Non-enzymatic ribonucleotide reduction in the prebiotic context.

DOI: 10.1002/chem.201405741

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& Prebiotic Chemistry

Non-enzymatic Ribonucleotide Reduction in the Prebiotic Context Ivan Dragicˇevic´,[a, b] Danijela Baric´,*[a] Borislav Kovacˇevic´,[a] Bernard T. Golding,[c] and David M. Smith*[a]

Abstract: Model studies of prebiotic chemistry have revealed compelling routes for the formation of the building blocks of proteins and RNA, but not DNA. Today, deoxynucleotides required for the construction of DNA are produced by reduction of nucleotides catalysed by ribonucleotide reductases, which are radical enzymes. This study considers potential non-enzymatic routes via intermediate radicals for the ancient formation of deoxynucleotides. In this context, several mechanisms for ribonucleotide reduction, in a putative H2S/HSC environment, are characterized using computa-

tional chemistry. A bio-inspired mechanistic cycle involving a keto intermediate and HSSH production is found to be potentially viable. An alternative pathway, proceeding through an enol intermediate is found to exhibit similar energetic requirements. Non-cyclical pathways, in which HSSC is generated in the final step instead of HSC, show a markedly increased thermodynamic driving force (ca. 70 kJ mol1) and thus warrant serious consideration in the context of the prebiotic ribonucleotide reduction.

Introduction

notion that the prebiotic formation of RNA might be feasible.[6, 11] The discontinuous model,[12, 13] in which the building blocks are produced and assembled separately, is associated with some difficulties.[14–16] There are, however, alternative proposals, involving a continuous model of RNA generation under appropriate prebiotic conditions.[13, 17, 18] The plausible presence of prebiotic RNA, coupled with its potential ability to act as an information carrier and a catalyst,[19] forms the basis of the “RNA world” hypothesis.[20] This hypothesis proposes that self-replicating[21] RNA molecules were precursors to current life. Whilst this concept may be widely accepted,[22] there is also support for the idea that the “RNA world” may not have been the first self-replicating system but rather arose from an earlier, unknown, pre-RNA precursor.[23, 18] Over time, DNA replaced RNA as the genetic polymer, while proteins replaced RNA as the primary biocatalysts. However, given the lack of evidence for the facile formation of DNA under prebiotic conditions, it is interesting to consider the possible origins of the requisite deoxyribonucleotide building blocks that are so ubiquitous today. Such an endeavour can be potentially aided by a consideration of modern-day ribonucleotide reduction. Most discussions of life’s origin focus on heterolytic chemistry even though today many radical processes in biology have been recognised.[24] This study takes today’s extant biology (ribonucleotide reductase mechanism) and extrapolates back, attempting to identify prebiotic radical chemistry, the possibility of which is explored using computational chemistry.

The primordial soup and the RNA world The idea that the key chemical components of living matter may have arisen in a “warm little pond” subject to light, heat, and electricity was expressed as early as 1871 in a letter by Charles Darwin.[1, 2] In the 1950s, Miller and Urey provided experimental support for this idea[3, 4] and initiated the field of prebiotic model chemistry.[5, 6] The Miller–Urey experiment exposed a mixture of CH4, NH3, H2O, and H2 to an electric discharge, which generated numerous amino acids that may have arisen from intermediates such as hydrogen cyanide and formaldehyde.[7] In a similar manner,[8] purine and pyrimidine bases, such as adenine, can be derived from hydrogen cyanide.[5, 6] Formaldehyde, on the other hand, can give rise to sugars,[9] including ribose, which accumulates in the presence of borate.[10] Deoxyribose, however, is not formed under such conditions.[5] The relatively simple pathways leading to the building blocks of life, such as adenine and ribose (but not deoxyribose!), from a tentative “primordial soup” has led to the [a] I. Dragicˇevic´, Dr. D. Baric´, Dr. B. Kovacˇevic´, Dr. D. M. Smith Division of Organic Chemistry and Biochemistry Rud¯er Bosˇkovic´ Institute, Bijenicˇka 54, 10000 Zagreb (Croatia) E-mail: [email protected] [email protected] [b] I. Dragicˇevic´ Department of Chemistry Faculty of Science and Education, University of Mostar Matice hrvatske bb, 88000 Mostar (Bosnia and Herzegovina) [c] Prof. B. T. Golding School of Chemistry, Newcastle University Newcastle upon Tyne, NE1 7RU (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201405741. Chem. Eur. J. 2015, 21, 1 – 13

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Full Paper Enzymatic ribonucleotide reduction: RNRs

begins with the previously mentioned (oxidative) abstraction of an H atom from C3’ by the radical form of Cys408. The second step involves the elimination of water from 2 and the accompanying proton redistribution between Cys119 and Glu410. The third step involves a second H transfer, from Cys419 to the delocalized keto radical 3 and the simultaneous formation of a reduced disulfide linkage between Cys119 and Cys419. The disulfide gives up its excess electron in the next step, which in combination with the proton transfer from Glu410 constitutes a proton-coupled electron transfer (PCET)[39] that is thought to be the rate-limiting step in the overall transformation.[38d] The third and final H transfer, from Cys408 to C3’, completes the ribonucleotide reduction and regenerates the original radical centre at Cys408. In order for the enzyme to complete the turnover, however, the disulfide bond still needs to be reduced. This is achieved by a pair of redox-active, C-terminal cysteine residues (731 and 736 in L. leichmannii), which interact with the thioredoxin system.[40] Notwithstanding the obvious role of RNRs in bridging the DNA and RNA worlds, it is interesting to consider why and how it came to pass that Nature chose this particular type of complex radical mechanism for ribonucleotide reduction.

