Amber-Compatible Parametrization Procedure for Peptide-like

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Amber compatible parameterization procedure for peptide-like compounds: application to 1,4- and 1,5-substituted triazole-based peptidomimetics Antoine Marion, Jerzy Góra, Oliver Kracker, Tanja Fröhr, Rafa# Latajka, Norbert Sewald, and Iris Antes J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.7b00305 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 9, 2017

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Journal of Chemical Information and Modeling

Amber compatible parameterization procedure for peptide-like compounds: application to 1,4- and 1,5substituted triazole-based peptidomimetics Antoine Marion,1 Jerzy Góra,2,3 Oliver Kracker,2 Tanja Fröhr,2 Rafał Latajka,3 Norbert Sewald,2,* and Iris Antes1,* 1

Center for Integrated Protein Science Munich at the Department of Biosciences, Technische Universität München, Emil-Erlenmeyer-Forum 8, D-85354 Freising, Germany

2

Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, Universitätsstraße 25, D-33615 Bielefeld, Germany

3

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wroclaw University of Science and Technology, Wybrzeze Wyspianskiego 27, PL-50-370 Wroclaw, Poland

*corresponding authors: Norbert Sewald: [email protected] Iris Antes: [email protected]

KEYWORDS. molecular mechanics, conformational space, force field, parameterization, peptidomimetics, peptidotriazolamers, triazolamers, AMBER, ff14SB

1 ACS Paragon Plus Environment


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ABSTRACT Peptidomimetics are molecules of particular interest in the context of drug design and development. They are proteolytically and metabolically more stable than their natural peptide counterparts, but still offer high specificity towards their biological targets. In recent years, 1,4and 1,5-substituted 1,2,3-triazole-based peptidomimetics have emerged as promising lead compounds for design of various inhibitory and tumor-targeting molecules, as well as for synthesis of peptide analogues. The growing popularity of triazole-based peptidomimetics and a constantly broadening range of their application generated a demand for elaborate theoretical investigations by classical molecular dynamics simulations and molecular docking. Despite this rising interest, accurate and coherent force field parameters for triazole-based peptidomimetics are still lacking. Here we report the first complete set of parameters dedicated to this group of compounds, named TZLff. This parameterization is compatible with the latest version of the AMBER force field (ff14SB) and can be readily applied for the modeling of pure triazole-based peptidomimetics as well as natural peptide sequences containing one or more triazole-based modifications in their backbone. The parameters were optimized to reproduce HF/6-31G* electrostatic potentials as well as MP2/cc-pVTZ equilibrium Hessian matrices and conformational potential energy surfaces through the use of a genetic algorithm-based search and least square fitting. Following the standards of AMBER, we introduce residue building units, thus allowing the user to define any given sequence of triazole-based peptidomimetics. Validation of the parameter set against ab initio- and NMR-based reference systems shows that we obtain fairly accurate results, which properly capture the conformational features of triazolebased peptidomimetics. The successful and efficient parameterization strategy developed in this


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work is general enough to be applied in a straightforward manner for parameterization of other peptidomimetics and, potentially, any polymeric assemblies.



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1. Introduction Peptidomimetics are regarded as molecules of particular interest in field of drug design and development. Through the replacement of peptide bonds by proteolytically and metabolically stable moieties they offer higher chemical and conformational stability, increased lipophilicity and better absorption in comparison to natural peptides. These properties considerably increase their potency in molecular biology, as well as in drug discovery. Various biomimetic oligomers that have protein-like functionality have been reported in the literature as an alternative to peptides

1-5

. The oligomers, which analogously to natural peptides adopt a particular periodic

conformation in solution, are often classified as peptidomimetic foldamers. The ability to adopt a stable secondary structure is a defining factor that allows for a comprehensive study of associated biological processes and facilitates their application as lead compounds in drug design. The 1,2,3-triazole ring exhibits several advantageous features making it a strong candidate for synthesis of peptidomimetics. Notably, it resists enzymatic degradation, hydrolysis and oxidation. On a molecular level, it is planar and it displays a dipole moment and electronic properties highly similar to those of a standard peptide bond. Furthermore, the triazole ring presents two hydrogen bond acceptor nitrogen atoms and one hydrogen atom that shows a certain degree of electrophilicity, thus mimicking, to a given extent, the hydrogen bonding pattern of a standard peptide bond. A thorough review focusing on the properties of the 1,2,3-triazoles and their emerging role in synthesis of peptidomimetics was published by Pedersen and Abel in 2011 6

.


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The common approach to organic synthesis of 1,2,3-triazole-based peptidomimetics follows the ‘click chemistry’ principles using copper-catalyzed azide-alkyne cycloaddition (CuAAC) or ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) 7. The copper-catalyzed protocol yields 1,4-substitued rings, while RuAAC results in 1,5-substituted moieties. The meaningful difference is that the 1,4-substituted triazole ring introduces a conformation that closely resembles the trans-peptide bond, whereas the 1,5-subsitued moiety tends to mimic the behavior of a cis-peptide bond, as clearly illustrated in Ref. 6. This additional benefit in conjunction with the fact that the cycloaddition process is compatible with the side chains of all amino acids in their protected forms shows the potency of the approach, as peptidomimetics of nearly any given composition can be synthesized that way. The replacement of each peptide bond in a polypeptide by a 1,2,3-triazole ring led to the creation of a novel class of peptidomimetic foldamers – triazolamers, whereas substitution of every second peptide bond produces structures that are referred to as peptidotriazolamers (Scheme 1). The synthesis protocol and characterization of 1,4-substituted triazolamers was first published by Angelo and Arora in 2005

8-9

. Subsequently, Hecht et al. reported the synthesis of

peptidotriazolamers by following the principles of ‘click chemistry’ in 2010 10. They highlighted the potency of this class of peptidomimetics to be fine-tuned in terms of their biodegradability and bioavailability and thus their usefulness in various fields of applications. Further studies by Ke et al. demonstrated that the ‘click’ reaction can be used as an effective way of synthesis of peptidotriazole-based organogelators

11

by providing NMR-based evidence that short

peptidotriazolamers show the tendency to dimerize in a head-to-tail manner with a β-strand conformation above a given molar concentration. The ability of 1,2,3-triazole rings to replace amide bonds in peptide sequences has been successfully demonstrated by Horne et al. through



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the synthesis of several mutants of a tetrameric α-helical coiled coil 12. By replacing one specific peptide bond in each of the four helices, the authors showed that the 1,2,3-triazole ring can effectively substitute a peptide bond, participating in a similar manner to the backbone conformational stability. Thermal denaturation assays further revealed that the absolute position of the 1,2,3-triazole ring in the sequence can drastically lower the melting temperature of the molecular assembly. In that case, the introduction of a triazole ring tends to destabilize the molecular structure thus emphasizing the relevance of these peptidomimetics for modulating protein stability. Since the introduction of the idea of triazole-based peptidomimetics as biologically active molecules different groups have reported the synthesis of these compounds as potential drug candidates

13-20

. In recent years, the number of reports of such molecules has been growing

exponentially, leading to the introduction of new therapeutic targets. Amongst others, triazolebased pseudo-peptides are currently studied as promising molecules in treatments against tumors (e.g., Smac mimetics 21-22, cryptophycin-52 analogues 23) and neurodegenerative diseases 24-25, as well as in tumor tracing and targeting 26-27, mitochondrial delivery systems 28 and as analogues of various biologically-active molecules 29-30. The growing popularity of triazole-based peptidomimetics and their constantly broadening range of applicability results in a demand for precise and elaborate theoretical research that would allow to evaluate their conformational preferences, as well as to examine the physiochemical properties of this class of molecules. Recently, Kann et al. reported an extensive conformational study of 1,4- and 1,5-peptidotriazolamers performed at a relatively high level of quantum mechanics (QM) with an implicit solvent model

31

. The investigation of the behavior of new

chemical moieties at a full QM level of theory provides important insights into the electronic


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structure of the compounds and is therefore necessary for a throughout understanding of the compounds properties. However, QM-level calculations are still computationally intensive and their practical applicability tends to reach a limit once the size and flexibility of the molecular system increases. One critical application is the characterization of the conformational space and dynamics of the compounds in solution. Current efforts in the development of efficient QM codes and methodologies capable of treating efficiently large molecular systems open a promising door towards an explicit description of the solvation of biological compounds using electronic structure methods only

32-39

. In this direction, recent milestones have been reached

with full QM geometry optimizations and ab-initio molecular dynamics (MD) of large biomolecular systems

40-41

. While remarkable, ab initio MD simulation of such systems is still

limited to a few tens to hundreds of picoseconds. The use of approximate QM models offers an alternative and allows to reach nanosecond scale simulations the development of the related QM potentials

45

42-44

, but requires attentive care in

. Therefore, it is not yet practically possible to

reach sufficient sampling of very large or flexible macromolecular systems at the QM level. Instead, other approaches based on the parameterization of classical models appear more appealing due to their lower computational expense. The drawback of such methodologies, however, is that parameters are often system specific and therefore the derivation of a new, dedicated parameter set is required for new classes of molecules. Among the classical models used in molecular modeling, molecular mechanics (MM) energy functions are usually the preferred choice for the sampling of (macro-)molecular conformational space using techniques such as molecular dynamics or molecular docking 46. Over the past decades, this has led to the development of a number of peptide-centered MM force fields (e.g., OPLS, AMBER, CHARMM, GROMOS, AMOEBA), which have been continuously updated and improved 47-48.



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Despite the rising interest in triazole-based peptidomimetics, accurate and coherent force field parameters are still lacking. Here we report the first complete set of MM parameters dedicated to model peptidotriazolamers and triazolamers. This new parameter set, named TZLff (triazole force field), is compatible with the latest version of the AMBER force field (ff14SB) for natural peptides and is available for download in the Supporting Information. In agreement with the AMBER standards, we introduced residue-based building units, thus allowing the user to define any given sequence of triazole-based peptidomimetic. The parameterization strategy that we designed for this work gathers different ideas from the field of parameter development and we detailed it in a comprehensive way. This strategy resulted in fairly accurate parameters for the present application and we postulate that it is general enough to be applied in a straightforward manner to the parameterization of other peptidomimetics and potentially to any polymeric (macro-)molecules.

2. Theory2.1 Parameterization strategy Considering the intrinsic relationship between triazole-based peptidomimetics and standard polypeptides, we designed the new models such that the inclusion of our new set of parameters into an existing amino acid force field is straightforward. Our overall goal was to allow for the description of pure isolated peptidomimetics, as well as triazole-based fragments within a polypeptide sequence or in interaction with other protein residues. We chose to build upon the last version of the AMBER force field, ff14SB

49

, which uses the following additive potential

energy function:


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𝐸𝐸!"#$% =

!

!!!

𝑞𝑞! 𝑞𝑞! + 𝜀𝜀𝑅𝑅!"

