amide N-H•••O/N-H•••O=C H-bonds. The experimental (thio)amideN-H•••S H-bond energy is ~ -30 kJ/mol, which is appreciably larger than previously anticipated for a SCHB. The concentration dependent NMR experiments provided precise determination of Gibbs free energy and H-bond enthalpy of the amide–N-H•••S H-bond that matched well with the bench mark calculations at the CCSD(T) level. Only consideration of



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

electronegativity of S is inadequate to explain why the amide-N-H•••S H-bonds are equally strong as the classical amide N-H•••O/N-H•••O=C H-bonds. As reported earlier4, 11 it is the combined effect of electronegativity, charge and polarizability of S that determines the strength of amide-N-H•••S H-bonds. We hope, this interesting outcome will definitely augment fundamental understanding of non-covalent interactions and to contrive and concede H-bonds beyond the concept of electronegativity. All the experiments were carried out in solution phase with 400MHz (9.41 Tesla) Bruker Avance-III Nanobay liquid state NMR spectrometer. The geometry optimization and frequency calculations were carried out at B97D-SMD/aug-cc-pVDZ level of theory. The Hbond enthalpies were estimated at CCSD(T)-SMD/aug-cc-pVDZ// B97D-SMD/aug-ccpVDZ. The natural bond orbital (NBO)56 analysis was carried out at MP2/aug-cc-pVDZ level. Turbomole 6.5,57 Gaussian0958 and NBO-6.059 were employed to perform all the computations. Further details of experimental and computational methods used in this study are provided in the Supporting Information. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Details of experimental and computational methods; Experimental H-bond enthalpies and free energies; CCSD(T) energetics; NMR spectra; Cartesian coordinates of the optimized model compounds AUTHOR INFORMATION Corresponding Author


Page 12 of 26

Page 13 of 26

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


*Email:[email protected]; Phone: +91-674-2494 186/185 ORCID Himansu S. Biswal: 0000-0003-0791-2259; Arindam Ghosh: 0000-0002-6963-9879; Dipak Kumar Sahoo: 0000-0002-6900-3897; Sanjeev Gautam: 0000-0002-5607-1305; V. Rao Mundlapati: 0000-0003-0559-9684 Author Contributions H.S.B. conceived the project and planned the experiments. V.R.M., A. G. and S.G. performed the experiments. H.S.B., V. R.M. and D. K. S. did the computation. V. R. M and H. S. B. carried out the protein structure analysis and wrote the manuscript. All the authors discussed the results and made comments on the manuscript. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT The authors acknowledge department of atomic energy (DAE), Govt. of India for the financial support. The authors thank Prof. A. C. Dash for many valuable discussions. REFERENCES 1.

Moore, T. S.; Winmill, T. F. CLXXVII.-The State of Amines in Aqueous Solution. J.

Chem. Soc. Trans. 1912, 101, 1635-1676. 2.

Biswal, H. S.; Shirhatti, P. R.; Wategaonkar, S. O−H•••O Versus O−H•••S Hydrogen

Bonding I: Experimental and Computational Studies on the P-Cresol•H2O and P-Cresol•H2S Complexes. J. Phys. Chem. A 2009, 113, 5633-5643.



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

3.

Biswal, H. S.; Wategaonkar, S. Sulfur, Not Too Far Behind O, N, and C: S-H•••π

Hydrogen Bond. J. Phys. Chem. A 2009, 113, 12774-12782. 4.

Biswal, H. S.; Wategaonkar, S. Nature of the N-H•••S Hydrogen Bond. J. Phys.

Chem. A 2009, 113, 12763-12773. 5.

Hansen, A. S.; Du, L.; Kjaergaard, H. G. Positively Charged Phosphorus as a

Hydrogen Bond Acceptor. J. Phys. Chem. Lett. 2014, 5, 4225-4231. 6.

Das, B.; Chakraborty, A.; Chakraborty, S. Effect of Ionic Charge on OH•••Se

Hydrogen Bond: A Computational Study. Comp. Theor. Chem. 2017, 1102, 127-138. 7.

Biswal, H. S.; Bhattacharyya, S.; Bhattacherjee, A.; Wategaonkar, S. Nature and

Strength of Sulfur-Centred Hydrogen Bonds: Laser Spectroscopic Investigations in the Gas Phase and Quantum-Chemical Calculations. Int. Rev. Phys. Chem. 2015, 34, 99-160. 8.

Biswal, H. S.; Gloaguen, E.; Loquais, Y.; Tardivel, B.; Mons, M. Strength of N-H•••S

Hydrogen Bonds in Methionine Residues Revealed by Gas-Phase IR/UV Spectroscopy. J. Phys. Chem. Lett. 2012, 3, 755-759. 9.

