Variable-Temperature NMR Spectroscopy, Conformational Analysis

Article pubs.acs.org/joc

Cite This: J. Org. Chem. 2018, 83, 2554−2569

Variable-Temperature NMR Spectroscopy, Conformational Analysis, and Thermodynamic Parameters of Cyclic Adenosine 5′-Diphosphate Ribose Agonists and Antagonists Sarah-Marie Saatori, Tanner J. Perez, and Steven M. Graham* Department of Chemistry, St. John’s University, 8000 Utopia Parkway, Queens, New York 11439, United States S Supporting Information *

ABSTRACT: Cyclic adenosine 5′-diphosphate ribose (cADPR) is a ubiquitous Ca2+-releasing second messenger. Knowledge of its conformational landscape is an essential tool for unraveling the structure−activity relationship (SAR) in cADPR. Variable-temperature 1H NMR spectroscopy, in conjunction with PSEUROT and population analyses, allowed us to determine the conformations and thermodynamic parameters of the furanose rings, γ-bonds (C4′−C5′), and βbonds (C5′−O5′) in the cADPR analogues 2′-deoxy-cADPR, 7-deaza-cADPR, and 8-bromocADPR. A significant finding was that, although the analogues are similar to each other and to cADPR itself in terms of overall conformation and population (ΔG°), there were subtle yet important differences in some of thermodynamic properties (ΔH°, ΔS°) associated with each of the conformational equilibria. These differences prompted us to propose a model for cADPR in which the interactions between the A2′−N3, A5″−N3, and H2−R5′ atoms serve to fine-tune the N-glycosidic torsion angles (χ).

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INTRODUCTION Beyond its role in redox metabolism, nicotinamide adenine dinucleotide (NAD+) has emerged of late as a regulator of numerous other biological pathways.1,2 NAD+ acts as an acyl acceptor in the decacylation of histone proteins in reactions catalyzed by silent information regulator-2 (Sir2) proteins (sirtuins), the donor in the mono- and polyadenosine diphosphoribosylation of proteins as catalyzed by mono-ADPribosyl transferases and poly-ADP-ribosyl polymerases (PARPs), a 5′-cap to some prokaryotic RNAs,3 and, relevant here, a substrate for cyclic adenosine 5′-diphosphate ribose (cADPR, Figure 1) synthases. At the phenotypic level, a recent mouse study suggested enhancing NAD+ levels can prolong lifespan.4 NAD+ levels can be depleted by the multifunctional transmembrane glycoprotein CD38 (and CD157), which is an NAD+ hydrolase, cADPR synthase, and cADPR transporter.5,6 Whereas CD38-deficient mice have greatly enhanced NAD+ levels compared to wild type, overexpression of CD38 in human kidney cells led to the downregulation of over 100 proteins involved in an array of cellular processes.7 Clearly, NAD+ is a major player in the regulation of cellular metabolism and homeostasis, but we wish to focus on one aspect in particular: the role of its Ca2+-releasing metabolite cADPR. First discovered in 1987,8 cADPR quickly emerged as a Ca2+releasing second messenger signaling molecule.9 Changes in intracellular Ca2+ levels regulate a host of cellular processes that have physiological significance, notably heart muscle contraction, fertilization, and insulin secretion.10,11 The end result of the formation of cADPR is Ca2+ efflux from the ryanodine receptor (RyR), a large (∼2 mDa total mass) tetrameric protein located in the sarco(endo)plasmic reticulum membrane. It is © 2018 American Chemical Society

