# Cosolvent Effects Question the Role of Water in Native Chemical Ligation

Julie Di Adamo<fnref n="1" />, Vangelis Agouridas<fnref n="1" />,<fnref n="2" />, Vincent Diemer<fnref n="1" />, Oleg Melnyk*<fnref n="1" />

*Supporting Information Placeholder*

**ABSTRACT:** Guanidine hydrochloride and acetonitrile strongly inhibit Native Chemical Ligation (NCL). These effects, considered alongside the cosolvents' impact on water structure, suggest that water actively participates in NCL.

<figure><img src="image_1.png" /></figure>

The Native Chemical Ligation (NCL<fnref n="1" />) between a C-terminal peptide thioester and an N-terminal cysteinyl (Cys) peptide enables the formation of a larger peptide featuring a native peptide bond at the Cys residue (Scheme 1). This reaction has become a widely used method for chemical protein synthesis, bioconjugate production, and other applications in the life sciences and beyond.<fnref n="2" /> NCL can be performed using peptide alkylthioesters I in the presence of arylthiol catalysts³ such as 4-mercaptophenylacetic acid (MPAA⁴), which leads to the in situ formation of a more reactive aryl peptide thioester of type II,⁵ or directly from aryl peptide thioester of type II (Scheme 1). The latter approach is particularly attractive owing to their ease of synthesis from peptide hydrazides.⁶

<figure><img src="image_2.png" /><figcaption>**Scheme 1.** Principle of the NCL reaction.</figcaption></figure>

A few studies have investigated the rate of NCL involving aryl thioesters of type II, as this information is essential for defining its scope of applications and for comparison with other ligation techniques.⁷ A striking observation from these reports is the wide variation in the measured reaction rates, i.e. from 0.26 M⁻<fnref n="1" /> s⁻¹⁷a to 4.05 ± 0.1 M⁻<fnref n="1" /> s⁻¹⁷b for a peptidyl alanyl aryl thioester, reflecting the absence of standardized experimental conditions across the studies. While the influence of parameters such as pH or catalysts' concentration is well understood and controlled in NCL, other factors that might explain such variations have been largely overlooked. This is notably the case for the addition of guanidine hydrochloride (Gn·HCl) or acetonitrile (ACN), which are nonetheless routinely employed as cosolvents to help

the solubilization of the peptide reactants,²b as in the above mentioned kinetic studies. In this work, we provide a systematic analysis that unveils the major effects of Gn·HCl and ACN on NCL kinetics and discuss their impact on reactivity. Urea, because of its tendency to decompose and to generate byproducts from peptidic material,⁸ is a less preferred protein denaturant and was excluded from the focus of our study.

The model reaction studied in this work is shown in Figure 1A. Peptidyl alanyl thioester 1 was reacted with a Cys peptide 2 in a buffer (pH 7.0) containing either Gn·HCl or ACN as cosolvents. The submillimolar peptide concentrations (thioester, 50 µM; Cys peptide, 60 µM) used for kinetic experiments were adapted to the precise monitoring of the reactions, including the initial fastest phase of the kinetics. MPAA and tris(2-carboxyethyl)phosphine (TCEP) were included as additives to prevent the formation of branched thioesters involving the internal Cys residue of the ligation product 3, and to suppress disulfide formation through oxidation, respectively. The reactions were analyzed by HPLC using UV detection at 215 nm. The peak areas for peptide thioester I were corrected for the contribution of the MPAA residue. The data were analyzed using Kintek kinetic explorer and the apparent second order rate constants are reported as value ± standard error of fit (SE).⁹

Preliminary experiments showed that sodium phosphate and 3-(N-morpholino)propanesulfonic acid (MOPS) buffers gave similar second-order rate constants; including in the presence of the studied cosolvents (see Supplementary Information). However, phosphate buffers are known to react with aryl thioesters and promote their hydrolysis and epimerization.¹⁰ Consequently, MOPS was used for the kinetic studies.