Alauddin, M.; Biswal, H. S.; Gloaguen, E.; Mons, M. Intra-Residue Interactions in

Proteins: Interplay between Serine or Cysteine Side Chains and Backbone Conformations, Revealed by Laser Spectroscopy of Isolated Model Peptides. Phys. Chem. Chem. Phys. 2015, 17, 2169-2178. 10. Mundlapati, V. R.; Ghosh, S.; Bhattacherjee, A.; Tiwari, P.; Biswal, H. S. Critical Assessment of the Strength of Hydrogen Bonds between the Sulfur Atom of Methionine/Cysteine and Backbone Amides in Proteins. J. Phys. Chem. Lett. 2015, 6, 13851389.


Page 14 of 26

Page 15 of 26

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


11. Mundlapati, V. R.; Sahoo, D. K.; Ghosh, S.; Purame, U. K.; Pandey, S.; Acharya, R.; Pal, N.; Tiwari, P.; Biswal, H. S. Spectroscopic Evidences for Strong Hydrogen Bonds with Selenomethionine in Proteins. J. Phys. Chem. Lett. 2017, 8, 794-800. 12. Wang, D.; Fujii, A. Structures of Protonated Hydrogen Sulfide Clusters, H+(H2S)n, Highlighting the Nature of Sulfur-Centered Intermolecular Interactions. Phys Chem Chem Phys 2017, 19, 2036-2043. 13. Takahashi, O.; Kohno, Y.; Nishio, M. Relevance of Weak Hydrogen Bonds in the Conformation of Organic Compounds and Bioconjugates: Evidence from Recent Experimental Data and High-Level ab initio MO Calculations. Chem. Rev. 2010, 110, 60496076. 14. Salonen, L. M.; Ellermann, M.; Diederich, F. Aromatic Rings in Chemical and Biological Recognition: Energetics and Structures. Angew. Chem. Int. Ed. 2011, 50, 48084842. 15. Lin, S.; Jacobsen, E. N. Thiourea-Catalysed Ring Opening of Episulfonium Ions with Indole Derivatives by Means of Stabilizing Non-Covalent Interactions. Nat. Chem. 2012, 4, 817-824. 16. Biedermann, F.; Schneider, H.-J. Experimental Binding Energies in Supramolecular Complexes. Chem. Rev. 2016, 116, 5216-5300. 17. Pinalli, R.; Brancatelli, G.; Pedrini, A.; Menozzi, D.; Hernández, D.; Ballester, P.; Geremia, S.; Dalcanale, E. The Origin of Selectivity in the Complexation of N-Methyl Amino Acids by Tetraphosphonate Cavitands. J. Am. Chem. Soc. 2016, 138, 8569-8580.



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

18. Alemán, C. On the Ability of Modified Peptide Links to Form Hydrogen Bonds. J. Phys. Chem. A 2001, 105, 6717-6723. 19. Lee, H. J.; Choi, Y. S.; Lee, K. B.; Park, J.; Yoon, C. J. Hydrogen Bonding Abilities of Thioamide. J. Phys. Chem. A 2002, 106, 7010-7017. 20. Sherman, D. B.; Spatola, A. F. Compatibility of Thioamides with Reverse Turn Features: Synthesis and Conformational Analysis of Two Model Cyclic Pseudopeptides Containing Thioamides as Backbone Modifications. J. Am. Chem. Soc. 1990, 112, 433-441. 21. Kim, N.-K.; Lee, H.-J.; Choi, K.-H.; Yu, J.-A.; Yoon, C.-J.; Park, J.; Choi, Y.-S. Substituent Effect of N,N-Dialkylamides on the Intermolecular Hydrogen Bonding with Thioacetamide. J. Phys. Chem. A 2000, 104, 5572-5578. 22. Miwa, J. H.; Patel, A. K.; Vivatrat, N.; Popek, S. M.; Meyer, A. M. Compatibility of the Thioamide Functional Group with β-Sheet Secondary Structure: Incorporation of a Thioamide Linkage into a β-Hairpin Peptide. Org. Lett. 2001, 3, 3373-3375. 23. Miwa, J. H.; Pallivathucal, L.; Gowda, S.; Lee, K. E. Conformational Stability of Helical Peptides Containing a Thioamide Linkage. Org. Lett. 2002, 4, 4655-4657. 24. Wildemann, D.; Schiene-Fischer, C.; Aumüller, T.; Bachmann, A.; Kiefhaber, T.; Lücke, C.; Fischer, G. A Nearly Isosteric Photosensitive Amide-Backbone Substitution Allows Enzyme Activity Switching in Ribonuclease S. J. Am. Chem. Soc. 2007, 129, 49104918. 25. Bachmann, A.; Wildemann, D.; Praetorius, F.; Fischer, G.; Kiefhaber, T. Mapping Backbone and Side-Chain Interactions in the Transition State of a Coupled Protein Folding and Binding Reaction. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 3952-3957.