The reduction of ribonucleotides in the DNA world is carried out by radical enzymes[24] known as ribonucleotide reductases (RNRs).[25, 26] All known RNRs activate their ribonucleotide substrates by abstracting the 3’-H atom using a cysteine-derived thiyl radical (CH2SC).[27] The primary difference between the three main classes of RNR lies in the machinery used to generate this thiyl radical. Class I enzymes initially produce a tyrosyl radical through the action of a bimetallic cluster containing iron and/or manganese.[28] Class II enzymes utilize the homolytic CoC cleavage of coenzyme B12,[29] while, in anaerobic organisms, Class III enzymes produce a glycyl radical with the help of an iron–sulfur protein and S-adenosylmethionine.[30] In addition to their important place as a link between the RNA and DNA worlds,[31] RNRs have also received significant interest as medicinal targets.[25, 32, 33] The different classes of RNRs are supposed to be the result of divergent evolution, perhaps from a common ancestor.[34] Class I enzymes are, in an evolutionary sense, likely to be the most recent. There is some debate as to whether the Class II or Class III enzymes most closely resemble the ancestral RNR, with compelling arguments for and against both cases.[34] For a discussion of the substrate mechanism, however, we have chosen Class II (Figure 1). This mechanism is practically identical to that of Class I but differs from Class III, which employs formate instead of two cysteines as the active site reductant. The proposition and understanding of the mechanism shown in Figure 1, which is now widely accepted for both Class I and Class II RNRs, emerged as the result of extensive experimental[26, 35] and theoretical[36–38] investigation. The pathway

Prebiotic radical chemistry?

Before considering the case of prebiotic ribonucleotide reduction, it is interesting to make a detour to the analogous enzymatic dehydration of vicinal diols. The enzyme diol dehydratase (DDH)[41] is related to Class II RNR in that it transforms 1,2diols using a radical mechanism,[42] which is initiated by the CoC cleavage of coenzyme B12 and involves the H-atom abstraction from a substrate carbon atom (1’!2’ in Figure 2).[43] The substrate-derived radical generated in this way (2’) undergoes a 1,2-OH shift before recapturing an H atom and eliminating water to produce the product aldehyde (3’).[44] Interestingly, the exposure of 1,2-diols to the COH radical, generated in aqueous solution either by oxidation with Fenton’s reagent[45] or by g-radiolysis,[46] yields detectable amounts of both 2’ and 3’ (among other products). If, therefore, the complex enzyme–coenzyme machinery in the case of DDH can be replaced by a simple COH radical, it is tempting to contemplate similar radical chemistry for the prebiotic conversion of a ribonucleotide into a deoxyribonucleotide.[47] More specifically, we wish to consider the scenario that radiFigure 1. Substrate mechanism of Class II RNR. PP signifies a diphosphate and B represents a generic base, such cals such as HSC, which could as adenine. The numbering system corresponds to the RNR enzyme from Lactobacillus leichmannii.[26e] &

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Full Paper Computational Methods Using the model systems shown in Figure 3, all stationary points were optimized at the B3LYP/6-31G(d) level of theory and their vibrational frequencies were calculated analytically. All presented structures were found to have the appropriate number of imaginary frequencies and, in the case of transition states, the single imaginary modes were confirmed to correspond to the correct reaction coordinate. Calculated vibrational zero-point energies were scaled by 0.9806.[58] The quaternary molecular complexes arising from a model system of the type shown in Figure 3 are necessarily associated with a large number of conformers. As the primary purpose of this study was to investigate the viability of reduction reactions, we did not engage in an exhaustive search for global minima on the potential energy surfaces. Rather, we began our investigations from a plausible conformation for the reactant complex (see the Supporting Information) and proceeded to map out potential chemical transformations. This involved performing intrinsic reaction coordinate (IRC) calculations to follow the reaction path in both directions from each transition state. In order to ensure maximum connectivity of the resulting reaction paths, we frequently iterated this procedure. In certain cases, however, the complex resulting from the optimization of the forward IRC path from one mechanistic step did not correspond exactly to the complex resulting from the optimization of the reverse IRC from the subsequent step. The differences in such cases were always related to minor librational rearrangements of the loosely bound fragments in the model. In such cases, for reasons of simplicity, we have chosen to present the energy of the more stable of the two alternative complexes.

Figure 2. Schematic depiction of the transformation of 1,2-diols into the corresponding aldehydes, as catalysed by diol dehydratase (R = H, CH3).

have arisen in, for example, Wchtershuser’s iron–sulfur world,[48] might be able to initiate the reduction of RNA. Of relevance is the claim that deoxy compounds are formed on treatment of ribonucleosides with FeS/H2S.[49] Pulse radiolysis of a thio-d-ribo-hexofuranitol system in a basic medium showed the generation of a thiyl radical, which subsequently abstracted an H atom to irreversibly produce a ribosyl-based carbon radical.[50] Encouraged by these preliminary results and inspired by the mechanism of RNR, we have constructed a model system with which to investigate the possibilities for non-enzymatic ribonucleotide reduction under potentially achievable prebiotic conditions. The model is shown in Figure 3 and supposes, firstly, that there is a sufficient concentration of H2S to allow an arrangement of the type shown in the Figure to be realized. This assumption is supported by the fact that hydrogen sulfide has been suggested to have played a prominent role in the chemistry of Earth’s early atmosphere and in prebiotic synthesis.[51, 52] Furthermore, H2S is commonly detected in interstellar space,[53] volcanic emissions,[54] and especially hydrothermal vents,[55] which may have been subject to conditions of extreme pressure.[56] Secondly, we assume the presence of HSC given that numerous methods have been described for the formation of this radical from H2S. These include exposure of H2S in aqueous media to ultrasonic irradiation, g-radiolysis and treatment with ferric species.[48, 57] With these considerations, it is not unreasonable to surmise that a locally significant concentration of HSC radicals could have arisen under appropriate conditions.