generally believed that cAPDR does not interact with the RyR directly but rather through the agency of some other protein. RyR activity is modulated by multiple protein ligands, including kinases, phosphatases, calmodulin, and the FK-506 binding protein FKBP12.6 (calstabin).12,13 Which of these ligands, if any, is the cognate protein binding partner for cADPR remains to be established, although a recent photoaffinity labeling study utilizing a cADPR analogue identified glyceraldehyde 3phosphate dehydrogenase (GAPDH) as a low affinity (18 μM) cADPR binding protein.14 The SAR in cADPR. The structure−activity relationship (SAR) in cADPR continues to evolve. Over 100 cADPR analogues are known, and although the SAR is far from settled, certain patterns have emerged. The parent cAPDR (4a, Figures 1 and 2) has an EC50 for Ca2+ release of 30−90 nM15,16 in the sea urchin homogenate (SUH) assay. Three early analogues, 2′deoxy-cADPR (2′-dA cADPR, 4b),16 7-deaza-cADPR (4c),17 and 8-bromo-cADPR (8-Br-cADPR, 4d)15 (Figure 2), are a potent agonist (EC50 58 nM), partial agonist (90 nM, with only 66% of the total Ca2+ released), and antagonist (IC50 ∼1000 nM), respectively. We wished to perform detailed conformational analyses on cADPR analogues, and we chose these three analogues for this variable-temperature NMR study because (1) they span the range of pharmacological activity, (2) are easily synthesized using the ‘chemoenzymatic’ approach (chemical synthesis of the appropriate NAD+ analogue and enzymatic cyclization with the commercially available Aplysia californica ADP ribosyl cyclase, Figure 1 and Scheme 1), and (3) are all Received: October 30, 2017 Published: January 24, 2018 2554

DOI: 10.1021/acs.joc.7b02749 J. Org. Chem. 2018, 83, 2554−2569

Article

The Journal of Organic Chemistry

Figure 1. Synthesis of cADPR from NAD+ and breakdown of cADPR to ADPR. ADP ribosyl cylase (ADPRC) from the mollusk Aplysia californica is a soluble, commercially available enzyme. CD38 and CD157 are transmembrane glycoproteins. cADPR can be hydrolyzed to ADPR in the continued presence of these enzymes; it can also undergo spontaneous hydrolysis with a half-life of 2.5 days at pH 7 at 37 °C.

Figure 2. Structures of cADPR analogues. On the basis of its name, cADPR for cyclic adenosine diphosphate ribose, we prefer to call the ribofuranose ring derived from adenosine (the ‘A’ in cADPR) the ‘A-ring’ and the other ribofuranose ring (the ‘R’ in cADPR) the ‘R-ring’. Others have called these rings the ‘southern’ and ‘northern’ ribose rings, respectively, but we fear this can lead to confusion in the subsequent discussion where the terms ‘north’ and ‘south’ refer to conformational descriptors of furanose ring geometries.

Scheme 1. Synthesis of cADPR Analoguesa

a Reagents and conditions: (a) (i) PO(OEt)3, POCl3, 0 °C, 2−4 h, (ii) H2O, 0 °C (iii) tri-n-octylamine, CH3OH; (b) (PhO)2POCl (diphenyl phosphoryl chloride, DPPC), tri-n-butylamine (TBA), dioxane, DMF; (c) (i) Ac2NMN, DMF, TBA, pyridine, (ii) NH3, CH3OH, 0 °C, 15 min; (d) ADPRC, HEPES buffer, pH 7.0, 2−3 h. Adenosine 5′-monophosphate (AMP, 2a), 2′-deoxyadenosine 5′-monophosphate (dAMP, 2b), and NAD+ (3a), each as their free acids, were obtained from commercial sources. Ac2NMN was prepared as described.39

feature of deaza analogues 4c and 9a, carbocyclic 10, and cATPR 8 is an improved resistance to hydrolysis, which has implications for NMR studies (vide infra). A-Ring and Adenine-Modified Analogues. Generally, substitution at the 8-position results in an antagonist. 8-AminocADPR, like 8-Br-cADPR 4d, is an antagonist15 in both the SUH assay and in Jurkat T cells (JTC).22 The 7-deaza alteration alters activity inconsistently. For example, 7-deazacADPR 4c is less effective at Ca2+ release than cADPR, whereas