In 10 mM MOPS buffer at pH 7.0, the reaction of peptide thioester 1a with Cys peptide 2 displayed an apparent second order rate constant of 41.4 ± 5.7 M⁻<fnref n="1" /> s⁻<fnref n="1" />, meaning an absolute second order rate constant of ~290 M⁻<fnref n="1" /> s⁻<fnref n="1" /> considering the fraction of Cys thiolate species (~15%¹¹) at pH 7. We substituted His7 residue in peptide 1a by an alanine residue and found that this analog, i.e. peptide 1b (Figure 1A), reacted as fast as peptide thioester 1a (63.8 ± 9.4 M⁻<fnref n="1" /> s⁻<fnref n="1" />). This result rules out the participation of His7 in the reaction, particularly through the formation

<footnote n="1">¹ Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, F-59000 Lille, France.</footnote>

<footnote n="2">² Centrale Lille, F-59000 Lille, France.</footnote>
of activated acylimidazole-type intermediates.¹² Substituting
MPAA-derived thioester **1a** by the corresponding thiophenyl
ester led to a similar rate (58.4 ± 11.9 M⁻¹ s⁻¹, see
Supplementary Information). Finally, replacing the C-terminal
Ala residue with Val as in peptide **1c** (Figure 1A) afforded an
apparent second order rate constant of 2.93 ± 0.80 M⁻¹ s⁻¹, con-
sistent with the greater steric hindrance of the Val residue com-
pared to Ala.¹³

We next examined the effect of Gn·HCl as cosolvent. Figure 1B
shows that the addition of Gn·HCl, up to 6 M, resulted in a sig-
nificant decrease in the second-order rate constant. The rate
constant determined in this work at 6 M Gn·HCl (5.9 ± 0.5 M⁻¹
s⁻¹) is consistent with the previously reported value (4.05 ± 0.1
M⁻¹ s⁻¹) for an MPAA thioester generated *in situ* from a peptide

alkyl thioester (0.1 mM) and excess MPAA (200 mM).<sup>7b</sup> Note
that 2 M Gn·HCl, which corresponds to a Gn·HCl molar frac-
tion x<sub>Gn·HCl</sub> = 0.04 (Figure 3A), already has a strong negative
impact on the NCL rate. Further addition of Gn·HCl, up to
x<sub>Gn·HCl</sub> = 0.16 (6 M Gn·HCl), led to a monotonic yet progres-
sively smaller decrease in the NCL rate. A similar inhibitory
effect of 6 M Gn·HCl was observed when peptide thioester **1c**
equipped with a C-terminal valine residue was reacted with Cys
peptide **2** (see Supplementary Information). These results can-
not be explained by the modest (~0.2 unit) decrease in the ap-
parent pK<sub>a</sub> of the Cys thiol induced by 6 M Gn·HCl,<sup>14</sup> indicating
that another mechanism underlies the marked effect of Gn·HCl
on the NCL rate.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Effect of increasing concentrations of Gn·HCl or ACN on the rate of NCL. A) Reaction studied. B) Effect of Gn·HCl. C) Effect of ACN. The dotted lines correspond to the fitting curves.</figcaption></figure>

Figure 1C shows that the apparent second-order rate constant of NCL also decreased significantly upon addition of ACN. The NCL rate dropped by approximately fourfold when the volumetric fraction of ACN was increased from 0 (41.4 ± 5.7 M⁻¹ s⁻¹) to 50% (12.4 ± 6.4 M⁻¹ s⁻¹), corresponding to a molar fraction x<sub>ACN</sub> = 0.5, which is considerably higher than x<sub>Gn·HCl</sub> at 6 M Gn·HCl (Figure 3A). Thus, the inhibitory effect of ACN on the NCL rate is less pronounced than that induced by Gn·HCl. The relationship between the NCL second-order rate constant and the ACN molar fraction (Figure 3A) also differs markedly from that observed for Gn·HCl. In this case, the rate constant decreases monotonically up to x<sub>ACN</sub> ≈ 0.3 and does not vary significantly above this value.

Given the inhibitory effects of Gn·HCl and ACN on NCL, we
recommend minimizing their molar fraction whenever possible
and employing detergents such as N-octylglucoside, which has
no impact on the NCL rate (data not shown), to improve peptide
solubility.