Improved single-point energies for the selected stationary points obtained with the B = Im system were evaluated using a variant of the G3(MP2)-RAD,[58] which differs from the original only in the use of the Gaussian 09[59] package for the evaluation of the ROCCSD(T) energies. The final energy obtained by G3(MP2)-RAD is given as sum of four terms:

E 0 ¼ E½ROCCSDðTÞ=6-31GðdÞ==UB3LYP=6-31GðdÞþ DEðG3MP2largeÞ þ DEðHLCÞ þ ZPVE sc

Here, the second term is an additive basis-set correction obtained as the difference between ROMP2(fc) energies calculated with a large (G3MP2large) and a modest basis set (6-31G(d)). The third term, the so-called higher-level correction, is an empirical improvement of total energy while the last term, ZPVEsc, is the same scaled (0.9806) zero point vibrational energy mentioned above. The use of the restricted open-shell formulation ensures that the components of the G3(MP2)-RAD energies are eigenfunctions of the spinsquared operator and are free from spin contamination. This has been shown to be advantageous with correlated methods, especially MP2.[58, 60]

Figure 3. Minimal models used to investigate the non-enzymatic reduction of ribonucleotides.

Single-point energies were additionally carried out using the B3LYP,[61] B3LYP-D3,[62] BMK[63] and M06-2X[64] DFT functionals, combined with Pople’s 6-311 + G(3df,2p) basis set. These calculations were initially used to establish the reliability of the DFT functionals, against the G3(MP2)-RAD results (for B = Im). This comparison was used to justify the subsequent application of selected functionals to the larger B = Ad system, where the G3(MP2)-RAD calculations are less tractable. All DFT calculations were carried out within the unrestricted formulation. While this does introduce small amounts of spin contamination into the calculations, the resulting DFT energies have been found to be insensitive to this shortcoming.[60b]

By characterizing the system shown in Figure 3, with stateof-the-art computational methods, we hope to shed light on the type of chemistry that may have served as a forerunner to the development of an ancestral RNR.

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Full Paper The effects of solvation were estimated using the isodensity polarizable continuum model,[65] with the density calculated with B3LYP/6311 + G(d,p)//B3LYP/6-31G(d) and an isodensity value of 0.004. This approach circumvents discontinuities that arise in the cavitation energy with the application of overlapping spheres for loosely bound complexes. All calculations were carried out with the Gaussian 09 software package.[59] The intermediates depicted in the mechanistic schemes below correspond to the dominant Lewis structure in each case. These structures were deduced on the basis of the spin and charge distributions (Table S7 in the Supporting Information) but do not always completely capture the full complexity of the occasionally delocalized electron distributions.

Figure 4. Mechanism of ribonucleotide reduction by H2S/HSC : keto pathway. The base, B, may be either imidazole (Im) or adenine (Ad) as per Figure 3.

with a unique and well-defined model system, the optimization of the quaternary complex M1 in the gas-phase (pictured in Figure S5 in the Supporting Information) nevertheless constitutes our starting point. The enthalpies at 0 K of the relevant stationary points, relative to M1, are presented as a schematic reaction profile in Figure 5. We have not attempted to calculate entropies or free energies of the investigated intermediates. Step 1: M1!M2: In the first step of the reaction, the HSC radical abstracts a hydrogen atom from C3’, resulting in the formation of the radical complex denoted as M2. At the G3(MP2)-RAD level of theory, this step is found to be endothermic by 21.4 kJ mol1 and associated with a relatively low (36.2 kJ mol1) barrier (see Figure 5 and Table S1 in the Supporting Information). The endothermicity corresponds very closely to the 24.7 kJ mol1 difference in the (0 K) G3(MP2)-RAD bond dissociation energies (BDEs) of H2S and the RNA model shown in Figure 3 (B = Im). The small difference between this radical stabilization energy (RSE) and the value shown in Figure 5 results from subtle differences in the hydrogen bonding energies between M1 and M2 and is consistent with results reported in references [36a], [37b] and [66]. Although the presence of a base, such as a carboxylate, near the 3’-OH group in an unconstrained model system can lower the H-abstraction barrier[38a] and alter the reaction energetics,[66] the enzyme is not thought to contribute significant catalysis to this step.[37a, 38b] In our non-enzymatic model, H2S’’ occupies an analogous position. However, because H2S is a rather weak base, its effect on the abstraction barrier is expected to be minor. Step 2: M2!M3K : The second step depicted in Figures 4 and 5 involves the elimination of water from M2 resulting in the formation of a delocalized keto radical (M3K), in a manner similar to the aforementioned DDH model systems.[45, 46] In analogy to the enzyme-catalysed pathway, the (mid-grey, Figure 4) proton adding to the hydroxyl leaving group originates from a sulfur atom (S’’ in this case). Interestingly, this