+

!"#

%$!

𝐴𝐴!"

!" 𝑅𝑅!" !!!

𝐾𝐾! 𝑅𝑅 − 𝑅𝑅!

!

− +

𝐵𝐵!"

! 𝑅𝑅!" !"#$%&

𝐾𝐾! 𝜃𝜃 − 𝜃𝜃!

!

+

!"!!"#$%&

𝑉𝑉! 1 + cos 𝑛𝑛𝑛𝑛 − 𝛾𝛾!

(1)

In Eq (1), non-bonded interactions are described as function of the interatomic distance between each atom pair (Rij) using Coulomb (with the atomic point charges qi) and Lennard-Jones potentials (with pairwise parameters Aij and Bij). Lennard-Jones parameters are derived from the value of the atomic van der Waals radius (ri) and potential well depth (εi) parameters. Harmonic potentials are placed on all bonds (R) and bond angles (θ) with their respective equilibrium values (R0 and θ0) and force constants (KR and Kθ). Bond dihedral and improper angles (φ) are modeled with a periodic potential where n is the periodicity of the function, γn the angle phase shift and Vn the associated force constant. While most of the parameters present in Eq. (1) can be adapted from the parent force field to describe the new residue moieties, some critical terms of the potential energy function require particular attention. Notably, the definition of the non-bonded parameters must be consistent with the original force field to ensure a proper balance between the different interactions in the molecular system. In addition, parameters related to the conformational preference of new structures must be specifically optimized, leading to a dedicated set of bond, angle, and, most importantly, dihedral angle parameters. Thus, the choice of reference data constitutes a pivotal part of our parameterization strategy. However, apart from the structural training set, the exact definition of the reference calculations used is mostly dictated by the kind of calculations performed for the fitting of the parent force field. While there exists still some flexibility in the choice of the reference set for bonded parameters (i.e., bonds, angles, dihedrals, and impropers), it is crucial to use the same strategy and level of theory as used in the original force field for the



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derivation of the atomic charges. Otherwise unbalanced results might be obtained once the new parameters are used together with original force field parameters for standard amino acids. All parameters developed in this work are accessible through the Supporting Information as an archive containing Amber compatible files (frcmod parameter files as well as library files containing residue charges and topologies). The archive also contains a configuration file (Amber leaprc) that helps setting up triazole-based peptidomimetics systems. In what follows, we first describe the algorithms developed for this work. Then, we summarize the definition of new residues and atom types and subsequently discuss our strategy to derive missing non-bonded and bonded parameters. A flowchart summarizing our parameterization protocol is depicted in Figure 1. 2.2 Description of the fitting procedure The parameterization was carried out by a series of ‘in-house’ python codes that drive the python application program interfaces (APIs) of sander and parmed, as implemented in AmberTools15. The codes are available at “https://www.bioinformatics.wzw.tum.de/fitff/scripts/” (protected by password during the review process: “Tzl-Fit”) and details of the applied algorithms are described in the following. The core of the scripts is a python class designed to store, create, and manage Amber’s topologies. At each step of the optimization process, the topology can either be rebuilt with current parameters using the tleap program of AmberTools or, if possible, directly modified without running an external program in order to speed-up the fitting algorithm. For each conformation in the data set, the MM energy can be evaluated by running a single point energy calculation with the sander API using the topology and parameters of the current optimization step. Alternatively, for large data sets, only relevant parts of the MM potential energy function (Eq. (1)) are computed in order to speed-up the overall process. In such a case,


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the code first identifies all internal degrees of freedom (i.e., bonds, angles, and dihedrals) that are impacted by the parameters to be optimized. Before starting the optimization, the MM energy of each conformation in the data set is computed by setting the value of all parameters to zero. Then, at each step of the optimization, only the terms of the potential related to the previously identified degrees of freedom are evaluated by our python script. As these functions are fast and straightforward to compute (i.e., harmonic and periodic potentials), this treatment considerably reduces the computational time since it limits to a minimum the number of calls to external programs and routines. This is particularly relevant if the optimization process involves a large number of evaluations of the objective (or fitness) function, as it is the case in a genetic algorithm (GA) scheme. For the parameterization attempts based on the fit of a Hessian matrix (see Subsection 2.3) the MM Hessian matrix of a given geometry is evaluated numerically at each step by central finite-differentiation of the gradient with a step of 10-3 Å. For each set of parameters and each variation of the geometry, the gradient is obtained by running a single point calculation using the sander API. The optimization routines use a genetic algorithm and/or a least square fitting step. The GA search is based on the Pyevolve python package (version 0.6rc1) 50 and the least square fitting is driven by the leastsq module as provided by the scipy package (version 0.17.1) 51. In this work, all GA runs were performed for 1000 generations with a population size of 500 individuals. Crossover and mutation rates were set to 0.8 and 0.1, respectively. The inverse of the energy root mean square deviation (RMSD) was used as fitness function to be maximized during GA search. Each GA-based parameter search was repeated 20 times to ensure convergence of the final solution. Least square fitting runs were performed using default settings of the leastq module until convergence and the search algorithm was re-initialized every 1000 calls of the objective



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function in order to force a new evaluation of the Hessian. The sum of square differences between reference and target data in the training set was used as objective function for least square fitting. Additionally, the parameters space was forced to remain in a realistic range using the following penalty function (χ! ) for each parameter (𝑃𝑃! ): χ! 𝑃𝑃!

𝑊𝑊! 𝑃𝑃! − 𝑃𝑃!!"# ! = 𝑊𝑊! 𝑃𝑃! − 𝑃𝑃𝑢𝑢𝑢𝑢 ! ! 0

𝑖𝑖𝑖𝑖 𝑃𝑃! < 𝑃𝑃!!"# 𝑢𝑢𝑢𝑢 𝑖𝑖𝑖𝑖 𝑃𝑃! > 𝑃𝑃! 𝑜𝑜𝑜𝑜ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒

(2)

In Eq. (2), 𝑃𝑃!!"# and 𝑃𝑃!!" are the lower and upper boundaries of a given parameter 𝑃𝑃! , and 𝑊𝑊! is

the corresponding weight of that penalty. As detailed later in this Section, the sum of all penalties was then added to the objective function and the boundaries were set to a suitable value depending on the parameterization case. In the present application, the value of the 𝑊𝑊! constant

was set to 1010 for all restraints, in order to ensure that these conditions bear the highest weight and, therefore, are fulfilled during the fitting process. 2.3 Definition of new residues and atom types Following the well-established residue definition of protein fragments used in AMBER and other force fields, we derived building blocks with the intention of covering a wide range of amino acid-like sequences (step 1 in Figure 1). Our residue definition for representative compounds is shown in Figures 2 and 3. Furthermore, considering the resemblance of triazole-based peptidomimetics with polypeptides, we adopt the same sequence orientation standards in the present work. Therefore, in the orientation adopted in Figures 2 and 3, the left and right-hand parts of the molecules are further referred to as N- and C-terminal ends. In peptidotriazolamers and triazolamers, the standard peptide bond is replaced by a 1,2,3-triazole ring (Scheme 1). Unlike standard peptide bonds, there is no straightforward way of splitting a


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triazole ring in two parts. Therefore, we chose to define the triazole ring as a single residue. Since two substitution sites are possible on the ring, we define 1,4- and 1,5-triazole rings as T14 (Figure 1.A) and T15 residues (Figure 1.B), respectively. Correspondingly, we define three methyl capping residues named after their substitution position in the triazole ring (i.e., MC1, MC4, and MC5). Amino acid-like 1,4- and 1,5-peptidotriazolamer residues are described in Figures 2.C and 2.D, respectively. Following our definition, a standard Ala-like unit is composed of one N-terminal amino acid-like residue (A4n or A5n), one triazole residue (T14 or T15), and one C-terminal amino acid-like residue (A4c or A5c). The nomenclature of the amino acid-like residues reflects their position in the sequence (N- or C-terminal), as well as the type of triazole ring that they are bound to (i.e., 1,4- or 1,5-substituted). Notice that amino acid-like peptidotriazolamer residues retain an NH and a CO group at the N- and at the C-termini, respectively, to ensure further connectivity with compatible residues (i.e., other peptidotriazolamers or standard amino acids). Figure 3 shows the definition of triazolamers-related residues. Therein, a standard Ala-like unit is made of one N-terminal triazole residue (T14 or T15), one amino acid-like residue (A44, A45, A54, or A55), and one C-terminal triazole residue (T14 or T15). In this case, the nomenclature of amino acid-like moieties is based on the character of the triazole ring (i.e., 1,4- or 1,5substituted) connected at the N- and at the C-terminal part of the residue. For both peptidotriazolamers and triazolamers, other amino acid-like residues are defined with any standard amino acid side chain by attaching the corresponding chemical function to the Cα-atom (CA atom name). For each standard amino acid, we define eight new residues allowing constructing all possible triazole-based peptidomimetics. The nomenclature of other amino acid-



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like residues follows the aforementioned convention, in which the first character of the residue name refers to the one letter identifier of the corresponding standard amino acid. Triazole-based peptidomimetics share structural similarities with standard polypeptides. Similarly to the Φ/Ψ Ramachandran definition in polypeptides

52

, the conformation of each

amino acid-like residue can be characterized by a combination of two backbone dihedral angles around the α-carbon. The specific definition of this pair of angles is described in Figures 2 and 3 for each residue on the left-hand side scheme of each subfigure. In what follows, we use the Φ/Ψ nomenclature for all amino acid-like residues, unless a particular differentiation is required. In such cases, the angles will be labeled with a specific subscript as defined in Figures 2 and 3. In the present work, we introduced a total of 13 new atom types (step 2 in Figure 1) to describe the 1,4- and 1,5-triazole rings (i.e., nI, nJ, nK, cL, cM, hT, and hU; Figures 2.A and 2.B), as well as the alpha carbons of different amino acid-like peptidotriazolamers (i.e., cU and cV; Figures 2.C and 2.D) and triazolamers (i.e., cW, cX, cY, and cZ; Figure 3). This allows for a full flexibility in our parameterization process while limiting the number of new parameters for parts of triazole-based peptidomimetics that are similar to standard peptides (i.e., NH and CO groups for peptidotriazolamers; all side chains of amino acid-like residues). Notably, all bonded parameters of the triazole rings, as well as each set of backbone torsion parameters for amino acid-like residues were specifically optimized. New non-bonded van- der-Waals parameters were also derived for nJ, nK, hT, and hU atom types, while the ff14SB parameters were found to be suitable for the remaining atom types, as described in the following subsection. 2.4 Derivation of non-bonded parameters


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As defined in standard AMBER force fields, each newly introduced residue in this work has a unit charge (0, -1, +1), depending on the characteristics of the side chain. As a result, the set of atomic charges of each residue is unique and cannot be transferred from one force field to another. As a consequence, we derived a dedicated set of atomic point charges for each residue introduced in this work (i.e., triazole rings, capping groups, and every variant of amino acid-like residues; step 3 in Figure 1). The derivation of atomic point charges was performed following the RESP procedure by an ‘in-house’ python script that drives the different modules of antechamber as available in AmberTools15. Note that the RED server

53

could have produced similar results,

but considering the large amount of residues that needed to be parameterized, a local management of the calculations was found to be more efficient. In accordance with the ff14SB parameterization protocol, the electrostatic potential calculations were carried out at the HF/631G* level following minimization at the same level of theory. The charges of T14 and T15 residues were derived from a single conformation of the systems MC4-T14-MC1 and MC5-T15-MC1, respectively (see Figure 2.A and 2.B). During this step, charges for the MC4, MC5, and MC1 capping methyl groups were also derived from these conformations. Since MC1 is common to both systems, the final set of atomic charges for this residue was averaged. N- and C-terminal amino acid-like residues of peptidotriazolamers were parameterized separately. To that end, the corresponding model compounds were built with one single amino acid-like residue, one triazole ring and the necessary capping residues: e.g., ACE-A4n-T14-MC1 or MC5-T15-A5c-NME. For triazolamers the model compounds were built in a similar manner as those presented in Figure 3, as the building unit is made of only one amino acid-like residue.