Page 16 of 26

Page 17 of 26

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


26. Culik, R. M.; Jo, H.; Degrado, W. F.; Gai, F. Using Thioamides to Site-Specifically Interrogate the Dynamics of Hydrogen Bond Formation in β-Sheet Folding. J. Am. Chem. Soc. 2012, 134, 8026-8029. 27. Newberry, R. W.; VanVeller, B.; Raines, R. T. Thioamides in the Collagen Triple Helix. Chem. Commun. 2015, 51, 9624-9627. 28. Walters, C. R.; Szantai-Kis, D. M.; Zhang, Y.; Reinert, Z. E.; Horne, W. S.; Chenoweth, D. M.; Petersson, E. J. The Effects of Thioamide Backbone Substitution on Protein Stability: A Study in α-Helical, β-Sheet, and Polyproline II Helical Contexts. Chem. Sci. 2017, 8, 2868-2877. 29. Bohon, J.; de los Santos, C. R. Structural Effect of the Anticancer Agent 6Thioguanine on Duplex DNA. Nucleic Acids Res. 2003, 31, 1331-1338. 30. Špačkova, N.; Cubero, E.; Šponer, J.; Orozco, M. Theoretical Study of the Guanine → 6-Thioguanine Substitution in Duplexes, Triplexes, and Tetraplexes. J. Am. Chem. Soc. 2004, 126, 14642-14650. 31. Bohon, J.; de los Santos, C. R. Effect of 6-Thioguanine on the Stability of Duplex DNA. Nucleic Acids Res. 2005, 33, 2880-2886. 32. Salon, J.; Jiang, J.; Sheng, J.; Gerlits, O. O.; Huang, Z. Derivatization of DNAs with Selenium at 6-Position of Guanine for Function and Crystal Structure Studies. Nucleic Acids Res. 2008, 36, 7009-7018. 33. Zhang, F.; Fu, L.; Wang, Y. 6-Thioguanine Induces Mitochondrial Dysfunction and Oxidative DNA Damage in Acute Lymphoblastic Leukemia Cells. Mol. Cell. Proteomics 2013, 12, 3803-3811.



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

34. Faustino, I.; Curutchet, C.; Luque, F. J.; Orozco, M. The DNA-Forming Properties of 6-Selenoguanine. Phys. Chem. Chem. Phys. 2014, 16, 1101-1110. 35. Kirsanova, O. V.; Sergeev, A. V.; Yasko, I. S.; Gromova, E. S. The Impact of 6Thioguanine Incorporation into DNA on the Function of DNA Methyltransferase Dnmt3a. Nucleosides, Nucleotides Nucleic Acids 2017, 36, 392-405. 36. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank. Nuc.l Acids Res. 2000; 28: 235-242. 37. Nisius, L.; Grzesiek, S. Key Stabilizing Elements of Protein Structure Identified through Pressure and Temperature Perturbation of Its Hydrogen Bond Network. Nat. Chem. 2012, 4, 711-717. 38. Panman, M. R.; Bakker, B. H.; den Uyl, D.; Kay, E. R.; Leigh, D. A.; Buma, W. J.; Brouwer, A. M.; Geenevasen, J. A. J.; Woutersen, S. Water Lubricates Hydrogen-Bonded Molecular Machines. Nat. Chem. 2013, 5, 929-934. 39. Peters, G. M.; Skala, L. P.; Davis, J. T. A Molecular Chaperone for G4-Quartet Hydrogels. J. Am. Chem. Soc. 2016, 138, 134-139. 40. Tsunoda, Y.; Takatsuka, M.; Sekiya, R.; Haino, T. Supramolecular Graft Copolymerization of a Polyester by Guest-Selective Encapsulation of a Self-Assembled Capsule. Angew. Chem. Int. Ed. 2017, 56, 2613-2618. 41. Schumacher, M. A.; Crum, M.; Miller, M. C. Crystal Structures of Apocalmodulin and an Apocalmodulin/SK Potassium Channel Gating Domain Complex. Structure 2004, 12, 849-860.