We would like to stress that attempts to use high concentrations
of MPAA (200 mM) in combination with ACN led to the partial
N-terminal acetylation of the Cys peptide **2** during NCL (HPLC

yield ~50%, see Supplementary Information). No acetylation
was observed with submillimolar concentrations of MPAA as
in our kinetic experiments, which are therefore not biased by
this side-reaction. Extensive experimentation using Cys peptide
**4** (Figure 2 and see Supplementary Information) showed that i)
acetylated peptide **5a** (34% yield) was found to be identical by
UPLC-MS and NMR to an authentic sample produced by solid
phase peptide synthesis, ii) acetylation proceeded with a half-
reaction time of ~20 min, iii) the use of deuterated acetonitrile
furnished deuterated peptide **5b**, and thus evidence that ACN is
the origin of the acetyl group, iv) neither thioimidate nor thia-
zoline intermediates could be detected, as observed by others
with activated nitriles¹⁵.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Acetylation of Cys peptide **4** in the presence of ACN or acetonitrile-d₃ (ACN-d₃) and MPAA. A) Reactions studied. B) Time-course of peptide acetylation using ACN.</figcaption></figure>

Coming back to cosolvent effects and with the aim to provide a unified picture of these apparently puzzling data, we searched the literature for the factors influencing the nucleophilic additions to carbonyl compounds, which is the type of reaction involved in the rate-limiting step of the studied NCL (II + III → IV, Scheme 1). Strikingly, several works demonstrated that nucleophilic additions to various types of carbonyl compounds are promoted by water.¹⁶ This effect is particularly well discussed for ester aminolysis¹⁶a-d as well as thioester aminolysis ¹⁷ and hydrolysis.¹⁸ By contrast, no such detailed mechanistic investigations exist for the thiol-thioester exchange reaction. Nevertheless, it is reported that thiol-thioester interchanges¹⁹ and, by extension, NCL²⁰ do not proceed in anhydrous organic solvents unless a strong base is added. In line with this, NCL proceeds in ionic liquids only when water is used as a cosolvent in high proportions.²¹ Taken together, these reports suggest an active role of water in NCL, meaning that any perturbation of water catalysis should negatively affect the rate of the reaction. This led us to examine the knowledge regarding the impact of Gn·HCl and ACN on water structure.

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Impact of Gn·HCl and ACN cosolvents on A) the second order rate constant of NCL, see also Figure 1. B) on water activity. Data taken from refs²².</figcaption></figure>

Water-ACN mixtures have been studied extensively using various in silico and experimental techniques.²³ When ACN is added to pure water, it initially occupies the cavities between water molecules. When the ACN molar fraction x<sub>ACN</sub> exceeds 0.2–0.3, the mixture becomes microheterogeneous, a state characterized by the coexistence of large clusters of water and ACN molecules. For example, Bertie and Lan identified x<sub>ACN</sub> = 0.3 as the threshold for the onset of microheterogeneity based on quantification of hydrogen bonding by infrared spectroscopy. Previous studies established that the dependence of water activity on x<sub>ACN</sub> in water-ACN mixtures exhibits an initial decrease up to x<sub>ACN</sub> ≈ 0.2, followed by a modest, monotonic decline up to x<sub>ACN</sub> ≈ 0.5 (Figure 3B).²⁴ Taken together, these insights shed light on the pattern presented in Figure 3A, which shows a decline of NCL rate up to x<sub>ACN</sub> ≈ 0.3 and little variation above this value.

Regarding Gn·HCl, both experimental and computational studies have shown that the guanidinium cation forms strong hydrogen bonds with water molecules.²⁵ Raman spectroscopy studies of aqueous Gn·HCl solutions revealed that 2 M Gn·HCl (x<sub>Gn·HCl</sub> = 0.04) already causes substantial disruption of the water hydrogen-bond network, with progressively smaller effects at higher concentrations.²⁶ Our data parallel that observation by showing that 2 M Gn·HCl has already a large impact on NCL rate (Figure 3A). Note that guanidinium cations can also accumulate at the surface of polypeptides by interacting with aromatic and hydrophobic side chains, thereby displacing water molecules from their hydration shells.²⁷ Therefore, if water actively participates in NCL, this provides a straightforward explanation for the large inhibitory effect of Gn·HCl via two possible mechanisms: perturbation of the water structure and competition with Gn·HCl for the hydration of the aryl thioester moiety.
Figure 4 proposes a mechanism to explain the cosolvent effects observed in this work, which should be viewed as a working hypothesis for future studies. Although water can act as a bi-functional catalyst in thioester hydrolysis and aminolysis, this role does not apply to the NCL capture step—the thiol–thioester exchange involving Cys thiolate—which proceeds without proton transfer. We therefore propose that the transition state of the thiol-thioester exchange step is stabilized by a water-mediated hydrogen bond network. Such a mechanism, which is reminiscent of the significant role of water in enzyme catalysis or in certain reactions,²⁸ is supported by the capacity of thioethers to hydrogen bond to water and other hydrogen bond donors.²⁹ A preliminary molecular dynamics simulation shows that tetrahedral intermediate VIII is surrounded by an array of water-mediated hydrogen bonds network involving for example the sulfur atoms, the α-amino group, the amide nitrogens and carbonyl groups and the oxyanion (see Supplementary Information). The disruption of such a network by the cosolvent, which is likely to be present in a late transition state,³⁰ might be at the origin of the effect of Gn·HCl and ACN on NCL rate reported here.