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For all amino acid-like residues, a multi-conformational RESP procedure was applied. For each case 16 conformations were generated, representing a grid with a 90° step on the Φ/Ψ conformational map. Each conformation was minimized with frozen backbone dihedrals and the electrostatic potential was calculated for the minimized geometry. For residues presenting side chains other than Gly- and Ala-like amino acids, relaxation of the side chain was performed prior to minimization. This conformational search was performed using a 40 ps simulated annealing simulation by gradually heating up the molecule to 400 K and subsequently cooling it down to 0 K afterwards. The position of backbone atoms was kept fixed during the simulations by means of belly type dynamics, while the side chains remained fully flexible. For Gly- and Ala-like residues, among the 16 initial conformations, only those bearing a relative energy lower than 10 kcal·mol-1 were selected to avoid artifacts during the RESP fit. For the other neutral and charged side chains, this cutoff was increased to 15 and 20 kcal·mol-1, respectively. The number of conformations used for each new amino acid-like residue parameterized in this work ranged from 8 to 15. The side chain of all standard amino acids (except Selenocysteine and Proline) were parameterized for each of the 8 types of systems described above and in Figures 2.C, 2.D, and 3 (i.e., X4n, X4c, X5n, X5c, X44, X45, X54, and X55, where X denotes the one letter amino acid identifier). Each side chain was considered in its standard protonation state. For His-like residues, protonation was considered either at the ε or at the δ position of the imidazole ring (one letter identifier H and J, respectively). Note that considering the modular protocol used in this work, extension of the residue set can be achieved in a straightforward manner. Most of the van der Waals parameters (Lennard-Jones potential) were adapted from the atom types that already exist in ff14SB (step 4 in Figure 1). Notably, all α-carbons of triazole-based peptidomimetics (i.e., cU, cV, cW, cX, cY, cZ) share the same parameters as the CT atom type


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in ff14SB. For the triazole rings, the nI atom type is equivalent to N in ff14SB, while cL and cM have the same parameters as CA in AMBER. However, for the N2, N3, and hydrogen atoms (H5 and H4) of the triazole rings, our efforts to identify well suited existing types failed as no wellbalanced interactions in the model systems could be obtained. Therefore, we derived new van der Waals parameters for the nJ, nK, hT, and hU atom types, based on the interaction energy surface between one N-methylacetamide (NMA) molecule and each of the two triazole rings (MC4-T14MC1 and MC5-T15-MC1, as depicted in Figure 2.A and 2.B). For each triazole ring, three interaction energy curves were calculated to reflect the specific interaction between NMA and each atom types of the triazole ring to be parameterized. The reference interaction energy surfaces were calculated at the MP2/cc-pVTZ level of theory through a rigid scan of the relevant interaction distances. For each triazole ring, all three atom types were parameterized simultaneously with a least square fitting procedure by minimizing the following objective function: ! ∆𝐸𝐸!"# =

!

!"

!! 𝑤𝑤! 𝐸𝐸!"#,! − 𝐸𝐸!"#,!

!

+

!

χ𝑚𝑚 𝑃𝑃𝑚𝑚

(3)

!"

with 𝐸𝐸!"#,! being the non-relaxed interaction energy between the two fragments calculated at the !! being the molecular mechanics MP2-level QM reference for the geometry point k, and 𝐸𝐸!"#,!

energy of the corresponding structure with the current set of parameters. In the first term of Eq.

(3), 𝑤𝑤! is the weight associated with a given data point k. In the present application, the weight of interaction energy values located around the minimum of each curve was set to 102 instead of

1 to ensure a good description of the minimum interaction distance. Positive values of the interaction energy at low distance were given a weight of 0, thus disregarded from the fitting process. The second term in Eq. (3) is the sum the penalty function of all parameters as described



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in Eq. (2). The lower (𝑃𝑃!"# ) and upper (𝑃𝑃!" ) boundaries of the atomic van der Waals radius (ri)

were set to 0.0 and 5.0 Å, respectively. For the potential energy well depth (εi), 𝑃𝑃!"# and 𝑃𝑃!"

were set to 0.0 and 2.0 kcal·mol-1, respectively. Final parameters for nJ and nK were obtained as

an average from the fit involving 1,4- and 1,5-triazole rings. The resulting set of Lennard-Jones parameters are presented in Table 1. 2.5. Derivation of bonded parameters for the triazole rings We performed a systematic parameterization of each degree of freedom for 1,4- and 1,5-triazole rings based on ab initio reference calculations (step 5 in Figure 1). The parameters associated with the T14 and T15 residues were derived from the two following model systems: MC4-T14MC1 and MC5-T15-MC1 (Figures 2.A and 2.B, respectively). To limit the number of new atom types and related parameters, T14 and T15 share the same atom types for non-hydrogen atoms. MC4 and MC5 also possess the same atom types, HC and CT, while MC1 has H1 and CT atom types. As a consequence, several bond, angle, and dihedral parameters are identically defined in both systems. In such cases, the corresponding parameters were optimized independently for each system and finally averaged. Note that the methyl capping groups are described with standard ff14SB atom types. The corresponding bonded parameters (i.e., HC-CT, H1-CT, HCCT-HC, and H1-CT-H1) were not optimized in this work and kept as they are set in ff14SB. The full set of new parameters for triazole rings is listed in Table 1. First, we parameterized the dihedral angles defining the rotation of the three methyl groups (MC1, MC4, and MC5). The parameters were optimized with a least square fitting procedure to match the potential energy scan performed at the MP2/cc-pVTZ level of theory. For each surface, only two values of the two dihedrals were considered according to the c3 symmetry of the rotation of these methyl groups, resulting in a four points set of training geometries. The


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periodicity (n) of each dihedral function was fixed to 3 and the angular phase shift (γ) to 0 or 180 degrees with respect to the MP2 minimum geometry. The final root mean square deviation with respect to the MP2 reference for MC4-T14-MC1 and MC5-T15-MC1 was as low as 0.12 kcal·mol-1 in both cases. For remaining missing bonded parameters, we used a strategy based on the Hessian matrix of the system. The main motivation behind that strategy relies on the fact that the new parameters are expected to reproduce the vibrational behavior of the molecule around its minimum. Using the harmonic approximation, the vibration of each normal mode is directly related to the Hessian matrix of the minimum structure. This approach resembles to some extent the method suggested by Burger et al. in Ref

54

or by Barone et al.

55

, which are both related to the early work by

Seminario 56. Here, we fit the upper triangle of the MM Hessian matrix to that calculated on the MP2/cc-pVTZ level of theory using structures minimized at the same level of QM theory. This approach allows easy fixing of some parameters of the system such that the least square fitting procedure can be focused on the remaining flexible values. As we shall discuss later, this relatively simple methodology leads to parameters reproduce well the MP2 reference. The optimization of the parameters was performed through a least square fitting procedure by minimizing the following objective function: ∆𝑯𝑯! =

! !!!

!"

𝑯𝑯!" − 𝑯𝑯!! !"

!

+

!


(4)

!"

in which 𝑯𝑯!" and 𝑯𝑯!! !" are the k,l elements of the QM reference and of the MM target Hessian

matrices, respectively. The MM Hessian matrix was evaluated numerically within our ‘in-house’ python code with a step of 10-3 Å in each Cartesian direction. Notice that the second summation in the first term of Eq. (4) contains only elements greater or equal to the element of the first sum,



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thus including solely the upper triangle of the matrices. This choice is due to the symmetry of the Hessian matrix and it prevents double weighting the non-diagonal elements with respect to the diagonal ones. The second term in Eq. (4) is the penalty function as defined in Eq. (2). Here, only a lower boundary was set for all parameters in order to prevent negative values. The equilibrium values for bonds and angles were set according to the optimized geometry of the MC4-T14-MC1 and MC5-T15-MC1 systems at the MP2/cc-pVTZ level. The equilibrium values for equivalent bonded parameters in both systems were averaged prior to the fit. Complementary angles were slightly adjusted to ensure that planarity remains after averaging. The resulting bond and angle parameters are listed in Table 1, while proper and improper torsions are reported in Table 2. 2.6. Derivation of backbone torsion parameters The optimization of backbone torsions is at the core of our parameterization effort (step 6 in Figure 1). These conformational degrees of freedom represent the key features of triazole-based peptidomimetics and peptide-like foldamers in general. Unlike torsions in rigid molecules such as those involved in the parameterization of the triazole rings (see previous subsection), flexible dihedral degrees of freedom are poorly described by the harmonic approximation of the Hessian matrix. This is due to the complexity of the related potential energy surface, which is likely to feature various minima associated with different relative energies. In such case, fitting of the related potential energy surface is often found more relevant 55, 57-58. Additionally, more complex potentials are commonly described using a Fourier expansion of the periodic term present in Eq. (1) up to the second, third or higher orders. As we shall discuss in the Results and Discussions section, the relative complexity of the potential energy surface associated with triazole-based peptidomimetics led us to expand this Fourier series up to the fourth order.