Results and Discussion Small model system (B = Im) RNR inspired mechanism We initially present results from the use of the minimal model shown in Figure 3, with the simpler base imidazole (B = Im). In addition to exploring the viability of the non-enzymatic ribose reduction, this system is used to perform high-level [G3(MP2)RAD] benchmark calculations to establish the suitability of DFT functionals for a transformation of this kind. For these purposes, we begin by investigating a pathway (Figure 4), inspired by the accepted mechanism of the RNR-catalysed reaction (Figure 1). We note that, although our model system is similar to the simplest models that have been applied to the enzymatic reaction (such as in ref. [36a]), there are important differences. Firstly, MeSH has been commonly used to represent the reactive part of the cysteine residue because it is indeed a more appropriate model for this purpose than H2S. H2S, on the other hand, may have reactivity patterns that MeSH does not. Furthermore, our goal is to investigate the feasibility of the reduction under potentially realizable yet relatively simple prebiotic conditions to establish if HSC/H2S may constitute a viable ancestral predecessor for today’s RNR mechanisms. To the best of our knowledge, this has not been attempted before. As mentioned above, we assume the presence of the HSC radical and a sufficient concentration of H2S to allow an assembly such as M1 to occur. While obviously the assembly of such a complex under dilute gas-phase conditions would not be expected to be favourable, we prefer to consider this complex as being representative of a plausible molecular arrangement (a snapshot as it were), should an RNA be present in the stated conditions. In order to carry out our proof-of-principle study &

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Full Paper (M4K). This step is exothermic (22.7 kJ mol1) and associated with a barrier of only 27.3 kJ mol1. The H2S’’ molecule does not play any active role in this transfer. Step 4: M4K !M5K : Steps 1 to 3 effectively serve to generate a keto group on the substrate, which is subsequently reduced in the remainder of the reaction. For this reason we henceforth refer to this pathway as the keto mechanism. The remaining reduction of the M4K keto group proceeds through the stepwise addition of two hydrogen atoms. The first of these transfers, which also constitutes the fourth mechanistic step, requires the transfer of an H-atom from H2S’’ to the carbonyl oxygen. In a conFigure 5. Energy profiles of the keto-mechanism of ribonucleotide reduction by H2S/HSC in the gas phase, calculatcerted fashion, the unpaired ed using G3(MP2)-RAD and three DFT functionals for the B = Im model system. Tabulated values are provided in electron, partially generated on Table S1 in the Supporting Information. S’’, combines with the unpaired electron on S’’’ to form the relasame sulfur atom simultaneously serves as a base to receive tively stable HSSH molecule. The radical centre in the resultthe (dark grey in Figure 4) proton from the 3’-OH group, in ing M5K intermediate is, therefore, localized on the C3’ atom contrast to the carboxylate base that fulfils this role in the [1(C3’) = 0.83]. enzyme. That the water elimination is essentially an acid–base The conclusion that the M4K !M5K transformation is preprocess is supported by an analysis of the charge and spin dominantly a radical process is supported by the fact that the densities (Table S7 in the Supporting Information) in the transispin density in TS4K is distributed such that the 1(S’’) = 0.38, tion structure (TS2K). Namely, the majority of the positive 1(S’’’) = 0.25 and 1(C3’) = 0.31 (Table S7 in the Supporting Information). Nevertheless, the changing charge distribution during charges in TS2K are associated with the mobile (dark grey and the course of the reaction, especially of the water molecule, inmid grey in Figure 4) protons, while there is virtually no spin dicates an element of charge polarization, characteristic of density on the active sulfur atom (S’’). The additional H2S’’’ PCET, in this mechanistic step. molecule behaves as a spectator in this mechanistic step and Based on the calculated activation barrier of 124.8 kJ mol1, simply reorients in M3K to preserve the H-bond network. Numerous attempts to involve this molecule more explicitly in step 4 is rate limiting in the overall transformation of M1! the proton transfer chain were unsuccessful. M6K, as is the analogous step in the enzyme-catalysed transformation.[38d] G3(MP2)-RAD predicts this step to be endothermic Although the activation energy for the second step is conby 29.6 kJ mol1. siderable, assuming a value of 92.9 kJ mol1 at the G3(MP2)RAD level of theory, this step is strongly exothermic, with M3K Step 5: M5K !M6K : The second phase of the reduction of the keto group and the fifth and final step in the overall reaccalculated to be 35.5 kJ mol1 lower in energy than M2. These tion is the transfer of a hydrogen atom from H2S’ to C3’. This values are similar to those found both in simple RNR modresults in the formation of the reduced ribonucleotide and the els[36, 37b] and in a substantially more complex one,[37c] despite restoration of the original HSC radical (M6K). The final step is calthe fact that one might expect the reaction to be more difficult in the absence of a carboxylate base. That said, the analyculated to be exothermic by 20.4 kJ mol1 with an associated sis of step 2 in the presence of a large model of RNR concludbarrier of only 10.7 kJ mol1. As with the first H-abstraction ed that the enzyme does not provide any significant electrostep (M1!M2), the difference between the G3(MP2)-RAD RSE static catalysis for the water elimination step.[37c] of the DNA model appearing in M6K (23.8 kJ mol1 at 0 K) and the exothermicity of the final step in Figure 4 is only minor. Step 3: M3K !M4K : The next step involves the quenching of This once again reflects subtle differences in the hydrogenthe newly formed radical centre at C2’ (in M3K) by the H2S’’’ bonding arrangements between M5K and M6K. molecule, which acted as a spectator in the previous step. The spin and charge distributions (Table S7 in the Supporting InforThe overall transformation: M1!M6K : From a thermochemmation) in TS3K are consistent with an H-transfer reaction, leadical point of view, the ribonucleotide reduction can be written, in short, as shown below in Equation (2). ing to the closed-shell keto intermediate and an HSC radical Chem. Eur. J. 2015, 21, 1 – 13

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Full Paper RNA þ 2 H2 S ! DNA þ HSSH þ H2 O