based on a deoxyribofuranose or ribofuranose scaffold and as such do not require substantial modification to the default parameters of PSEUROT18 (see Results and Discussion), the program used to convert 3-bond vicinal 1H−1H coupling constants to (deoxy)ribofuranose conformations. Compounds 4b−4d, along with 3-deaza-cADPR (9a, EC50 ∼1 nM),19 and cyclic aristeromycin diphosphate ribose (cArisDPR, 10, EC50 80 nM)20 typify one class of cADPR analogues, the ‘A-ring’modified analogues (see the legend to Figure 2).21 A common 2555


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Figure 3. Structures of ring-deleted cADPR analogues.

8-bromo-7-deaza-cADPR is a more effective antagonist than 8bromo-cADPR 4d.23 Removal of the A-ring 2′-OH from the antagonist 8-NH2 cADPR led to a 30-fold drop in potency, whereas the same deletion in antagonist 8-Br cADPR 4d had no effect on activity.24 In terms of modifications to the A-ring furanose, the Potter group found the A-ring 2′-OH could be deleted (2′-dA cADPR, 4b) with essentially no loss in activity, but deletion of the A-ring 3′-OH resulted in an ∼100-fold drop in agonistic activity and replacing the 3′-OH with a methoxy group resulted in a weak antagonist.16 Additionally, analogue potencies cannot be compared across different assays and organisms, as agonists in a SUH assay for example may not show the same behavior in a JTC assay.22 cIDPR Analogues. A second class of cADPR analogues (Figure 2), the N1-cyclic inosine diphosphate ribose (N1cIDPR, 5) analogues, has been studied extensively by the Potter group.25,26 The requisite dinucleotide precursor, nicotinamide hypoxanthine dinucleotide (NHD+), is a substrate for ADPRC but cyclizes at N7 rather than N1. Recognizing the cyclase’s preference for a syn-oriented base and knowing that bulky C8substituents favor a syn orientation, they rationalized that 8bromo-NHD+ might adopt a conformation more suitable for ADPRC-catalyzed cyclization at N1. The initially formed 8bromo-N1-cIDPR was subsequently converted to N1-cIDPR (5), as well as to 8-phenyl-, 8-azido-, and 8-amino-cIDPR analogues. The cIDPR analogues show greatly enhanced hydrolysis resistance, are membrane permeable, and 5 behaved almost identically to cADPR in a JTC assay. cIDPR 5 is also an inhibitor (IC50 = 276 μM) of the cADPR hydrolase activity of soluble human CD38.27 R-Ring and Adenine-Modified Analogues. The analogues discussed above were all prepared via the chemoenzymatic approach: chemical synthesis of a nicotinamide dinucleotide precursor and subsequent cyclization using ADPRC. Although some R-ring-modified cADPR analogues have been prepared using this strategy28 (requiring the preparation of a nicotinamide mononucleotide (NMN) analogue), total chemical synthesis is generally required. In the R-ring-modified series (Figure 2), cyclic adenosine diphosphate carbocyclic ribose (cADPcR, 6, EC50 15 vs 50 nM for cADPR)29,30 and cyclic adenosine diphosphate thioribose (cADPtR, 7, EC50 39 vs 210 nM for cADPR),31 prepared by the Shuto group, are illustrative. The carbocyclic 6 series again showed hydrolysis resistance; both series interestingly showed that the 8-amino analogues were agonists. Pyrophosphate-Modified Analogues. Comparatively speaking, pyrophosphate-modified cADPR analogues are rare