Page 18 of 26

Page 19 of 26

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


42. Valerie, N. C. K.; Hagenkort, A.; Page, B. D. G.; Masuyer, G.; Rehling, D.; Carter, M.; Bevc, L.; Herr, P.; Homan, E.; Sheppard, N. G.; Stenmark, P.; Jemth, A.-S.; Helleday, T. NUDT15 Hydrolyzes 6-Thio-Deoxygtp to Mediate the Anticancer Efficacy of 6Thioguanine. Cancer Res. 2016, 76, 5501-5511. 43. Oliva, R.; Cavallo, L.; Tramontano, A. Accurate Energies of Hydrogen Bonded Nucleic Acid Base Pairs and Triplets in tRNA Tertiary Interactions. Nucleic Acids Res. 2006, 34, 865-879. 44. Hobza, P. Calculations on Noncovalent Interactions and Databases of Benchmark Interaction Energies. Acc. Chem. Res. 2012, 45, 663-672. 45. Desiraju, G. R. Hydrogen Bridges in Crystal Engineering: Interactions without Borders. Acc. Chem. Res. 2002, 35, 565-573. 46. Sheu, S.-Y.; Yang, D.-Y.; Selzle, H. L.; Schlag, E. W. Energetics of Hydrogen Bonds in Peptides. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12683-12687. 47. Frey, J. A.; Leutwyler, S. Binding Energies of Hydrogen-Bonded Cis-Amide and Nucleobase Dimers: An Evaluation of DFT Performance. Chimia 2005, 59, 511 - 516. 48. Annat, G.; MacFarlane, D. R.; Forsyth, M. Transport Properties in Ionic Liquids and Ionic Liquid Mixtures: The Challenges of NMR Pulsed Field Gradient Diffusion Measurements. J. Phys. Chem. B 2007, 111, 9018-9024. 49. Bayley, P. M.; Best, A. S.; MacFarlane, D. R.; Forsyth, M. Transport Properties and Phase Behaviour in Binary and Ternary Ionic Liquid Electrolyte Systems of Interest in Lithium Batteries. ChemPhysChem 2011, 12, 823-827.



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

50. Forsyth, M.; Yoon, H.; Chen, F.; Zhu, H.; MacFarlane, D. R.; Armand, M.; Howlett, P. C. Novel Na+ Ion Diffusion Mechanism in Mixed Organic–Inorganic Ionic Liquid Electrolyte Leading to High Na+ Transference Number and Stable, High Rate Electrochemical Cycling of Sodium Cells. J. Phys. Chem. C 2016, 120, 4276-4286. 51. Johnson Jr, C. S. Diffusion Ordered Nuclear Magnetic Resonance Spectroscopy: Principles and Applications. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 34, 203-256. 52. Cohen, Y.; Avram, L.; Frish, L. Diffusion NMR Spectroscopy in Supramolecular and Combinatorial Chemistry: An Old Parameter—New Insights. Angew. Chem. Int. Ed. 2005, 44, 520-554. 53. Biswal, H. S.; Gloaguen, E.; Mons, M.; Bhattacharyya, S.; Shirhatti, P. R.; Wategaonkar, S. Structure of the Indole−Benzene Dimer Revisited. J. Phys. Chem. A 2011, 115, 9485-9492. 54. Mollner, A. K.; Casterline, B. E.; Ch’ng, L. C.; Reisler, H. Imaging the State-Specific Vibrational Predissociation of the Ammonia−Water Hydrogen-Bonded Dimer. J. Phys. Chem. A 2009, 113, 10174-10183. 55. Newberry, R. W.; Raines, R. T. A Prevalent Intraresidue Hydrogen Bond Stabilizes Proteins. Nat. Chem. Biol. 2016, 12, 1084 –1088. 56. Weinhold, F.; Landis, C. R. Valency and Bonding: A Natural Bond Orbital DonorAcceptor Perspective 2005. 57. Furche, F.; Ahlrichs, R.; Hättig, C.; Klopper, W.; Sierka, M.; Weigend, F. Turbomole. WIREs Comput. Mol. Sci. 2014, 4, 91-100.


Page 20 of 26

Page 21 of 26

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


58. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; et al. Gaussian 09, Revision C.01; Gaussian, Inc.: Wallingford, CT, 2011. 59. Glendening, E. D.; Landis, C. R.; Weinhold, F. NBO 6.0: Natural Bond Orbital Analysis Program. J. Comp. Chem. 2013, 34, 1429-1437.



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

50x50mm (300 x 300 DPI)


Page 22 of 26

Page 23 of 26

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


83x96mm (300 x 300 DPI)



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

175x129mm (300 x 300 DPI)


Page 24 of 26

Page 25 of 26

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


83x105mm (300 x 300 DPI)



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

83x69mm (300 x 300 DPI)


Page 26 of 26

Thioamide, a Hydrogen Bond Acceptor in Proteins and Nucleic Acids

Recommend Documents