The deviation from G3(MP2)-RAD, in terms of the exothermicity of step 2, is larger for the DFT approaches, with B3LYPD3 exhibiting a significant discrepancy of 31 kJ mol1, whereas the other two functionals both show the same deviation from G3(MP2)-RAD (8.7 kJ mol1), but in different directions. For the barrier of the same step, the B3LYP-D3 functional again performs poorly while, of the other two functionals, it appears that M06-2X offers a less severe underestimation of this quantity. It is clear from Figure 5 that, for the rest of the profile, the performance of the B3LYP-D3 functional remains quite poor, as evidenced by an average absolute deviation from G3(MP2)RAD of 30 kJ mol1. The energies calculated by B3LYP-D3 (and B3LYP, see Table S1 in the Supporting Information) are substantially and systematically lower than those obtained by the higher-level method. On this basis, neither B3LYP functional will be adopted as a method of choice for our calculations in this work. BMK appears to be a better choice than M06-2X for a comparison of the energetics of the intermediates involved in step 3 (M3K, TS3K, and M4K) with the starting complex M1. However, in terms of the barrier and exothermicity of this step alone, both the BMK and M06-2X functionals exhibit comparative performance. Step 4, which involves the transfer of an H-atom to the keto group, sees the BMK and M06-2X functionals underestimate the barrier by more than 10 kJ mol1. On this occasion, however, it is M06-2X that gives superior performance for the comparison with M1. The DFT approaches also underestimate the endothermicity of step 4 but BMK does so in a more dramatic fashion than M06-2X. For the final step, M06-2X shows very good agreement with G3(MP2)-RAD for the barrier and the exothermicity, as well as the relative positions of the intermediates compared to M1. While BMK’s performance for the barrier and exothermicity for step 5 is acceptable, the disagreement with respect to the stability of M5K (relative to M1) is compounded so that the BMK profile diverges more appreciably from the reference G3(MP2)RAD curve. Overall, both the BMK and M06-2X functionals reproduce the main energetic trends of the G3(MP2)-RAD reaction profile. While some steps are better treated by BMK (e.g., step 3), M06-2X offers better performance for other steps (e.g., step 4). Given that the average absolute deviation from G3(MP2)-RAD profile amounts to 5.8 kJ mol1 for M06-2X and 12.9 kJ mol1 for BMK, it seems that, on the whole, the former is more preferable. Therefore, for the remainder of this work, we shall present and discuss the M06-2X values in the text. For reasons of completeness, the BMK and B3LYP-3D results are presented in Supporting Information (Tables S1–S5 and Figures S1–S4).

ð2Þ

In this context, RNA and DNA represent the B = Im models shown in Figures 3 and 4. At the G3(MP2)-RAD level of theory, this reaction is calculated to be exothermic by 8.2 kJ mol1. Interestingly this is significantly less exothermic than the analogous reaction involving MeSH, which has been previously used to model the reduction in the context of the enzyme (see, for example, ref. [36a]). RNA þ 2 CH3 SH ! DNA þ H3 CSSCH3 þ H2 O

ð3Þ

Namely, using G3(MP2)-RAD, Equation (3) is found to be exothermic by 51.2 kJ mol1 (substitution on the methyl group as in for example, cysteine is not expected to alter this value significantly). The difference can be primarily assigned to the fact that the SH bond in CH3SH is 18.5 kJ mol1 weaker than in H2S (see also ref. [66]) while the SS bond in H3CSSCH3 is 6.2 kJ mol1 stronger than that in HSSH (all values at 0 K). In the model system investigated in Figure 5, the overall transformation is exothermic by 27.6 kJ mol1. In light of the above discussion, it can be concluded that the changes in the hydrogen-bonding network between M1 and M6K are more significant than those noted for steps 1 and 6 in the preceding section. This can be understood in terms of the interactions arising with the eliminated water as well as differences in the H-bonding capacities between H2S and HSSH (see also Figure S5 in the Supporting Information).[36a] Such interactions are also likely to increase the fundamental exothermicity of the overall reaction (of 8.2 kJ mol1) in solution, so it is relatively safe to conclude that the reduction of RNA with H2S/HSC is indeed associated with a thermodynamic driving force. The rate-limiting step in Figure 5 is the H transfer to the carbonyl oxygen of M4K, which is coupled with the formation of HSSH. The barrier of about 120 kJ mol1, although relatively high, is within the range that could be expected under standard (or elevated) conditions of temperature and pressure. The combination of the kinetic and thermodynamic considerations therefore indicates that the non-enzymatic reduction of ribonucleotides could indeed be feasible under conditions conducive to the assembly of a complex such as M1. Comparative performance of DFT functionals Apart from investigating the viability of the overall reduction, the model system with B = Im was used to examine the performance of four DFT functionals (B3LYP, B3LYP-D3, BMK and M06-2X) against the higher-level G3(MP2)-RAD methodology. Only three of these functionals are shown in Figure 5, while the B3LYP results can be found in Table S1 in the Supporting Information. In the first, facile, hydrogen abstraction (step 1), BMK and M06-2X functionals perform well with respect to the endothermicity, while the B3LYP-D3 overestimates the stability of M2. In terms of the barrier, the situation is similar, with M06-2X functional exhibiting slightly better performance when compared to BMK. &

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Large model system (B = Ad) Keto mechanism As mentioned above, Figure 5 demonstrates that the M06-2X functional exhibits the most satisfactory performance for the keto mechanism, relative to G3(MP2)-RAD, for the small model system (B = Im). For this reason, we can be confident that this 6



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Figure 6. Energy profiles of the reduction of ribonucleotide by H2S/HSC according to the keto-mechanism in gasphase and in water, for B = Ad. The energies are obtained with the M06-2X/6-311 + G(3df,2p) model using structures and scaled ZPVE obtained with B3LYP/6-31G(d). The effect of water solvation, DEsolv, is estimated using IPCM(B3LYP/6-311 + G(d,p) with the same structures. Tabulated values are provided in Table S2 in the Supporting Information. See also Figure S1 in the Supporting Information.