(Figure 2). Phosphorothioate 13 (see Ring-Deleted Analogues below and Figure 3) is one example; cyclic adenosine triphosphate ribose (cATPR, 8, 20-times more potent than cADPR)32 and two analogues33 where the pyrophosphate is replaced by methylenebisphosphonate are others. The latter analogues, cADPR[CH2] (9b, EC50 111 vs 59 nM for cADPR) and 3-deaza-cADPR[CH2] (9c, EC50 302 vs ∼1 nM for 3deaza-cADPR 9a), as well as cATPR (8) were prepared via the chemoenzymatic approach. Ring-Deleted Analogues. Some cADPR analogues (Figure 3) have been prepared that entirely lack an A-ring, R-ring, or both. Members of this class of analogues are the cyclic inosine diphosphate ribose ethers (cIDPRE 11 and their 8-substituted congeners 12),34 phosphorothioate 13 (one of the diastereomers is an agonist and the other an antagonist),35 cyclic triazole diphosphate ribose ether (cTDPRE 14),36 and cyclic inosine diphosphate diethers (cIDPDE 15). These ‘Rring-deleted’ analogues, prepared by the Zhang group, often retain agonistic activity. Especially remarkable is that ‘no ring’ analogue 15 is only 3-fold less potent an agonist than cADPR. The ‘no ring’ 15 notwithstanding, deletion of the A-ring is deleterious to activity. A-ring-deleted N9-butyl-cIDPRE (16), like cIDPR 5, is an inhibitor of the cADPR hydrolase activity of soluble human CD38.27 cAcyloDPR analogue 17, prepared by the Potter group, was found to be inactive in the SUH system. Rationale. Despite this wealth of pharmacological data, a key question in the SAR of the cADPR system remains mostly unanswered: in making cADPR analogues, do the alterations to the cADPR scaffold change the structure (more precisely, its conformation), or is the structure largely preserved, and consequently what changes is its interaction with the receptor? If a new conformation is adopted, has an analogue become better or worse suited to the receptor binding site? Or is cADPR merely a scaffold upon which groups can be deleted from or appended to, allowing for better (or worse) contacts in the as yet unknown receptor binding site? In an extensive study by Moreau,22 a strong correlation was found between activity and the conformation of the ‘southern’ ribose ring (the ‘A-ring’ in our terminology),21 at least in the sea urchin system. Kudoh37 found cADPR (1a), cADPcR (6), and a 6-analogue to be similar in A-ring structure in an NOE-restrained MD simulation. Less is known about the conformation of the R-ring, and other than our earlier38,39 and recent40 work on cADPR and its A-ring 2′-OMe and 3′-OMe analogues, even less is known regarding the conformation about the pyrophosphate backbone and the glycosidic bonds. Detailed structural information on the conformational landscape is lacking for 2556


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Figure 4. 1D 1H NMR spectra (400 MHz) of the cADPR analogues at 298 K. (A) cADPR (4a) reference spectrum (600 MHz).40 (B) 2′-dA-cADPR (4b). The peaks in the A2′ signal marked with stars are from the 13C satellite peak of the triethylammonium signal. (C) 7-deaza-cADPR (4c). (D) 8Br-cADPR (4d). Primes in the 1′, 2′, 3′, and 4′ signals are omitted for clarity. Note the generally overlapped A3′/R2′/R4′ region in each analogue, the overlapped A5″/R3′ signals of 2′-dA cADPR (4b), and the overlapped A5″/R5′/A4′ of 8-Br cADPR (4d). The 8-bromo sample was contaminated with a small amount of its hydrolysis product 8-Br AppRib. Samples were 10−20 mM in D2O containing TMSP as an internal standard.