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The parameterization was performed against a fairly high level of QM theory (MP2/cc-pVTZ) that was used to calculate the reference potential energy surface of each amino acid-like residue template defined in this work (i.e., X4n, X4c, X5n, X5c, X44, X45, X54, and X55). Each surface consists of a 20° relaxed scan of the two main backbone dihedral angles (Φ/Ψ as defined in Figures 2 and 3). As for the derivation of atomic point charges, the model systems were built to include only one amino acid-like residue, thus limiting the conformational degrees of freedom to only two torsions per surface. In a similar manner as it was performed for ff99SB and further adapted in ff14SB, Gly-like residues were first used to parameterize the main backbone parameters and then additional backbone/side-chain dihedral parameters were optimized to fit the corresponding surfaces of Ala-like residues. In ff14SB, the backbone dihedral parameters are set only for C-N-Cα-C and N-Cα-C-N torsions, while other backbone torsions involving the oxygen and hydrogen atoms of the amide group are set to zero. Additionally, for non-glycine residues, two other torsions are parameterized involving the beta-carbon of the side chain (i.e., C-N-Cα-Cβ and Cβ-Cα-C-N). We adopted a similar definition here, by parameterizing only the main backbone torsions (i.e., the dihedral angles described in Figures 2 and 3, and the corresponding ones involving CB atoms). Although the phase parameter (γ) of the periodic potential can adopt any real value between 0 and 180 degrees, it is advisable to limit the range of accessible values to discreet values of 0 and 180 degrees only. Such treatment allows defining a single set of parameters that is compatible with both enantiomers of compounds presenting a chiral center (i.e., the backbone alpha carbon atom in the present case). The optimization of dihedral parameters (i.e., Vn and γn in Eq. (1)) was performed by fitting the MM relative energy (∆𝐸𝐸!!! ) to the corresponding QM reference relative energy (∆𝐸𝐸!!! ), at each

21

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point (k) of the surface. The MM geometries were optimized prior to the parameter optimization with frozen backbone dihedrals to prevent artifacts coming from small variation in other internal degrees of freedom. In initial search was performed using a genetic algorithm as described in Section 2.2 and followed by a least square fitting refinement. For the GA runs, the alleles of force constants (Vn) were formed of values ranging from 0.0 to 4.0 kcal·mol-1 with a step of 0.5 kcal·mol-1, while the phase parameters (γn) were allowed only values of 0 and 180 degrees. The GA fitness function to be maximized was the inverse of the weighted root mean square deviation (RMSD-1) of the relative energy:

𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 !!

1 ∆𝐸𝐸 = 𝑁𝑁

!

!"

𝑤𝑤! ∆𝐸𝐸!

−

! ∆𝐸𝐸!!!

!! !

(5)

For least square fitting the following weighted sum of squares was used as objective function: ∆∆𝐸𝐸 ! =

!

!"

𝑤𝑤! ∆𝐸𝐸!

− ∆𝐸𝐸!!!

!

+

!


(6)

In Eqs. (5) and (6), 𝑤𝑤! is the weighting factor associated with the kth point of the surface and !"

calculated as 𝑤𝑤! = exp (−0.2∆𝐸𝐸! ). Such procedure was shown to be favorable in other

applications 58 and ensures that the parameterization focusses on physically accessible regions of the potential energy surface, thus limiting the impact of the highly energetic conformations. The penalty function (χ! ) was set to ensure that the force constant parameters of the periodic

potentials remain between 0.0 and 4.0 kcal·mol-1. The parameters resulting from this stage of the parameterization are listed in Tables 3 and 4 for peptidotriazolamers and triazolamers, respectively.


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3. Methods All quantum mechanical (QM) geometry optimizations, single point, and electrostatic potential (ESP) calculations were performed with the Gaussian09 program (version D.01) 59. Calculations of the ESP were performed at the HF/6-31G* level, on geometries optimized at the same level with a tight convergence criterion. Hessian matrices were evaluated at the MP2/cc-pVTZ level after geometry optimization performed at the same level of theory with a very tight convergence criterion. Relaxed potential energy scans were carried out at the HF/6-31G* level and followed by MP2/cc-pVTZ single point calculations. Molecular mechanics (MM) calculations were based on different modules of the Amber14/AmberTools15 suite 60. The new set of parameters derived in the present work was used for all triazole-based peptidomimetics, while standard amino acids were described with the ff14SB force field

49

. GAFF parameters

61

were used for capping

residues of the synthetized compounds (BOC, ALL and BNE for pep4Tzl and pep5Tzl; see experimental details in Figure S1 of the Supporting Information) and their corresponding atomic charges were derived following the RESP procedure 62. 3.1 Simulated annealing-based refinement of reference NMR structures Two previously synthesized 1,4- and 1,5-peptidotriazolamers were modeled in this work: BocGly-Ψ(1,4-triazole)-Phe-Gly-Ψ(1,4-triazole)-Val-OAll (referred to as pep4Tzl) and Boc-ValΨ(1,5-triazole)-Ala-Leu-Ψ(1,5-triazole)-Val-OBn (referred to as pep5Tzl)

63

. Reference

structures for those two molecules were derived from NMR spectra (see Figure S1 of the Supporting Information for NMR measurement protocol and assignment of signals) by means of simulated annealing with restraints set according to NOE inter-hydrogen atoms distances. NMR restraints files were generated with the standard scripts of AmberTools15 only for backbone and



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beta-hydrogen atoms (i.e., side chain hydrogen atoms were not restrained during the dynamics). The lower and upper limits of the harmonic restraints were respectively set to -10% and +10% of the corresponding NOE distance with a force constant of 60 kcal·mol-1·A-2. The temperature was progressively increased from 0 K to 600 K and finally gradually reduced to 0 K along a total of 1 ns long simulations. The force constant of each restraint was reduced to 6 kcal·mol-1·A-2 during the first 150 ps in order to avoid unrealistic forces due to the difference between the initial and target structures. The final conformation of the molecule after the last step of the simulation at 0 K was further retained as reference NMR structure. 3.2 Molecular dynamics simulations Molecular dynamics (MD) simulations were carried out with both the MPI and the CUDA versions of the pmemd program in Amber14. Five molecular systems were simulated: two Glylike peptidotriazolamers containing one 1,4- or one 1,5-triazole ring (further referred to as 4Tzl and 5Tzl), two peptidotriazolamers (pep4Tzl and pep5Tzl; see Figure S1 of the Supporting Information) and three α-helical coiled coil (PDB-IDs: 1GCL, 1U9F, and 1U9H

12

). Small

molecules (i.e., 4Tzl, 5Tzl, pep4Tzl, and pep5Tzl) were solvated in a pre-equilibrated cubic box of solvent of 50Å edge length at 300 K, by deleting the overlapping solvent molecules based on the sum of atomic van der Waals radii. Three solvents were considered: water, chloroform, and dimethyl sulfoxide (DMSO). Water simulations (4Tzl and 5Tzl) used the Tip4P-Ew 64 force field with an initial density of 1000 kg·m-3. For the two synthetized peptidomimetics, we performed the simulation in the same solvent as that used for recording the NMR spectra. Pep4Tzl was simulated in chloroform (initial density of 1490 kg·m-3) with parameters taken from Ref. 65 while the simulation of pep5Tzl was performed in DMSO (initial density of 1100 kg·m-3) with parameters from Ref.

66

. The α-helical coiled coils were modeled in their tetrameric form. The 24 ACS Paragon Plus Environment

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structures were solvated in Tip4P-Ew water molecules in a truncated octahedron and neutralized using the tleap program with a buffer region of 12 Å around the protein atoms. The missing residues in 1GCL, 1U9F, and 1U9H were manually added to the sequences as well as N-methyl caps at the C-terminal part of each helix. Heat up of each molecular system was performed gradually in the NVT ensemble following a protocol similar to that described in Ref. 67. After reaching the target temperature (300 K if not otherwise stated), the systems were equilibrated in the NPT ensemble before production runs. In the case of 1U9F, specific inter-atomic distances were restrained during the heat up and released during the NPT MD equilibration phase (1 ns at 300K). In accordance with the crystal structure, these distances involve triazole rings weakly bound to the standard amino acids backbone. The restraints were necessary to avoid breaking of these interactions due to unphysical motions of the whole system during heat up. Production runs were then performed for each system for 200 ns or 150 ns in the NPT ensemble. For 1U9F, an additional run of 200 ns at 270 K was performed to evaluate the temperature dependency of the protein stability. For 1GCL and 1U9H additional 200 ns of simulations were performed at 270K, 330K, and 360K. During all simulations a time step of 2 fs was used and SHAKE bond length constraints were applied to all bonds involving hydrogen atoms. Temperature and pressure were controlled with Langevin dynamics (collision frequency of 4.0 ps-1) and isotropic position scaling, respectively. The structures of small molecules (i.e., 4Tzl, 5Tzl, pep4Tzl, and pep5Tzl) were clustered over the last 100 ns of the corresponding MD simulations. The clustering analysis was performed based on the non-hydrogen atoms of the molecule of interest using a hierarchical agglomerative algorithm as implemented in the cpptraj program of AmberTools15. The minimum distance between each cluster was set to 1 Å and 2 Å for small (i.e., 4Tzl and 5Tzl) and larger (i.e.,



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pep4Tzl and pep5Tzl) peptidotriazolamers, respectively. The RMSD of α-helical coiled coils was calculated with respect to their respective crystal structure based on the backbone atoms (i.e. C, N, CA, N1, N2, N3, C4, and C4). The last four C-terminal residues of each helix were omitted to lower the impact of these highly flexible regions.

4. Evaluation of the parameters 4.1. Non-bonded interactions As we described in the parameterization strategy section, we performed a detailed analysis and specific parameterization of the interactions between triazole rings and polypeptide backbone atoms. For this the interaction energy between either two N-methylacetamide (NMA) molecules or one NMA and one triazole ring was computed for different intermolecular distances and orientations. The results are presented in Figure 4 for the 1,4-triazole ring and in Figure S2 of the Supporting Information for the 1,5-triazole ring. The inset of each subfigure shows a representative structure of the corresponding model system and highlights the distance that was scanned. The interaction energy (Eint) between two molecules of NMA interacting along a typical backbone-backbone hydrogen bond (N-H···O, as depicted in the inset of the figure) is given in Figure 4.A. The minimum of this interaction predicted at the MP2/cc-pVTZ level is located at a distance of 1.8 Å with an energy of -7.5 kcal·mol-1. Similarly, figures 4.B, 4.C, and 4.D show three different energy profiles for the interaction between NMA and the 1,4-triazole ring with respect to the intermolecular distance. The comparison of MP2 interaction energy curves in Figure 4 (i.e., red curves in the plots) shows that the interaction of the nitrogen atoms of the triazole ring (N2 and N3) with the backbone hydrogen atom of NMA (Figure 4.C and 4.D) is