(for B = Ad). For this task, we selected the IPCM method using densities obtained from B3LYP/6311 + G(d,p) calculations. The difference between the gas-phase and solution-phase energies obtained in this way is taken as the solvation energy. This energy is subsequently added to the solid curve in Figure 6, which results in the corresponding dashed curve. It is immediately apparent that the energetic effects of continuum solvation in this profile are minor, ranging from a minimum of about 2 kJ mol1 (M5K) to a maximum of around 12 kJ mol1 (TS4K). The energies of the two high-energy transition states (TS2K and TS4K) are both reduced by the polar environment. However, the magnitude of these reductions is relatively minor on the energetic scale of the overall reaction.

functional will offer comparable accuracy in the context of the Enol mechanism larger model system (B = Ad), where the G3(MP2)-RAD calculations are considerably less feasible. Figure 6, therefore, conDuring the course of our investigation of the keto mechanism, tains the schematic reaction profile for the keto mechanism shown schematically in Figure 4, it became clear that an alterwith B = Ad, obtained using M06-2X (solid curves). The direct native mechanism for non-enzymatic reduction of ribonucleoticomparison to the B = Im system for the same functional is des was possible. This alternative mechanism, which shares its provided in the Supporting Information as Figure S10. Overall, first step (M1!M2) with the keto mechanism, is shown schethe replacement of the smaller imidazole base with the adematically in Figure 7. nine alternative has only a minor effect on the reaction profile Because this pathway essentially involves the generation of the keto mechanism. All local minima and their connecting and subsequent reduction of an enol intermediate (see below), transition structures for B = Ad are completely analogous to we have chosen to refer to it henceforth as the enol mechathose found for B = Im. Apart from a small inductive effect arising from the electronic differences between the adenine and imidazole substituents, the main differences arise from the fact that N3 of adenine can act as an H-bond acceptor (e.g., for the eliminated water) more readily than N3 of imidazole. This can be seen by comparing the structures in Figure S5 (B = Im) to those in Figure S6 (B = Ad) in the Supporting Information. To estimate the effects of a polar environment, we have calculated the solvation energies of the intermediates and transition states on the keto pathway Figure 7. Mechanism of ribonucleotide reduction by H2S/HSC : enol pathway, B = Ad. Chem. Eur. J. 2015, 21, 1 – 13

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Full Paper related to M5K. Indeed the only difference is a minor rearrangement in the H-bond network (compare Figures S6 and S7 in the Supporting Information). This difference arises from our attempts to ensure maximum continuity across each reaction profile, as mentioned in the Computation Methods. The barrier for this step is also relatively high, assuming a value of 86.8 kJ mol1 in the gas-phase. This is lower than the value for the analogous step in the keto mechanism (110.9 kJ mol1, Figure 6), implying that the SS coupled H addition to the C=C bond is more favourable than to the C=O bond. Step 3 is calculated to be exothermic by Figure 8. Energy profiles of the reduction of ribonucleotide by H2S/HSC according to the enol-mechanism for 15.0 kJ mol1 in the gas phase. B = Ad. The energies are obtained with the M06-2X/6-311 + G(3df,2p) model using structures and scaled ZPVE obStep 4: M4E !M5E : The final tained with B3LYP/6-31G(d). The effect of water solvation, DEsolv, is estimated using IPCM(B3LYP/6-311 + G(d,p) with the same structures. Tabulated values are provided in Table S3 in the Supporting Information. See also Figstep involves the transfer of an ure S2 in the Supporting Information. H atom from H2S’ to C2’ and is analogous to step 5 in the keto mechanism. Indeed the barrier (14.3 kJ mol1) and exothermicity (11.2 kJ mol1) are virtually nism. The schematic reaction profile for the enol mechanism, including the relevant energies, is presented in Figure 8. the same as the analogous values shown in Figure 6. Although Step 2: M2!M3E : Like the keto mechanism, the second the enol mechanism is, overall, slightly more exothermic than the keto alternative, this difference is again related to the apstep of the enol mechanism involves the loss of water. Howevpearance of varying conformers resulting from reaction-path er, instead of a standard acid/base elimination, as shown in continuity. Figure 4, the generation of M3E has significantly more radical The overall transformation: M1!M5E : As with the keto character. It may be described as being initiated by an H-atom transfer from H2S’’’ to O2’, which triggers the homolytic cleavmechanism, the pathway involving the generation of an enol intermediate and its subsequent reduction appears to be a poage of the C2’O2’ bond and the formation of a closed-shell tentially viable exothermic reaction. In a manner closely analoenol intermediate (M3E). This interpretation, which is consistent with the relocation of the radical centre to S’’, is supported by gous to its predecessor, the enol mechanism is characterized an analysis of the charge and spin densities in TS2E (1(C2’) = by two energy-demanding steps corresponding to the genera0.19, 1(C3’) = 0.36, 1(O3’) = 0.13 and 1(O2’) = 0.21, see Table S7 tion of a double bond and a subsequent H-atom addition, in the Supporting Information, although there is some degree which is coupled to SS bond formation. Neither of the barriof charge reorganization). As with the keto mechanism, the reers for these steps is, however, sufficiently large to preclude leased water molecule assumes the role of an H-bond donor this mechanism on a sufficiently long time scale. to N3 of the adenine substituent (Figure S7 in the Supporting We have also evaluated the effect of solvent on the enol Information). mechanism and the corresponding results are shown with the The barrier to the generation of the enol intermediate (M3E) dashed curves in Figure 8. As with the keto mechanism, the effect of a polar environment is minimal. The only species with is calculated to be 116.1 kJ mol1, which makes it rate-limiting appreciable solvent effects are the two high-energy transition step for the enol mechanism. The same step is exothermic in states. Thus, although the overall effect of electrostatic solvathe gas-phase by some 7.4 kJ mol1. tion is small, it serves to increase the viability of the enol Step 3: M3E !M4E : In order to complete the ribonucleotide mechanism. reduction, the newly formed C3’=C2’ double bond in M3E needs to be sequentially reduced, in much the same way as the C3’=O3’ functionality in M4K (Figure 4). The first hydrogen A connection between the keto and enol mechanisms atom is delivered, in step 3, by H2S’’ to C2’. In analogy with the Based upon the fact that enol and keto intermediates are genketo mechanism, this H-atom addition occurs in concert with erally known to readily interconvert (tautomerize), we investithe formation of HSSH and results in the M4E intermediate, gated a potential connection between the two mechanisms. which has the unpaired electron localized on C3’ and is closely &