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Water-mediated hydrogen-bond network surrounding tetrahedral intermediate VIII as revealed by a molecular dynamics simulation. The snapshot at 4000 ps shows water molecules located within 4 Å of VIII.</figcaption></figure>

In conclusion, the apparent second-order rate constant of NCL in neutral water for an unhindered peptidyl alanyl aryl thioester is approximately 40 M⁻¹ s⁻¹. Guanidinium chloride and acetonitrile, when used as cosolvents, markedly inhibit the reaction rate by 5-7 fold, with the strongest effect observed for guanidinium chloride. Our kinetic data and the known effects of these additives on water structure support an active role of water in NCL.

## ASSOCIATED CONTENT

Data Availability Statement

The data underlying this study are available in the published article and in its Supporting Information.

## Supporting Information Statement

The Supporting Information is available free of charge at ACS Publications website.

Experimental procedures and characterization data for all compounds (pdf file). All the data collected during this study can be found in the data source file associated with this article (excel file).

## AUTHOR INFORMATION

### Corresponding Authors

Dr Oleg Melnyk – CNRS

Center of Infection and Immunity of Lille, CNRS UMR9017, Inserm U1019, 59021, Lille, France.

Orcid 0000-0002-3863-561

Email: [Oleg.melnyk@ibl.cnrs.fr](mailto:Oleg.melnyk@ibl.cnrs.fr)

### Authors

Julie Di Adamo – Univ Lille

Center of Infection and Immunity of Lille, CNRS UMR9017, Inserm U1019, 59021, Lille, France.

Orcid 0000-0002-5735-4630

Email: [julie.di-adamo.etu@univ-lille.fr](mailto:julie.di-adamo.etu@univ-lille.fr)

### Vangelis Agouridas

Centrale Lille, F-59000 Lille, France.

Orcid: 0000-0003-0911-1527;

Email: [vangelis.agouridas@ibl.cnrs.fr](mailto:vangelis.agouridas@ibl.cnrs.fr)

### Dr Vincent Diemer – CNRS

Center of Infection and Immunity of Lille, CNRS UMR9017, Inserm U1019, 59021, Lille, France.

Orcid 0000-0002-5735-4630

Email: [Vincent.diemer@ibl.cnrs.fr](mailto:Vincent.diemer@ibl.cnrs.fr)

## Author Contributions

JD performed the experiments, VA analyzed the kinetic data, OM and VD conceived the study. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

## Notes

The authors declare no competing financial interest.

## ACKNOWLEDGMENT
This project has received financial support from the French Na-
tional Research Agency (ANR-23-CE07-0016). We gratefully
acknowledge CNRS and IDRIS for providing access to the Jean
Zay supercomputer (AD010816084) for conducting the molecular
dynamics simulations.

REFERENCES

(1) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Synthesis of Proteins by Native Chemical Ligation. *Science* **1994**, *266*, 776-779.

(2) (a) Agouridas, V.; El Mahdi, O.; Cargoët, M.; Melnyk, O. A Statistical View of Protein Chemical Synthesis Using Ncl and Extended Methodologies. *Bioorg. Med. Chem.* **2017**, *25*, 4938-4945.
(b) Agouridas, V.; El Mahdi, O.; Diemer, V.; Cargoet, M.; Monbaliu, J.-C. M.; Melnyk, O. Native Chemical Ligation and Extended Methods. Mechanisms, Catalysis, Scope and Limitations. *Chem. Rev.* **2019**, *12*, 7328-7443.
(c) Sun, Z.; Liu, H.; Li, X. Precision in Protein Chemical Modification and Total Synthesis. *Chem* **2024**, *10*, 767-799.
(d) *Total Chemical Synthesis of Proteins*; Wiley-VCH, 2021.
(e) Conibear, A. C.; Watson, E. E.; Payne, R. J.; Becker, C. F. W. Native Chemical Ligation in Protein Synthesis and Semi-Synthesis. *Chem. Soc. Rev.* **2018**, *47*, 9046-9068.