proceeded uneventfully, but the phosphorylation of 8bromoadenosine (1d) to the corresponding 5′-phosphate 2d proved problematic, as described below. Monitoring the conversion of 1d to 2d by reversed-phase (RP) HPLC analysis indicated the formation of two products with significantly different retention times (4.1 and 4.8 min with baseline separation) by RP HPLC but rather similar UV spectra (λmax 262 and 264 nm, respectively). Partial separation was achieved by ion-exchange chromatography (Sepharose Q resin eluted with formic acid). The early eluting fractions (early-2d) were sufficiently pure to be pooled, as were the later fractions (later-2d), as judged by RP HPLC analysis of the individual fractions. The 1H NMR spectra of early-2d and later2d were remarkably similar with H2 signals at 8.36 and 8.34 ppm, H1′ signals at 6.14 (d, J = 5.6 Hz) and 6.13 ppm (d, J = 5.6 Hz), and H2′ signals at 5.21 ppm (t, J = 5.5 Hz) and 5.25 ppm (t, J = 5.5 Hz). Ultimately, we determined the acidic conditions of the Yoshikawa phosphorylation promoted an exchange of the 8-bromo substituent for chlorine (a phenomenon we were unaware of at the time), which had been previously noted in the literature.42 Comparison of the 13 C NMR chemical shifts for C8,43 and ultimately LCMS data (early-2d, 140 ppm, m/z MH+ 382; later-2d, 129 ppm, MH+ m/z 426), showed that early-2d was 8-Cl-AMP 2e and later-2d was the desired 8-Br-AMP 2d.44 With the desired monophosphates in hand, we then focused on the coupling step, joining the adenosine 5′-monophosphates 2b−2d to NMN via pyrophosphate bond formation to form NAD+ analogues 3b−3d. As in our previous synthesis of cADPR analogues, we chose,39 as had others,16 to use the Michelson45 procedure in which diphenyl phosphoryl chloride

the entire cADPR family, and missing completely is any thermodynamic data (ΔGo, ΔHo, ΔSo) on the conformational equilibria; data could begin to explain why certain conformations are favored. Do conformational changes in one part of cADPR drive changes in another, that is, are the molecular motions coupled? This work, a variable-temperature NMR spectroscopy investigation, aims to fill part of that gap by conducting a detailed conformational analysis and thermodynamic study on the known cADPR analogues 2′-dA-cADPR (4b), 7-deaza-cADPR (4c), and 8-Br-cADPR (4d).

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RESULTS AND DISCUSSION Synthesis. The known cADPR analogues 4b−4d were synthesized using a chemoenzymatic approach consisting of 5′phosphorylation of an adenosine analogue (1) to give an AMP analogue (2), formation of a pyrophosphate bond by activation of the 5′-phosphate of 2 to nucleophilic attack by NMN, thus providing the requisite NAD+ analogue (3), and cyclization using the commercially available Aplysia ADPRC (Scheme 1) to yield a cADPR analogue (4). Starting from 7-deazaadenosine (1c) or 8-bromoadenosine (1d), 5′-selective phosphorylation was performed by the Yoshikawa method (TEP, 2 equiv POCl3, 0 °C; water was omitted).41 Upon completion of the reaction (2−4 h), the reaction mixture was slowly dripped via cannula into ice-cold, stirred, dry ether. After a brief centrifugation, the solid pellet was stirred in ice-cold water. Ion-exchange chromatography (formic acid gradient) afforded the AMP analogues as their free acids. In our hands, the reaction was almost completely selective for 5′-phosphorylation, and only trace amounts of inorganic phosphate could be detected by 31P NMR. The synthesis and purification of 7-deaza-5′-AMP (2c) 2557


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Table 1. 1H−1H and 1H−31P (400 MHz) Coupling Constants (J, Hz)a of the cADPR A-Ring for 4b−4d and PSEUROT 6.3 Output (deg) for cADPR Analogues 4b−4d A-ring

T (K)

J1′2′

J1′2″

J2′2″

J2′3′

J2″3′

J3′4′

J4′5′

J4′5″

J5′5″

J5′P

J5″ P

4b 2′-dA

277 298 318 338 353 277 298 318 338 353 277 298 318 338 353 PN 22.5 359.1 352.2 20.3

6.85 6.68 6.51 6.19 ndb 6.38 6.28 6.13 5.97 5.84 5.41 5.37 5.27 5.15 nd ψNm 35i 38 35 35