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Full Paper volvement of HSC in the protontransfer chain is unconventional, the spin (1(S’’’) = 0.96) and charge distributions (Table S7 in the Supporting Information) are fully consistent with the mechanism shown in Figure 9. A slightly different arrangement of the various species has been chosen Figure 9. Mechanism of ribonucleotide reduction by H2S/HSC involving elements of both the keto- and enol-mechin Figure 9, compared to Figanisms and their interconversion (via TSK–E) for B = Ad. ures 4 and 7. This only affects the schematic representations. The more familiar structural representations can be found in Figure S8 in the Supporting InforOne possible manifestation of this connection is shown in mation. Figure 9, where the overall transformation initially proceeds via Despite the fact that highest energy steps of both the keto the keto mechanism to form M4K (Figure 4). (step 4) and enol (step 2) mechanisms are circumvented by This intermediate is then transformed into the closed-shell using the reaction sequence outlined in Figures 9 and 10, the enol species M3E by tautomerization. The subsequent reductautomerization-like transition state (TSK–E) is associated with tion of this species to M5E continues as in the enol mechanism (Figure 7). The potential advantage of the Figure 9 sequence is a high barrier (133.1 kJ mol1) so no net energetic benefit arises from this particular crossover sequence. that, of the two high-barrier steps required for both mechaAlternative reaction sequences, like M1!M2!M3E !M4K ! nisms, the higher energy one would be avoided in each case. M5K !M6K, can be envisaged such that the tautomerization The schematic reaction profile for the crossover pathway is barrier would be lowered in the forward direction (to provided in Figure 10. However, as the transformations from 113.4 kJ mol1). However, such a sequence contains the rateM1!M4K and from M3E !M5E have been already covered in the previous sections, we discuss here only the details of the limiting steps from both the keto (M4K !M5K) and enol (M2! M4K !M3E conversion (Figure 9). M3E) mechanisms, so is unlikely to be preferred over either of As shown in Figure 9, the intermediary proton-transfer sites the mechanisms individually. Nevertheless, these results do not for TSK–E (the only one of its kind we were able to characterize) preclude the participation of tautomerization in an experimental context or in an alternate model system. are H2O, H2S’’ and CS’’’H, all of which receive and donate a proton during the course of the reaction. Although the inAn alternative H-atom donor

Figure 10. Energy profiles of the reduction of ribonucleotide by H2S/HSC involving elements of both the keto- and enol-mechanisms and their interconversion (via TSK–E) for B = Ad. The energies are obtained with the M06-2X/6311 + G(3df,2p) model using structures and scaled ZPVEs obtained from B3LYP/6-31G(d). Tabulated values are provided in Table S4 in the Supporting Information. See also Figure S3 in the Supporting Information. Chem. Eur. J. 2015, 21, 1 – 13

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All reaction mechanisms considered up to this point were formulated so as to produce the same HSC radical that initiated them, which is a desirable condition for an enzyme-catalysed pathway. However, in the prebiotic context, it is interesting to consider reduction reactions that do not necessarily adhere to this condition. An attractive variant in this respect comes from the realization that the HSSC radical is considerably more stabilized than the HSC radical.[67] Indeed, the G3(MP2)RAD RSE (difference in the BDEs of HSSH and H2S at 0 K) of HSS· amounts to 70.4 kJ mol1, meaning that the SH bond in HSSH has a BDE of the order of 305 kJ mol1.[68] While, therefore, using the HSSC radical to initiate the reac-


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cussed herein may have been driven to take place. This is especially true in the context of the recent observation of a chemically competent thiosulfuranyl radical for the reactions of the E. coli Class III RNR in the absence of formate.[69] While putative ribonucleotide reduction is obviously not the only way for HSSC to form in an environment containing H2S/ HSC, it is interesting that, in the mechanisms presented above, HSSH is always formed in the second to last step. In other words, the final quenching of the DNA-related radical would represent the first possible opportunity for HSSC formation. Whether this sequence of events is truly viable in an experimental context remains to be seen. Whatever the case, HSSC represents an interesting observational target for any future pulsed radiolysis experiments related to the H2S-based, nonenzymatic reduction of ribonucleotides.