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comparable to a standard backbone-backbone hydrogen bond (Figure 4.A), with a minimum located at a distance of 2.0 Å and an interaction energy of -8 to -9 kcal·mol-1. However, the interaction between the hydrogen atom of the 1,4-triazole ring and the backbone oxygen atom of NMA (Figure 4.B) appears to be less stable, with a minimum located at a distance of 2.1 Å and an interaction energy of about -5 kcal·mol-1. This result is in agreement with the chemical character of the triazole ring hydrogen atom: i.e., compared to the hydrogen atom in an amide group, the hydrogen of the 1,4-triazole ring is slightly less electrophilic as it is bound to a carbon atom, which is less electronegative than the nitrogen atom of a standard peptide bond. Interactions involving the hydrogen atom of a 1,4-triazole ring are therefore expected to be weaker than usual hydrogen bonds between peptide backbone atoms. Next to the QM reference data Figure 4 shows the interaction energy profiles calculated for each system at the MM level. The interaction between two NMA molecules calculated with the ff14SB parameters is in good agreement with the MP2 reference, both for the position and depth of the minimum (blue cure in Figure 4.A). In a first attempt to describe the atoms in the triazole ring, we tried to assign existing Lennard-Jones (LJ) parameters, as available in ff14SB. For the H5 atom in the triazole ring, we assigned either the Amber atom types H (ri= 0.600 Å, εi= 0.0157 kcal·mol-1; hydrogen atom linked to the nitrogen atom in a peptide) or H4 (ri= 1.409 Å, εi= 0.0150 kcal·mol-1; bond hydrogen atom linked to a carbon atom in an imidazole group). While the H type overestimates the interaction, the H4 type critically underestimates it (green and yellow curves in Figure 4.B, respectively). Similarly, we assigned the N Amber type (ri= 1.824 Å, εi= 0.1700 kcal·mol-1; common to all nitrogen atoms in ff14SB) to atoms named N2 and N3 in the triazole ring. As shown by the green profiles in Figures 4.C and 4.D, the minimum of interaction predicted with these parameters is located at the correct intermolecular distance with



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respect to the MP2 reference. However, the strength of the interaction is underestimated by about 2 kcal·mol-1. Existing LJ parameters clearly fail at reproducing the balance between all four types of interactions considered here, as predicted by the MP2 reference calculations. In a macromolecular assembly, this could affect the relative stability of interactions involving triazole rings with respect to standard hydrogen bonds. This analysis shows that new non-bonded parameters are required to properly describe the interactions of triazole rings in a protein environment. Most of the LJ parameters used in the Amber force fields are derived from OPLS, in which the parameters were developed to reproduce experimental macroscopic properties 68-70. Other strategies consist in adding gas phase intermolecular interaction data obtained from ab-initio calculations to the training set, in order to improve the description of intermolecular potentials while ensuring a reproduction of macroscopic properties

71-72

. Here, we chose to base our parameterization solely on gas phase

QM interaction energies. The interaction energy profiles calculated with our new set of parameters are reported in Figures 4.B, 4.C, and 4.D (blue curves), and show a good agreement with the MP2 reference data. Such an approach is convenient from a computational point of view and allows to specifically tune local properties of the molecular system. However, as discussed in Ref.

60

and orther references therein, an important drawback of such a strategy is that many-

body effects are disregarded in the parameterization. Additionally, it is noteworthy that the ε parameters obtained here for nJ and nK atom types are an order of magnitude larger than more standard values (Table 1). It is however not clear if using a different parameterization strategy would have led to more usual values of the parameters. As a consequence of these observations, the LJ parameters developed in the present work are not expected to yield correct macroscopic properties of pure triazole liquids and are most likely not transferable to other molecular systems.


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Nevertheless, we are confident that they will provide a well-balanced description of non-bonded interactions in peptide-based macromolecular assemblies. These local properties are less sensitive to many-body effects and should be reasonably well predicted by our new set of parameters. The corresponding results for the analysis and parameterization of the 1,5-triazole ring are provided in Figure S2 of the Supporting Information. While the interactions involving the nitrogen atoms of the 1,5-triazole ring highly resemble those discussed for the 1,4-triazole ring, the interaction involving the hydrogen atom of the ring appears to be much weaker (i.e., minimum at 2.2 Å and interaction energy of about -3 kcal·mol-1; Figure S2.B). Therefore, a different set of parameters was derived for this particular hydrogen atom (H4 in T15 with the corresponding atom type hU), with which the MP2 reference can be reproduced well. 4.2 Bonded parameters of the triazole rings As detailed in the parameterization strategy section, parameters for new bonds, angles and torsions (proper and improper) were optimized to properly model the 1,4- and 1,5-triazole rings. We performed this parameterization based on the harmonic approximation of vibrational normal modes derived from the equilibrium Hessian matrix of each ring. A common way to assess the quality of such a protocol is to calculate the vibrational spectrum of the target molecules obtained with the molecular force field and to compare it to the corresponding ab initio reference calculations 54-55. In Figure S3 of the Supporting Information, we present such a comparison. As depicted in the figure, the vibrational spectrum obtained with our parameterization reproduces the ab initio reference quite well. Only a slight deviation in the high frequency region of the spectrum can be observed. This region of the spectrum is related to the vibrational stretch of the methyl groups C-H bonds. As the corresponding parameters were fixed to their respective value



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in ff14SB during the fit and therefore not optimized in this work, it was to be expected that they do not necessarily reproduce the MP2 reference. On the other hand, the last vibrational mode, which corresponds to the stretch of the C-H bond in the triazole ring, is reproduced correctly by the new set of parameters. Although these parameters are not of extreme importance for the overall force field developed in this work, such a careful parameterization will allow to fully trust intramolecular motions along a molecular dynamics simulation. 4.3 Backbone torsion parameters The parameters for the backbone torsional degrees of freedom of triazole-based peptidomimetics were specifically optimized. As described in the parameterization strategy section, we first derived primary backbone parameters from Gly-like models (involving only backbone atoms) and further optimized a secondary set from Ala-like models (involving the beta carbon atom of the side chain). This parameterization was performed for all eight new types of amino acid-like residues (four for peptidotriazolamers and four for triazolamers), resulting in 16 surfaces to be fitted. The results of the fits are listed in Table S1 of the Supporting Information and depicted in Figures 5 and 6 for Ala-like models. Additionally, the corresponding potential energy surfaces for Gly-like models are reported in Figures S4 and S5 of the Supporting Information. The weighted and non-weighted root mean square deviations (RMSD) in Table S1 are quite low, with none of the surfaces presenting an RMSD higher than 1.0 kcal·mol-1 with respect to the MP2 reference. It should be noticed that the RMSD values of each surface prior to the parameterization of the dihedral angle potentials were already low (i.e., maximum RMSD of 1.75 kcal·mol-1) and that they already captured the general features of the ab initio reference surface (i.e., main minima and maxima). As the shape of the conformational potential energy surface of peptidomimetics and standard peptides is mainly governed by the Coulomb and van


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der Waals non-bonded interactions, the low RMSD of the original, unfitted surfaces indicates that our set of non-bonded parameters is well suited for this system. Correspondingly, the values of the force constants (V1-4) are very low and act as a small correction to the surface (see Tables 3 and 4). This is especially true for the force constant for the fourth Fourier term of the periodic potential expansion (V4), which has a very low value in most cases due to the symmetry of the parameterized bond and thus the Fourier series could have been limited to the third order for most of the systems. However, as the weight of this term is not negligible in some other cases, we consistently included it in all the backbone torsions parameterized in this work. A comparison of the potential energy surfaces obtained with our new set of parameters with respect to their corresponding MP2 reference is depicted in Figure 5 for the Ala-like peptidotriazolamer systems. A very good agreement between the parameterized and reference surfaces can be seen, confirming the quality of the fits as observed from the RMSD values listed in Table S1 of the Supporting Information. Figure 5 shows clearly that the surfaces associated with the conformational preferences of peptidotriazolamers are significantly different from that of a standard alanine dipeptide. While the surface of A4n (Ala-like 1,4-peptidotriazolamer at the N-terminal end) still shares some similarities with a standard peptide, all others demonstrate that peptidotriazolamers bear a unique conformational space. The figure also depicts some characteristics of the two termini of such type of peptidomimetics (i.e., the N- and C-terminal parts, AXn and AXc, respectively). The C-terminal region presents a very symmetrical surface, with equal probability for two different basins, as opposed to the N-terminus. Similarly, Figure 6 presents the MP2 and force field calculated surfaces of the four Ala-like triazolamer systems. Here, the character of the conformational space is significantly different from that of a standard polypeptide as the surfaces are rough and present many local minima.



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Comparison of the conformational potential energy surfaces at the QM and MM level demonstrates that our new set of parameters successfully reproduces the MP2 reference. Therefore, the present force field (TZLff) is expected to appropriately reproduce the conformational preferences of triazole-based peptidomimetics. 4.4. Conformational preferences of Gly-like 1,4- and 1,5-peptidotriazolamers As a first evaluation of the new parameter sets, we re-investigated the work by Kann et al.

31

about the conformational preferences of the simplest Gly-like peptidotriazolamers presenting a 1,4- or a 1,5-triazole ring. Using our residue definition and nomenclature, these peptides named as 4Tzl and 5Tzl in the work by Kann et al. correspond to ACE-G4n-T14-G4c-NME and ACEG5n-T15-G5c-NME, respectively. As we briefly discussed in the Introduction, the conformational space of these two model systems was investigated in Ref.

31

by means of a

systematic search followed by geometry optimizations at various levels of QM theory. While no solvent model was considered during the initial search, an implicit solvent model was then introduced to differentiate between the conformational preferences in water, DMSO, and 1decanol. Despite the significant insights obtained by this study, it also has clear limitations due to the neglect of explicit solute-solvent interactions and dynamics. Here, we performed 200 ns long simulations of the 4Tzl and 5Tzl peptidotriazolamers in gas phase as well as in a box of explicit water. Peptidotriazolamer conformations were clustered from the last 100 ns of each trajectory and further analyzed. Table 5 lists a description of all clusters presenting a relevant population (i.e., higher that 1% of the simulation). Correspondingly, Figures S6 and S7 of the Supporting Information depict the representative structures of each cluster with a population higher than 10%, from the 4Tzl and 5Tzl simulations (gas phase and water), respectively.


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The population analysis of the structural clusters in Table 5 for the Gly-like 1,4peptidotriazolamer (4Tzl) in gas phase shows that this peptidomimetic is very flexible, with four clusters featuring a population higher than 10% (Figure S6.A). Clusters 1 and 2 are mirror images of each other and correspond to a folded conformation stabilized by intramolecular interactions between the N- and C-terminal amide bonds. Clusters 3 and 4 have a slightly lower population and show a more extended conformation with the N-terminal amide bond not participating in any interaction while the C-terminal end forms an interaction with the center of the triazole ring through the amide hydrogen atom. The structures represented by clusters 1/2 and clusters 3/4 directly correspond to the most stable conformations described by Kann et al. (named as 4Tzl-2 and 4Tzl-1 in Ref.