6.96 7.01 7.07 7.22 nd

14.20 14.19 14.22 14.32 nd

6.30 6.31 6.40 6.44 nd 5.06c 5.12 5.18 5.28 5.28 5.13 5.20 5.24 5.28 nd ψSm 31.5 27.3 30.7 28.8

3.58 3.78 3.96 4.22 nd

2.83 2.97 3.29 3.42 nd 2.65c 2.81d 2.95 3.09 3.20 3.44 3.50 3.53 3.57 nd RMSg 0.099 0.128 0.062 0.045

2.84 2.97 3.12 3.37 nd 2.64 2.52 2.56 2.71 2.81 2.34 2.29 2.28 nd nd

7.45 7.21 7.00 6.72 nd 7.23 7.00 6.74 6.69 6.47 6.17 6.10 5.92 nd nd

10.81 10.89 10.89 10.81 nd 10.9 11.0 11.0 11.0 11.0 11.14 11.10 11.00 nd nd

3.14 3.37 3.54 4.37 nd nd 3.57 3.74 3.78 3.98 2.93 3.31 3.61 nd nd

3.45 3.51 3.66 4.07 nd nd 3.48 3.46 3.51 3.58 4.47e 4.34 4.58e nd nd

4c 7-deaza

4d 8-Br

PS 6.3f 4ah 4b 4c 4d

PS 177.1 170.7 159.1 173.7

Values ± 0.04 Hz. Only J1′2′, J2′3′, and J3′4′ are used in PSEUROT. Furanose ring values are from the normal 1D 1H spectrum, J4′5′, J4′5″, and J5′5″ values from the 1H{31P} spectrum. J5′P determined by subtraction of Σ(J4′5′ + J5′5″) from Σ(J4′5′ + J5′5″ + J5′5P), the latter being the width of 5′-signal. J5″P determined analogously. bNd = not determined due to sample decomposition. cValue from 1D TOCSY with irradiation at A2′. dValue from A4′. e Value determined directly from A5″. fErrors in PS6.3 output: cADPR (4a) PN ± 4.3°, PS ± 2.6°, ψSm ± 0.7°; 2′-dA cADPR (4b): PN ± 4.2°, PS ± 2.3°,ψSm ± 0.6°; 7-deaza cADPR (4c): PN ± 2.1°, PS ± 1.6°, ψSm ± 0.4°; 8-Br cADPR (4d): PN ± 2.4°, PS ± 1.6°, ψSm ± 0.4°. gRoot-mean-square error of the observed vs PSEUROT-calculated J values in Hz. hData for cADPR (4a) from ref 40. iUnderlined values restrained in PSEUROT 6.3. Note the irregularity in the J5″P values for 4d (bold). a

NMR Spectroscopy. 1H NMR (400 MHz) spectra for the triethylammonium salts of each of the cADPR analogues 4b− 4d were recorded at 277, 298, 318, 338, and 353 K, though the lability of the R ring C1′-N1 bond in 4b and 4d limited data collection at the higher temperatures. The 1H NMR spectra at 298 K of cADPR analogues 4b−4d, as well as the 600 MHz spectrum (298 K) of cADPR (4a) for reference, are shown in Figure 4. (See pages S23, S27, S28, and S34 for the spectra of each analogue at each of the temperatures.) Samples were 10− 20 mM in D2O containing TMSP as an internal standard. The spectra were all first-order (confirmed by spectral simulation, not shown), allowing the three-bond vicinal coupling constants (3J) needed for the conformational analysis (see Population Analysis) using PSEUROT to be extracted directly from the spectra. The information needed to perform conformational analysis on cADPR/analogues resides in eight signals (ten for 2′-dA analogue 4b): the A-ring H1′ (henceforth A1′) and R1′ (d, J1′2), A3′ and R3′ (dd, J2′3′, and J3′4′), A5′ and R5′ (dt, J5′5″, J4′5′, J5′P), A5″ and R5″ (ddd and dt, J5′5″, J4′5″, J5″P), plus A2″ (J1′2″ and J2″3′) for deoxy-4b. The 1′2′, 2′3′, and 3′4′ (and 1′2″ and 2″3′ for 4b) couplings are used to determine the furanose conformations via PSEUROT, and the 5′/5″ couplings, via ‘sum rules’, are used to determine the γ- and β-bond conformations (see Population Analysis). The H4′ signals (broad multiplets due to coupling with H3′, H5′, H5″, and sometimes phosphorus) and H2′ signals (apparent triplet due to the near equivalence of J1′2 J2′3′) are less than ideal. A combination of routine 1D 1H- and phosphorus-decoupled H1 (1H{31P}) NMR were used to extract the coupling constants, as the needed signals were generally well-enough resolved. As we had done previously,38−40 additional information was obtained