ð4Þ

The exothermicity of Equation (4) [using G3(MP2)-RAD and B = Im] is greater than that of Equation (2) by precisely the RSE of 70.4 kJ mol1 and, therefore, corresponds to 78.6 kJ mol1. Figure 11 compares the reaction profiles for the final hydrogen abstraction, when carried out using H2S and HSSH. Although the barriers for the latter are marginally higher than for the former, the marked increase in exothermicity is evident. Using M06-2X and the B = Ad model system, the overall exothermicity is found to increase by 84.9 kJ mol1. While this is even slightly more than that expected from the higher level of theory with B = Im, it is not inconsistent with the previously discussed differences arising from modifications in the H-bonding network (cf. Figure S9 in the Supporting Information). Importantly, this kind of termination step is unlikely to arise in (enzymatic) models employing MeSH (or cysteine) instead of H2S because H3CSSCH3 (or any other disubstituted disulfide) is not an efficient H-atom donor in the same way as HSSH. As mentioned above, a net reaction of the form shown in Equation (4) cannot easily be transformed into a closed cycle. For that matter, even closing the reaction shown in Equation (2) requires the action of an external reductant. Nevertheless, the large exothermicity associated with Equation (4) represents an intriguing possibility as to how the reductions dis-

Conclusion

To investigate DNA formation in a prebiotic context, we have used computational means to characterize possible pathways for non-enzymatic ribonucleotide reduction. The model systems constructed assumed the availability of H2S and conditions conducive to the formation of HSC. By characterizing a simplified model system, with imidazole as the model base, we were able to determine that the overall transformation of RNA to DNA model compounds was associated with a small thermodynamic driving force, which can be accentuated by specific interactions with the environment. Furthermore, the simplified system was used to demonstrate that the BMK and, even more so, the M06-2X DFT functionals exhibit acceptable agreement with the higher-level G3(MP2)-RAD methodology. The performance of the popular B3LYP functional for this particular application was, however, not satisfactory. The pathway referred to as the keto mechanism, which was inspired by knowledge of ribonucleotide reductases, was found to be potentially viable with a rate-limiting barrier of the order of 110 kJ mol1. An alternative mechanism, involving the formation and subsequent reduction of an enol intermediate, was found to be associated with a very similar energy demand. Pathways involving elements of both these mechanisms are possible through the inclusion of a tautomerization step. In the models investigated herein, this inclusion did not offer any energetic advantage. This is not to Figure 11. Comparison of energy profiles for H-atom re-abstraction from M5K by H2S and HSSH for B = Ad. The ensay, however, that tautomerizaergies (relative to M1) are obtained with M06-2X/6-311 + G(3df,2p) using structures and scaled ZPVEs obtained tion unconstrained by the bounfrom B3LYP/6-31G(d). Tabulated values are provided in Table S5 in the Supporting Information. See also Figure S4 daries of our model systems in the Supporting Information. &

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Full Paper could not play an important role in transformations of this kind. An interesting feature of both the keto and enol mechanisms relates to the potential destructive quenching of the RNA radical intermediates by, for example, the reductive H2S environment. Once the reaction is initiated by the formation of M2, the sequence of steps is such that every rearrangement that results in a ribose-based radical is followed by a quenching-like H transfer from H2S. In this sense, the potentially reductive environment is actually a necessity rather than a hindrance. Finally, we investigated a modification of the keto and/or enol mechanisms in which the final H transfer is effected by HSSH instead of H2S. This substitution drastically increases the overall thermodynamic driving force for the reaction and, as such, represents an appealing component of a hypothesis involving the non-enzymatic reduction of ribonucleotides under prebiotic conditions. Overall, this study points the way toward experimental models that could replicate ribonucleotide reduction in a prebiotic context.

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Received: October 20, 2014 Revised: February 2, 2015 Published online on && &&, 0000

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Full Paper

FULL PAPER & Prebiotic Chemistry

Ancient DNA formation: Although most discussions of life’s origin focus on heterolytic chemistry, many radical processes have been recognised in modern biology. Inspired by the radical mechanism catalysed by today’s ribonucleotide reductase enzymes, this study extrapolates back, exploring the possibility of prebiotic radical chemistry with the aid of computational techniques (see scheme).

Chem. Eur. J. 2015, 21, 1 – 13

www.chemeurj.org


I. Dragicˇevic´, D. Baric´,* B. Kovacˇevic´, B. T. Golding, D. M. Smith* && – && Non-enzymatic Ribonucleotide Reduction in the Prebiotic Context

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Prebiotic chemical evolution in the astrophysical context.

Prebiotic chemistry: geochemical context and reaction screening.

The origin and evolution of ribonucleotide reduction.

Ribonucleotide reduction and the possible role of cobalamin in evolution.

Assay of ribonucleotide reduction in nucleotide-permeable hamster cells.

Nonenzymatic NADPH-dependent reduction of 2,6-dichlorophenol-indophenol.

Analysis of the electron paramagnetic resonance spectrum of the cobalamin intermediate in ribonucleotide reduction.

NiO-Graphene Nanocomposite.

Reduction of the small subunit of Escherichia coli ribonucleotide reductase by hydrazines and hydroxylamines.

The nonenzymatic decomposition of guanidines and amidines.

Unearthing the mechanism of prebiotic nitrile bond reduction in hydrogen cyanide through a curious association of two molecular radical anions.

Inhibition of intracellular pyrimidine ribonucleotide reduction by deoxycytidine, arabinosylcytosine and hydroxyurea.

A model for the role of multiple cysteine residues involved in ribonucleotide reduction: amazing and still confusing.

Issues in applying a harm reduction approach to breastfeeding in the context of maternal HIV.

Prebiotic oligosaccharides in premature infants.

A Prebiotic Formula Improves the Gastrointestinal Bacterial Flora in Toddlers.

Nonenzymatic polyubiquitination of expressed proteins.

Planetary science: Prebiotic chemistry on the rocks.

The interaction of adeninylalkylcobalamins with ribonucleotide reductase.

Selection of Prebiotic Molecules in Amphiphilic Environments.

Prebiotic phosphorylation of nucleosides in formamide.

Hydrated electrons and prebiotic evolution.

Health benefits of prebiotic fibers.

NMR studies of prebiotic polypertides.