(3) Dawson, P. E.; Churchill, M. J.; Ghadiri, M. R.; Kent, S. B. H. Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives. *J. Am. Chem. Soc.* **1997**, *119*, 4325-4329.

(4) Johnson, E. C.; Kent, S. B. H. Insights into the Mechanism and Catalysis of the Native Chemical Ligation Reaction. *J. Am. Chem. Soc.* **2006**, *128*, 6640-6646.

(5) Diemer, V.; Firstova, O.; Agouridas, V.; Melnyk, O. Pedal to the Metal: The Homogeneous Catalysis of the Native Chemical Ligation Reaction. *Chemistry* **2022**, *28*, e202104229.

(6) (a) Zheng, J. S.; Tang, S.; Qi, Y. K.; Wang, Z. P.; Liu, L. Chemical Synthesis of Proteins Using Peptide Hydrazides as Thioester Surrogates. *Nat. Protocols* **2013**, *8*, 2483-2495.
(b) Fang, G.-M.; Li, Y.-M.; Shen, F.; Huang, Y.-C.; Li, J.-B.; Lin, Y.; Cui, H.-K.; Liu, L. Protein Chemical Synthesis by Ligation of Peptide Hydrazides. *Angew. Chem. Int. Ed.* **2011**, *50*, 7645-7649.

(7) (a) Saito, F.; Noda, H.; Bode, J. W. Critical Evaluation and Rate Constants of Chemoselective Ligation Reactions for Stoichiometric Conjugations in Water. *ACS Chem. Biol.* **2015**, *10*, 1026-1033.
(b) Diemer, V.; Ollivier, N.; Leclercq, B.; Drobecq, H.; Vicogne, J.; Agouridas, V.; Melnyk, O. A Cysteine Selenosulfide Redox Switch for Protein Chemical Synthesis. *Nat. Commun.* **2020**, *11*, 2558.
(c) Gless, B. H.; Schmied, S. H.; Bejder, B. S.; Olsen, C. A. Förster Resonance Energy Transfer Assay for Investigating the Reactivity of Thioesters in Biochemistry and Native Chemical Ligation. *JACS Au* **2023**, *3*, 1443-1451.

(8) (a) Stark, G. R. On the Reversible Reaction of Cyanate with Sulfhydryl Groups and the Determination of NH₂-Terminal Cysteine and Cystine in Proteins. *J. Biol. Chem.* **1964**, *239*, 1411-1414.
(b) Baumruck, A. C.; Tietze, D.; Steinacker, L. K.; Tietze, A. A. Chemical Synthesis of Membrane Proteins: A Model Study on the Influenza Virus B Proton Channel. *Chem. Sci.* **2018**, *9*, 2365-2375.

(9) Johnson, K. A. Fitting Enzyme Kinetic Data with Kintek Global Kinetic Explorer. *Methods Enzymol.* **2009**, *467*, 601-626.

(10) (a) Gill, M. S.; Neverov, A. A.; Brown, R. S. Dissection of Nucleophilic and General Base Roles for the Reaction of Phosphate with *p*-Nitrophenyl Thiolacetate, *p*-Nitrophenyl Thiolformate, and Phenyl Thiolacetate. *J. Org. Chem.* **1997**, *62*, 7351-7357.
(b) Malins, L. R.; Cergol, K. M.; Payne, R. J.

Chemoselective Sulfenylation and Peptide Ligation at
Tryptophan. *Chem. Sci.* **2013**, *5*, 260-266.

(11) Maguire, O. R.; Zhu, J.; Brittain, W. D. G.; Hudson, A. S.; Cobb, S. L.; O'Donoghue, A. C. N-Terminal Speciation for Native Chemical Ligation. *Chem. Commun.* **2020**, *56*, 6114-6117.

(12) Heller, M. J.; Walder, J. A.; Klotz, I. M. Intramolecular Catalysis of Acylation and Deacylation in Peptides Containing Cysteine and Histidine. *J. Am. Chem. Soc.* **1977**, *99*, 2780-2785.