31

, respectively). The relative stability of these

conformations is however hard to assess from the calculations by Kann et al. as the energy differences are only of a few tenths of kcal·mol-1. According to the analysis of the simulations in explicit water (Table 5 and Figure S6.B), only two conformations of 4Tzl (mirror images of each other) could be sampled in solution with equal population of about 50%. Enhanced sampling techniques would be necessary to assess if other local minima are accessible at this temperature. The obtained structures correspond to the 4th most stable conformation suggested by Kann et al. for this molecule in implicit water (4Tzl-4 in Ref. 31). However, as the energy difference of that conformation in the Kann study to the highest ranked structure is only 1.09 kcal·mol-1

31

, a clear statement about the relative stability of the

different conformers is not possible. The results of our simulation in explicit solvent clearly show the impact of water on the conformational preferences of 1,4-peptidotriazolamers. The favored structures depicted in Figure S6.B feature a linear conformation at the N-terminal region with an interaction between the amide hydrogen and the N3 nitrogen of the triazole ring. The C-



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terminal region has an orientation similar to that observed in gas phase with an interaction between the amide hydrogen and the triazole ring. The solvation of the hydrophilic moieties of the molecule (i.e., amide bonds and triazole ring) is likely to be responsible for shielding any interactions between the termini of the peptidotriazolamers, thus leading to the stabilization of more extended conformations. Table 5 and Figure S7.A show a similar analysis for the Gly-based 1,5-peptidotriazolamer (5Tzl) in gas phase. It can be seen that the number of conformations accessible for 5Tzl is higher than that of 4Tzl. This observation and the obtained conformations of 5Tzl in gas phase are in agreement with the work by Kann et al. The two main clusters (Figure S7.A) feature a folded structure in which the two amide bonds interact with each other in an anti-parallel manner. The two following clusters are slightly less populated and also present a folded conformation with an intramolecular hydrogen bond between the two termini of the molecule. As also observed by Kann et al., the relative stability of the conformers is significantly affected by the presence of solvent, while the number of possible conformers remains very high (Table 5 and Figure S7.B). The two most stable conformations observed in gas phase are still present in solution but with lower probability. The main conformation in water is extended with the Cterminal amide hydrogen interacting with the center of the triazole ring. This is also likely to be the result of solvation of both termini, which destabilizes intramolecular interactions and thus favors extended conformations. Overall, our predictions for the predominant conformations of Gly-like 1,4- and 1,5peptidotriazolamers in the gas phase and in water generally agree with the previous work by Kann et al. However, some deviations are observed regarding the number of accessible conformations in water and their relative stability. Such deviations were to be expected and


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result from the different description of the solvent in the present study and in Ref. 31: explicit in the former and implicit in the latter. An explicit description of solute/solvent interaction is likely to be more realistic, thus emphasizing the relevance of precise force field development to model triazole-based peptidomimetics. Nevertheless, the agreement between those two studies is clear and shows the potential of the new force field parameters in describing the conformational properties of this family of compounds.

5. Results and Discussion 5.1. Structural prediction of synthetized 1,4- and 1,5-peptidotriazolamers After successful evaluation of the new parameter sets, we applied them for the prediction of the conformation of two peptidotriazolamers previously synthetized: i.e., Boc-Gly-Ψ(1,4-triazole)Phe-Gly-Ψ(1,4-triazole)-Val-OAll and Boc-Val-Ψ(1,5-triazole)-Ala-Leu-Ψ(1,5-triazole)-ValOBn further referred to as pep4Tzl and pep5Tzl, respectively. The two molecules are representatives of 4-units long 1,4- and 1,5-peptidotriazolamers. In our nomenclature and residue definition, these compounds correspond to: BOC-G4n-T14-F4c-G4n-T14-V4c-ALL (pep4Tzl) and BOC-V5n-T15-A5c-L5n-T15-V5c-BNE (pep5Tzl), with BOC, ALL, and BNE being capping residues corresponding to the Boc, Allyl, and Benzyl protecting groups, respectively. NOE distances were derived from the analysis of the NMR spectra of pep4Tzl and pep5Tzl, measured in DMSO and in chloroform, respectively (see Figure S1 of the Supporting Information). We performed two molecular dynamics simulations for each compound: first a simulated annealing-based MD simulation in gas phase with the NMR restraints to obtain a reference structure and second a standard, unconstrained 200 ns long molecular dynamics study



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in a box of explicit solvent (DMSO or chloroform according to the experimental conditions). The structures of the last 100 ns of the unconstrained MD simulation were clustered based on the root mean square deviation of all non-hydrogen atoms. In Figure 7, we present a comparison between the NMR reference structures and the representative structure of the main cluster extracted from the unconstrained MD trajectories in explicit solvent for pep4Tzl and pep5Tzl (Figures 7.A and 7.B, respectively). Therein, the NMR reference structure is shown in orange, while the most stable conformation obtained from MD is represented in cyan. As it can be visually observed, the non-constrained dynamics of the peptidotriazolamers reproduces the structure derived from NMR measurements for both compounds in a satisfactory manner. The RMSDs of pep4Tzl and pep5Tzl were calculated for the backbone atoms (i.e., atom names C, CA, N, N1, N2, N3, C4, C5, as defined in Figure 2) with respect to their reference structure and were found to be as low as 0.99 and 0.30 Å, respectively. Analysis of the conformational cluster populations showed a high stability of the final structures. The main conformations of pep4Tzl and pep5Tzl were observed for 83 % and 45 % of the simulated structures, respectively. For pep5Tzl, a second, highly populated cluster was present for 41 % of the simulation time. This cluster has a similarly small backbone RMSD of 0.99 Å with respect to the NMR structure. Both compounds feature a folded conformation with intramolecular interactions between the Nand the C-terminal regions. These conformations of the molecules are in agreement with the analysis of the Gly-like peptidotriazolamers discussed above, although a direct comparison is not possible due to the different solvents used in the simulations. Ke et al. reported an analysis of the structural features of Gly-containing 1,4-petidotriazolamers based on experimental observations and density functional theory calculations

11

. They determined a β-strand conformation for a 36


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molecule resembling pep4Tzl, as opposed to the folded structure predicted in the present work. Interestingly, the authors also emphasized that such an extended conformation corresponds to a dimer of the molecule, which only forms at relatively high molar concentrations (i.e., 50-133 mM). According to these observations, it appears that the NMR experiment for pep4Tzl was performed at a concentration for which the molecule most likely exists as a monomer (i.e., about 30 mM). The stability of an extended conformation in a β-strand is ensured by intermolecular interactions between two molecules. However, these intermolecular interactions are replaced by intramolecular ones in an isolated molecule leading to a fold in the molecular structure. It is noteworthy that in the case of pep4Tzl, the turn in the backbone conformation is characterized by the presence of a Gly-like residue, which correlates with the turn inducing effect of Gly in standard peptide sequences

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. Thus these two effects can explain the peptidotriazolamer

conformations obtained in this study. Further simulations of systems containing multiple molecules would be interesting to study the dimerization process, but this goes beyond the scope of the present work. Overall, these results indicate a great potential of our force field parameters for future NMR refinement and conformational dynamics analysis of triazole-based peptidomimetics. 5.2. Thermal stability of α -helical coiled coil structures As a final test case, we investigated the stability of peptide structures in which one peptide bond is replaced by a 1,4-triazole ring. The crystal structure of such a molecular system was reported by Horne et al. about a decade ago 12. In that work, the authors synthetized several variants of a tetrameric α-helical coiled coil consisting of four identical α-helices with a single point mutation per helix. They showed that the position of the mutation in the sequence drastically affects the thermal stability of the protein. We chose two structures from Ref. 12: one in which the peptide



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bond located in the middle of the sequence is replaced by a 1,4-triazole ring (PDB-ID 1U9F) and another in which the mutation is placed closer to the C-terminal end of each helix (PDB-ID 1U9H). As a control simulation, we also considered the wild-type protein (PDB-ID 1GCL). The melting point of these structures measured experimentally drops from >96°C for the wild-type to 61°C for 1U9H and 36°C for 1U9F, making those systems good candidates to assess the propensity of our force field to predict the stability of such assemblies. A representation of the 1U9F structure after 200 ns of MD simulation at 270 K is given in Figure 8.A Modeling this kind of a hybrid molecular structure is possible due to our modular derivation of the present force field (TZLff). While the standard amino acids are modeled as ff14SB residues, the peptidotriazolamer-based residues are described as a G4n-T14-L4c moiety (i.e., an N-terminal Gly-like residue, a 1,4-triazole ring, and a Leu-like C-terminal residue, respectively; see Parameterization strategy section for details). As shown in Figure 8.B and discussed in Ref 12

, the triazole ring successfully substitutes the standard peptide bond in the sequence and

participates in backbone stabilization. The N-terminal amide hydrogen atom and that of the triazole ring (H5) point in the same direction and interact with the backbone oxygen atom of residue Ile12 (residue indices are given according to the 1U9F crystal structure). Similarly, the N2 nitrogen atom of the triazole ring is involved in a stable hydrogen bond with the backbone hydrogen atom of the standard amino acid Ile18. We performed two simulations of 1U9F in water to evaluate the stability of the protein at low temperature (i.e., 270 K) and at a temperature closer to its melting point (i.e., 300 K). The corresponding time series are plotted in Figure 9. In Figures 9.A and 9.B we monitored the distance characteristic for the interaction of the triazole ring hydrogen atom with the backbone amide oxygen of Ile12 (RH5-O as defined in Figure 8.B) throughout the simulations at 270 K and


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300 K, respectively. The plots follow the RH5-O distance in each of the four helices. The stability of the backbone/triazole ring interaction is clearly affected by the increase in temperature. At 270 K (Figure 9.A), all four helices show a very stable interaction with a slightly higher flexibility for helix 2. The characteristic backbone/triazole ring distance in helices 1, 3, and 4 fluctuates around an average value close to the one of the crystal structure, as represented in Figure 8.B for helix 1. However, Figure 8.C shows that for helix 2 at 270 K this interaction is broken after 200 ns, favoring standard backbone/backbone hydrogen bonds. Monitoring of the RH5-O distance during the simulation at 300 K (Figure 9.B) demonstrates that the interaction between the triazole ring and the backbone breaks sequentially in each helix and that eventually, none of these interactions remain after 140 ns. In Figure 9.C we report the fluctuation of the backbone RMSD of the protein with respect to the crystal structure along the two simulations (i.e., at 270 K and 300 K in blue and red, respectively). The low and stable value of the RMSD for the simulation at 270 K (about 1.0 Å) shows that the structure is stable and does not suffer from the breaking/forming of any backbone/triazole ring interaction. However, following the RMSD at 300 K shows that the protein stability is directly affected by the breaking of this interaction in all four helices. The RMSD rises from 2.0 Å to more than 4.0 Å after 125 ns, at which point the last backbone/triazole ring interaction breaks (Figure 9.B). In Figures S9.A and S9.B of the Supporting Information, we report the RMSD time series along 200 ns of dynamics at 270K, 300K, 330K, and 360K in water for 1GCL and 1U9H, respectively. Selected representations of the structures are also shown in the Figure (at 300K for 1GCL, and at 300K and 360K for 1U9H). The structure of the wild-type (1GCL) remains stable at all studied temperature and bears a low RMSD oscillating around 1.5 Å with respect to the crystal structure.



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In the case of 1U9H, the RMSD profiles at temperatures from 270K to 330K are similar and show a stable oscillation around a value between 2.0 and 3.0 Å. The high RMSD value of this system compared to 1GCL and 1U9F is due to the more flexible character of the C-terminal region of the helices. However, as shown by the representation of the coil at 300K in Figure S8.B, the secondary structure of the protein is not affected. At higher temperature (360K), the RMSD is still stable but reaches a higher value of 4.0 Å. As depicted in Figure S8.B, this increase is due to the unfolding of the C-terminal part of one helix. However, the integrity of the N-terminal helices does not appear to be affected by the increased temperature. Our results are in agreement with the thermal stability assay performed in Ref. 12 for 1U9F and in a lower extent for 1U9H. In the case of 1U9F, which has the mutation in the middle of the sequence, the interaction between the standard backbone and the triazole ring is stable at low temperature and distance-wise close to the crystal structure. Correspondingly, the overall structure of the protein remains in a stable conformation. However, at a temperature closer to the melting point, the backbone/triazole ring interactions are too weak to be retained and eventually break in all helices, leading to the unfolding of the protein. In contrary, the wild-type protein (1GCL) is stable at all temperature evaluated in this work while a mutation at a less critical position (1U9H) slightly affects the overall integrity of the macromolecule. These observations point towards a direct involvement of the triazole ring mutation in the protein stability and is in agreement with the experimental observations showing that the stability of the protein depends on the position of the triazole ring in the sequence 12. While the stability of the peptidic region of the structures is due to the good quality of the ff14SB force field, the proper description of the impact of triazole-based mutations relies our present parameterization. As discussed in the previous section, the interaction between the hydrogen


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atom of a 1,4-triazole ring and a backbone oxygen atom is weaker than a standard backbone/backbone hydrogen bond. This correlates with the observations based on the MD simulations of 1U9F, as interactions between standard peptide backbone are favored at higher temperatures. This last result shows that our set of parameters allows for a well-balanced description of all interactions in hybrid protein/peptidotriazolamer systems. It also demonstrates the potential application of triazole-based peptidomimetics to modulate the stability of biomolecular assemblies.

6. Conclusions We performed the first complete force field parameterization of 1,4- and 1,5-substituted triazolebased peptidomimetics. The parameterization is compatible with the AMBER ff14SB force field and follows AMBER’s approach to parameter development. The resulting parameters are accessible as Supporting Information. The accurate results obtained using the new force field with respect to reference ab initio data demonstrate the success of our parameterization strategy and makes us confident about the applicability of the parameters for molecular simulations studies of this family of compounds. To the best of our knowledge, we report here the first molecular dynamics simulations of peptidotriazolamer compounds in solution. The simulation of model compounds showed that our parameterization yields accurate results regarding the conformational preferences of peptidotriazolamers in different solvents, compared to reference data from NMR measurements. Additionally, we validated our parameterization results against data from previous studies. In all cases we were able to successfully reproduce the preceding results and observations to a highly



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satisfactory degree. Our results also provide deeper insights into the effect of explicit solute/solvent interactions on the conformational preferences of triazole-based peptidomimetics. Finally, the precise parameterization of intermolecular interactions involving triazole rings and amide atoms allowed us to successfully simulate the thermal stability of an α-helical coiled coil presenting triazole-based point mutations. The parameterization strategy developed in this work inherits from other well-tested methodologies. We believe that the general workflow that we designed can be adapted to other molecular scaffolds in order to develop accurate parameters for any polymeric molecular assemblies similarly to the presented case of triazole-based peptidomimetics.

Acknowledgments Financial support from Deutsche Forschungsgemeinschaft (SE 609/10-1 to N.W., SFB 749/C08 to I.A.) and Heinrich Hertz Foundation (fellowship awarded to J.G.) is gratefully acknowledged. The co-operation on the project was financially supported by the Leading National Research Centre’s (KNOW, Poland) financing program for the Wroclaw Centre of Biotechnology for years 2014-2018, the BioNam: Bionanomaterials project, as well as the statutory activity subsidy from the Polish Ministry of Science and Higher Education (PMSHE) for the Faculty of Chemistry of Wrocław University of Science and Technology. Computational resources were provided by Wroclaw Centre for Networking and Supercomputing (http://wcss.pl), grant No. 197, the supercomputing facilities of the Leibniz-Supercomputing-Center in Garching, and by the Deutsche Forschungsgemeinschaft (SFB 749/C08).


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Author Contributions A.M. and J.G. performed parameterization and the computational studies. J.G., O.K., T.F., and R.L. performed the experiments. A.M., N.S., and I.A. designed the study. N.S. and I.A. supervised the project. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources Financial support was received from the Deutsche Forschungsgemeinschaft (SE 609/10-1, SFB 749/C08), the Heinrich Hertz Foundation (fellowship awarded to J.G.), the Leading National Research Centre’s (KNOW, Poland) financing program for the Wroclaw Centre of Biotechnology for years 2014-2018, the BioNam: Bionanomaterials project, the Polish Ministry of Science and Higher Education (PMSHE) for the Faculty of Chemistry of Wrocław University of Science and Technology, Wroclaw Centre for Networking and Supercomputing (http://wcss.pl), grant No. 197.



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Figures.

Scheme 1. Schematic representation of polypeptides, peptidotriazolamers, and triazolamers, for the two triazole-based peptidomimetics only the 1,4-subsitututed triazole rings are shown.


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Figure 1. Flowchart of the applied parameterization strategy. On the left-hand side, the general pipeline that can be transferred to the parameterization of other non-standard residues involved in polymeric sequences is shown. On the right-hand side, practical illustrations are provided for each step, based on the specific parameterization performed in this work. Each step should be performed in the presented sequential order, as the derivation of new parameters is dependent on those defined at the previous steps: e.g., variations in point charges (step 2) could affect the value of the torsional parameters (step 6). “AA-like” stands for amino acid-like.



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Figure 2. Atom names, atom types, and residue definition of 1,4- and 1,5-triazole rings (A. and B., respectively) as well as 1,4- and 1,5-triazole based peptidotriazolamers (C. and D., respectively). In each subfigure, the left representation shows the atom names in blue, while on the right side the corresponding atom types are depicted in red. The gray dashed lines in the right scheme indicate the residue definitions with the corresponding name of the structure in residue nomenclature given below. ACE and NME are standard ff14SB acetyl and N-methyl residues, while all other residues are newly introduced in the present work. Atom types of the form XX are standard ff14SB types, while new atom types defined in this work follow a xX nomenclature, as detailed in the main text. For peptidotriazolamers (C. and D.), the definition of the backbone dihedral angles is given below the left scheme.


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Figure 3. Atom names, atom types, and residue definition of triazolamers. A.: aminoacid-like triazolamer connected to a 1,4-triazole ring at both the N- and C-terminal ends. B.: aminoacidlike triazolamer connected to a 1,4- and to a 1,5-triazole ring at the N- and at the C-terminal end, respectively. C.: aminoacid-like triazolamer connected to a 1,5- and to a 1,4-triazole ring at the N- and on the C-terminal end, respectively. D.: aminoacid-like triazolamer connected to a 1,5triazole ring at both the N- and C-terminal ends. Nomenclature and coloring rules follow the definition given in Figure 2.



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Figure 4. Representation of the intermolecular interaction energy (in kcal·mol-1) between two Nmethyl acetamide (NMA) molecules (A.) and between one NMA and one 1,4-triazole ring (B., C., and D.). The inset in each subfigure shows the system used for the calculation and highlights the specific coordinate that was scanned in yellow. Surfaces calculated at the MP2/cc-pVTZ level of QM theory are depicted in red with cross shaped markers, while the corresponding surfaces obtained with the new set of parameters developed in this work are shown in blue with round shaped markers. Profiles obtained using other existing Lennard-Jones (LJ) parameters are reported in yellow and green, with diamond and triangle shaped markers.


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Figure 5. Comparison of conformational potential energy surfaces (kcal·mol-1) for peptidotriazolamers calculated with the MP2/cc-pVTZ reference method and with the final set of parameters developed in this work. Subfigures A. and B. correspond to Ala-like 1,4peptidotriazolamer at the N- and C-terminal end, respectively. C. and D. show the corresponding data for Ala-like 1,5-peptidotriazolamers. The corresponding molecular system is given in each subfigure with a definition of the Φ/Ψ dihedral angles.



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Figure 6. Comparison of conformational potential energy surfaces (kcal·mol-1) for triazolamers calculated with the MP2/cc-pVTZ reference method and with the final set of parameters developed in this work. Subfigures A., B., C. and D. represent each possible combination of triazole rings (1,4- or 1,5-substituted) at each end of an Ala-like triazolamer. The corresponding molecular system is given in each subfigure with a definition of the Φ/Ψ dihedral angles.


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Figure 7. Comparison of the representative structures obtained by standard molecular dynamics (main structural cluster of the last 100 ns, cyan carbon atoms) and from the NMR reference data (orange carbon atoms) for the two synthetized peptidotriazolamers: Boc-Gly-Ψ(1,4-triazole)Phe-Gly-Ψ(1,4-triazole)-Val-OAll (pep4Tzl; A.) and Boc-Val-Ψ(1,5-triazole)-Ala-Leu-Ψ(1,5triazole)-Val-OBn (pep5Tzl; B.). Lewis schematic representations of the molecules are given in each subfigure.



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Figure 8. Representation of the triazole-modified α-helical coiled coil (PDB-ID 1U9F) after 200 ns of molecular dynamics in water at 270 K. In subfigure A. a global cartoon representation of the protein is provided with the triazole-modified region shown in licorice.

For a better

differentiation, the helices (identical sequence) are colored as pairs. Subfigures B. and C. are magnifying representations of the structures around the triazole ring in helices 1 and 2, respectively. Side chains are omitted and their direction is simply indicated by their beta carbon atom. In addition, the values for characteristic distances involved in the stabilization of the protein backbone are given.


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Figure 9. Time series along 200 ns molecular dynamics trajectories in water of the triazolemodified alpha-helical coiled coil (PDB-ID 1U9F) simulated in water at 270K and 300K. Subfigures A. and B. show the fluctuations in the distances that characterize the interaction between the triazole ring and the backbone of the protein in each helix at 270K and 300K, respectively. Subfigure C. monitors the evolution of the protein backbone RMSD with respect to the crystal structure along the simulations at 270K and 300K (blue and red colored lines, respectively).