(13) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Protein Synthesis by Native Chemical Ligation: Expanded Scope by Using Straightforward Methodology. *Proc. Natl. Acad. Sci. U. S. A.* **1999**, *96*, 10068-10073.

(14) Garcia-Mira, M. M.; Sanchez-Ruiz, J. M. Ph Corrections and Protein Ionization in Water/Guanidinium Chloride. *Biophysical journal* **2001**, *81*, 3489-3502.

(15) (a) Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.; Berthelette, C. A Generally Applicable Method for Assessing the Electrophilicity and Reactivity of Diverse Nitrile-Containing Compounds. *Biorg. Med. Chem. Lett.* **2007**, *17*, 998-1002.
(b) Shalayel, I.; Coulibaly, S.; Ly, K. D.; Milet, A.; Vallée, Y. The Reaction of Aminonitriles with Aminothiols: A Way to Thiol-Containing Peptides and Nitrogen Heterocycles in the Primitive Earth Ocean. *Life* **2018**, *8*, 47.

(16) (a) Gordon, M.; Miller, J. G.; Day, A. R. Effect of Structure on Reactivity. II. Influence of Solvents on Ammonolysis of Esters. *J. Am. Chem. Soc.* **1949**, *71*, 1245-1250.
(b) Satterthwait, A. C.; Jencks, W. P. Mechanism of the Aminolysis of Acetate Esters. *J. Am. Chem. Soc.* **1974**, *96*, 7018-7031.
(c) Jencks, W. P. General Acid-Base Catalysis of Complex Reactions in Water. *Chem. Rev.* **1972**, *72*, 705-718.

(d) Singleton, D. A.; Merrigan, S. R. Resolution of Conflicting Mechanistic Observations in Ester Aminolysis. A Warning on the Qualitative Prediction of Isotope Effects for Reactive Intermediates. *J. Am. Chem. Soc.* **2000**, *122*, 11035-11036.
(e) Antonczak, S.; Ruiz-Lopez, M. F.; Rivail, J. L. Ab Initio Analysis of Water-Assisted Reaction Mechanisms in Amide Hydrolysis. *J. Am. Chem. Soc.* **1994**, *116*, 3912-3921.
(f) Williams, I. H.; Spangler, D.; Femec, D. A.; Maggiora, G. M.; Schowen, R. L. Theoretical Models for Solvation and Catalysis in Carbonyl Addition. *J. Am. Chem. Soc.* **1983**, *105*, 31-40.
(g) Wolfe, S.; Kim, C.-K.; Yang, K.; Weinberg, N.; Shi, Z. Hydration of the Carbonyl Group. A Theoretical Study of the Cooperative Mechanism. *J. Am. Chem. Soc.* **1995**, *117*, 4240-4260.

(17) Yang, W.; Drueckhammer, D. G. Computational Studies of the Aminolysis of Oxoesters and Thioesters in Aqueous Solution. *Org. Lett.* **2000**, *2*, 4133-4136.

(18) Saa, J. M.; Frontera, A. On the Role of Water as a Catalyst in Prebiotic Chemistry. *ChemPhysChem* **2020**, *21*, 313-320.

(19) Bongiardina, N. J.; Long, K. F.; Podgórski, M.; Bowman, C. N. Substituted Thiols in Dynamic Thiol-Thioester Reactions. *Macromolecules* **2021**, *54*, 8341-8351.

(20) Dittmann, M.; Sauermann, J.; Seidel, R.; Zimmermann, W.; Engelhard, M. Native Chemical Ligation of Hydrophobic Peptides in Organic Solvents. *J. Pept. Sci.* **2010**, *16*, 558-562.

(21) Baumruck, A. C.; Yang, J.; Thomas, G.-F.; Beyer, L. I.; Tietze, D.; Tietze, A. A. Native Chemical Ligation of Highly Hydrophobic Peptides in Ionic Liquid-Containing Media. *J. Org. Chem.* **2021**, *86*, 1659-1666.

(22) (a) French, H. T. Vapour Pressures and Activity Coefficients of (Acetonitrile + Water) at 308.15 K. *J. Chem. Thermodyn.* **1987**, *19*, 1155-1161.
(b) Schrier, M. Y.; Schrier, E. E. Osmotic and Activity Coefficients of Aqueous Guanidine Hydrochloride Solutions at 25 °C. *J. Chem. Eng. Data* **1977**, *22*, 73-74.

(23) (a) Bertie, J. E.; Lan, Z. Liquid Water-Acetonitrile Mixtures at 25 °C: the Hydrogen-Bonded Structure Studied through Infrared Absolute Integrated Absorption Intensities. *J.*
Phys. Chem. B **1997**, *101*, 4111-4119. (b) Kovacs, H.; Laaksonen, A. Molecular Dynamics Simulation and NMR Study of Water-Acetonitrile Mixtures. *J. Am. Chem. Soc.* **1991**, *113*, 5596-5605. (c) Chen, J.; Sit, P. H. L. Ab Initio Study of the Structural Properties of Acetonitrile-Water Mixtures. *Chem. Phys.* **2015**, *457*, 87-97. (d) Wakisaka, A.; Abdoul-Carime, H.; Yamamoto, Y.; Kiyozumi, Y. Non-Ideality of Binary Mixtures Water-Methanol and Water-Acetonitrile from the Viewpoint of Clustering Structure. *J. Chem. Soc., Faraday Trans.* **1998**, *94*, 369-374, 10.1039/A705777F.

(24) Blandamer, M. J.; Engberts, J. B. F. N.; Gleeson, P. T.; Reis, J. C. R. Activity of Water in Aqueous Systems; a Frequently Neglected Property. *Chem. Soc. Rev.* **2005**, *34*, 440-458, 10.1039/B400473F.

(25) (a) Mason, P. E.; Neilson, G. W.; Enderby, J. E.; Saboungi, M.-L.; Dempsey, C. E.; MacKerell, A. D., Jr.; Brady, J. W. The Structure of Aqueous Guanidinium Chloride Solutions. *J. Am. Chem. Soc.* **2004**, *126*, 11462-11470. (b) Scott, J. N.; Nucci, N. V.; Vanderkooi, J. M. Changes in Water Structure Induced by the Guanidinium Cation and Implications for Protein Denaturation. *J. Phys. Chem. A* **2008**, *112*, 10939-10948.

(26) Hédoux, A.; Krenzlin, S.; Paccou, L.; Guinet, Y.; Flament, M.-P.; Siepmann, J. Influence of Urea and Guanidine Hydrochloride on Lysozyme Stability and Thermal Denaturation; a Correlation between Activity, Protein

Dynamics and Conformational Changes. *Phys. Chem. Chem. Phys.* **2010**, *12*, 13189-13196, 10.1039/C0CP00602E.

(27) (a) Dempsey, C. E.; Piggot, T. J.; Mason, P. E. Dissecting Contributions to the Denaturant Sensitivities of Proteins. *Biochemistry* **2005**, *44*, 775-781. (b) Mason, P. E.; Brady, J. W.; Neilson, G. W.; Dempsey, C. E. The Interaction of Guanidinium Ions with a Model Peptide. *Biophysical J.* **2007**, *93*, L04-L06.

(28) (a) Xia, W.; Chen, J.; Jiang, L.; Lu, M.; Liu, M.; Yang, M.; Li, C.; Liu, Y. Visualize Transient Water-Mediated Hydrogen Bonds Facilitating the Formation of Enzymatic near-Attack Conformers. *JACS Au* **2025**. (b) Vik, E. C.; Li, P.; Maier, J. M.; Madukwe, D. O.; Rassolov, V. A.; Pellechia, P. J.; Masson, E.; Shimizu, K. D. Large Transition State Stabilization from a Weak Hydrogen Bond. *Chem. Sci.* **2020**, *11*, 7487-7494, 10.1039/D0SC02806A.

(29) (a) Chand, A.; Biswal, H. S. Hydrogen Bonds with Chalcogens: Looking Beyond the Second Row of the Periodic Table. *J. Indian Inst. Sci.* **2020**, *100*, 77-100. (b) Kojasoy, V.; Tantillo, D. J. Impacts of Noncovalent Interactions Involving Sulfur Atoms on Protein Stability, Structure, Folding, and Bioactivity. *Org. Biomol. Chem.* **2023**, *21*, 11-23, 10.1039/D2OB01602H.

(30) Wang, C.; Guo, Q.-X.; Fu, Y. Theoretical Analysis of the Detailed Mechanism of Native Chemical Ligation Reactions. *Chem. Asian J.* **2011**, *6*, 1241-1251.