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Tables Table 1. Final set of the non-bonded and harmonic parameters for the 1,4- and 1,5-triazole rings. All parameters are given in units that are compatible with standard inputs of AMBER. Refer to Figures 1.A and 1.B for the definition of atom types. ri (Å)

Atom nJ

a

nK

1.573

a

1.568

εi (kcal·mol-1)

Angle nI-CT-H1

1.974

a

b

1.232

0.002

cM-nI-CT

hUc

0.814

0.138

cL-nK-nJa

hT

Θ0 (degree)

48.874

109.183

64.306

119.650

a

1.854

nJ-nI-CT

KΘ (kcal·mol-1·rad-2)

a

cL-cM-nIa nK-cL-cM

nI-CT

a

cL-nK

a

cL-cM nK-nJ

a

a

316.427 388.316 441.264

R0 (Å)

128.800 108.650

59.289

103.750

91.188

109.100

a

134.080

106.950

nJ-nI-cMa

90.830

111.550

nK-nJ-nI KR (kcal·mol-1·Å-2)

Bond

a

50.515 103.798

b

1.446

HC-CT-cL

40.533

110.700

1.356

b

28.779

122.850

b

22.994

133.400

b

1.383

hT-cM-nI

cL-cM-hT

367.806

1.325

CT-cL-nK

59.852

121.900

nJ-nIa

330.239

1.347

CT-cL-cMb

25.690

129.000

cM-nIa

437.919

1.354

HC-CT-cMc

cM-hT CT-cL

b

b

442.680

38.585

110.933

1.075

CT-cM-cL

c

38.926

133.100

c

318.576

1.489

CT-cM-nI

60.826

123.150

CT-cMc

308.763

1.486

cM-cL-hUc

19.803

128.900

cL-hUc

446.719

1.076

hU-cL-nKc

30.515

122.000

a

Parameters common to both 1,4- and 1,5-triazole rings. b Parameters for 1,4-triazole ring only. c Parameters for 1,5-triazole ring only.


Page 59 of 65

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Table 2. Final set of dihedral and improper parameters for the 1,4- and 1,5-triazole rings. All parameters are given in units that are compatible with standard inputs of AMBER. Refer to Figures 2.A and 2.B for the definition of atom types. V (kcal·mol-1) γ (degree) Periodicity n

Dihedral nJ-nI-CT-H1a cM-nI-CT-H1

a

0.052

180

3

0.052

0

3

a

2.104

180

2

nK-nJ-nI-CTa

11.177

180

2

cL-nK-nJ-nIa

12.716

180

2

9.251

180

2

cL-cM-nI-CT

cL-cM-nI-nJ

a a

9.307

180

2

nK-nJ-nI-cMa

10.789

180

2

nK-cL-cM-nI

nJ-nK-cL-cMa HC-CT-cL-nK

10.566

180

2

b

0.000

0

3

b

0.000

180

3

9.096

180

2

HC-CT-cL-cM CT-cL-nK-nJb CT-cL-cM-hT

b

0.000

180

2

b

5.578

180

2

b

0.000

180

2

nK-cL-cM-hTb

2.805

180

2

CT-cL-cM-nI

hT-cM-nI-CT nJ-nI-cM-hT

b

3.424

180

2

c

0.056

180

3

c

0.056

0

3

CT-cM-cL-hUc

0.003

180

2

c

6.158

180

2

5.250

180

2

HC-CT-cM-cL HC-CT-cM-nI CT-cM-cL-nK CT-cM-nI-nJ

c

CT-cM-nI-CT

c

0.000

180

2

hU-cL-cM-nIc

1.923

180

2

c

6.450

180

2

hU-cL-nK-nJ

V (kcal.mol-1) γ (degree) Periodicity n

Improper nJ-CT-nI-cMa

0.000

180

2

nK-CT-cL-cMb

2.571

180

2

0.400

180

2

0.921

180

2

1.671

180

2

cL-hT-cM-nI

b

nK-hU-cL-cM cL-CT-cM-nI a

c

c

Parameters common to both 1,4- and 1,5-triazole rings. b Parameters for 1,4-triazole ring only. c Parameters for 1,5-triazole ring only.



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

Page 60 of 65

Table 3. Final set of backbone dihedral parameters for amino acid-like 1,4- and 1,5peptidotriazolamers. Each dihedral is described by a Fourier series truncated at the fourth order. The periodicity parameter n takes values from 1 to 4 and the corresponding force constants and angle phase shifts are labeled accordingly (Vn and γn, respectively). Force constants are given in kcal·mol-1 and phase shifts in degrees. Refer to Figures 2.C and 2.D for the definition of atom types. V1

γ1

V2

γ2

V3

γ3

V4

γ4

0.811

0

0.750

0

0.235

0

0.039

0

N -cU-cL-cM

0.471

0

0.066

180

0.134

0

0.108

0

cM-nI-cU-C

0.224

0

0.275

180

0.449

0

0.143

0

nI-cU-C -N

0.977

180

1.746

180

0.202

180

0.051

0

C -N -cU-CT

0.452

180

0.298

0

0.058

0

0.054

180

CT-cU-cL-cM

0.149

0

0.149

180

0.100

180

0.029

180

cM-nI-cU-CT

0.198

0

0.290

180

0.089

180

0.006

0

CT-cU-C -N

0.654

0

0.387

180

0.042

0

0.007

180

X5n/X5c C -N -cV-cM

0.340

0

0.533

0

0.383

0

0.205

0

N -cV-cM-nI

0.739

180

0.000

0

0.307

0

0.128

0

cM-nI-cV-C

0.404

0

0.502

180

0.396

0

0.149

0

nI-cV-C -N

0.849

180

1.844

180

0.304

180

0.054

180

C -N -cV-CT

0.314

180

0.210

0

0.220

0

0.007

0

CT-cV-cM-nI

0.144

180

0.355

180

0.048

0

0.093

0

cM-nI-cV-CT

0.095

0

0.358

180

0.103

0

0.028

0

CT-cV-C -N

0.551

0

0.411

180

0.149

0

0.041

180

Residue

Dihedral

X4n/X4c C -N -cU-cL


Page 61 of 65

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Table 4. Final set of backbone dihedral parameters for amino acid-like triazolamers residues. Each dihedral is described by a Fourier series truncated at the fourth order. The periodicity parameter n takes values from 1 to 4 and the corresponding force constants and angle phase shifts are labeled accordingly (Vn and γn, respectively). Force constants are given in kcal·mol-1 and phase shifts in degrees. Refer to Figures 3 for the definition of atom types. V1

γ1

V2

γ2

V3

γ3

V4

γ4

cM-nI-cW-cL

0.327

180

0.139

180

0.396

0

0.051

0

nI-cW-cL-cM

1.459

180

0.091

180

0.079

0

0.087

0

cM-nI-cW-CT

0.353

0

0.174

180

0.101

180

0.000

0

CT-cW-cL-cM

0.292

0

0.238

180

0.141

180

0.024

0

cM-nI-cX-cM

0.296

180

0.040

180

0.208

0

0.061

0

Residue Dihedral X44

X45

X54

X55

nI-cX-cM-nI

0.094

0

0.338

0

0.126

0

0.121

180

cM-nI-cX-CT

0.504

0

0.112

180

0.014

180

0.046

0

CT-cX-cM-nI

0.110

180

0.211

180

0.000

180

0.001

180

cM-nI-cY-cL

0.174

0

0.297

180

0.339

0

0.014

180

nI-cY-cL-cM

1.532

180

0.223

180

0.031

180

0.031

0

cM-nI-cY-CT

0.196

0

0.199

180

0.053

180

0.064

0

CT-cY-cL-cM

0.222

0

0.202

180

0.102

180

0.033

0

cM-nI-cZ-cM

0.221

180

0.092

180

0.249

0

0.035

180

nI-cZ-cM-nI

0.108

180

0.046

0

0.106

0

0.145

180

cM-nI-cZ-CT

0.495

0

0.175

180

0.009

0

0.019

0

CT-cZ-cM-nI

0.179

180

0.156

180

0.034

0

0.038

0



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Page 62 of 65

Table 5. Analysis of the structural clusters of Gly-like 1,4- and 1,5-peptidotriazolamers (4Tzl and 5Tzl, respectively) obtained from the MD simulations in gas phase and in water. Clustering was performed based on the non-hydrogen atoms among the last 100 ns of the respective simulations. Only structural clusters having a population greater than or equal to 1% are listed. A structural analysis of each conformation is given by the value of the main backbone dihedral angles (degrees). The definition of dihedral angles is given in Figures 2.C and 2.D. System 4Tzl in gas phase

4Tzl in water 5Tzl in gas phase

5Tzl in water

Cluster

Population (%)

Φ4n

Ψ4n

Φ4c

Ψ4c

1

26.4

79

-101

87

19

2

25.2

-90

103

-78

-19

3

15.7

77

-79

-106

-33

4

15.2

-78

82

84

26

5

6.8

160

-167

102

36

6

5.4

-178

-179

-81

-40

7

4.5

-120

147

78

37

1

50.5

179

177

-69

-7

2

49.4

-178

-174

69

12

1

40.1

81

80

-81

-44

2

24.8

-68

-74

75

37

3

13.1

-163

55

-99

-121

4

10.1

152

-57

99

72

5

4.0

78

-89

-87

-28

6

2.5

-59

110

-121

30

7

2.3

-82

94

80

31

8

1.3

89

-95

-59

150

9

1.0

-93

83

44

-137

1

37.9

151

170

69

19

2

19.4

142

-92

86

13

3

15.3

144

-168

-72

-27

4

13.1

-124

99

-84

-24

5

4.6

-89

173

-64

-22

6

4.4

-128

70

74

5

7

3.5

119

-66

-71

-15

8

1.6

-78

-84

-109

9


Page 63 of 65

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Supporting Information The supporting information consists of three files: 1. TZLff_SI.pdf: Figure S1. Description of the NMR assignment for pep4tzl and pep5TZL. Figure S2. Intermolecular interaction energy for systems involving a 1,5-triazole ring. Figure S3. Vibrational spectrum of the 1,4- and 1,5-triazole rings. Figure S4. Conformational potential energy surfaces (kcal·mol-1) of Gly-like peptidotriazolamers. Figure S5. Conformational potential energy surfaces (kcal·mol-1) of Gly-like triazolamers. Figure S6. Representative structures of a Gly-based 1,4-peptidotriazolamer (4Tzl). Figure S7. Representative structures of a Gly-based 1,5-peptidotriazolamer (5Tzl). Figure S8. RMSD time series along 200 ns molecular dynamics trajectories in water of PDB-IDs 1GCL and 1U9H. Table S1. RMSD, in kcal·mol-1 of the conformational potential energy surface of peptidotriazolamers and triazolamers. 2. TZLff.zip: All files and information necessary for the setup of triazolamer-based simulations with AMBER. 3. PDBs.zip: NMR and MD representative structures of pep4tzl and pep5tzl.



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Page 64 of 65

Table of Content


Page 65 of 65

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82x44mm (300 x 300 DPI)


Amber-Compatible Parametrization Procedure for Peptide-like

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