(DPPC, (PhO)2POCl) is used to activate the 5′-phosphate of a nucleotide toward nucleophilic attack via the formation of a P1nucleoside-5′-P2-diphenyl pyrophosphate (mixed anhydride, Scheme 1). Nucleophilic attack by the 5′-phosphate group of a second nucleotide displaces the weak base leaving group diphenyl phosphate from the mixed anhydride. In principle, either nucleotide could be the activated nucleotide and the other the attacking nucleophile; in practice, we chose to activate the AMP analogues 2b−2d and use 2′,3′-di-O-acetyl NMN (Ac2NMN; the acetyl groups enhance the solubility of NMN in organic solvents) as the attacking nucleophile so as to minimize the exposure time of the somewhat fragile NMN glycosidic bond to the reaction conditions. The free acids of AMP analogues 2b−2d were thus converted to their tri-noctylammonium salts to improve their solubility and reacted with DPPC. The mixed anhydride was precipitated into ether; the supernatant was decanted and dried briefly under vacuum, and a solution of Ac2NMN was then added. Ion-exchange chromatography as for 5′-AMP analogues 2c and 2d afforded NAD+ analogues 3b−3d in modest yield. Interestingly, the least challenging aspect of the synthesis was the enzymatic cyclization of NAD+ analogues 3b−3d to cADPR analogues 4b−4d. Cyclization reactions were typically run on a 50 μmol (30−40 mg) scale in ∼6 mM HEPES buffer at room temperature with a substrate concentration of ∼0.85 mM.46 All three NAD+ analogues were readily converted to the corresponding cADPR analogues over the course of several hours in conversions exceeding 60% as monitored by RP HPLC. Ion-exchange chromatography on the Sepharose-Q column utilizing either TEAB or TEAA buffers gave the cADPR analogues 4b−4d as their triethylammonium salts in good yields. 2558


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Table 2. 1H−1H and 1H−31P (400 MHz) Coupling Constants (J, Hz)a of the cADPR R-Ring for 4b−4d and PSEUROT 6.3b Output (deg) for 4b−4d R-ring

T (K)

J1′2′

J2′3′

J3′4′

J4′5′

J4′5″

J5′5″

JP5′

JP5″

4b 2′-dA

277 298 318 338 353 277 298 318 338 353 277 298 318 338 353 PN 340.0 337.4 343.2 337.2

4.00 4.04 4.06 4.02 ndd 4.00 4.04 4.06 4.07 4.06 3.98 4.03 4.07 4.10 nd ψNm 40i 40 40 40

5.07 5.05 5.08 5.13 nd 5.00 5.02 5.03 5.05 5.04 5.06 5.08 5.10 5.09 nd PS 215.8 216.2 212.2 215.4

2.11 2.17 2.25 2.36 nd 2.45 2.54 2.61 2.67 2.74 2.23 2.23 2.28 2.28 nd ψSm 36.6 36.6 34.5 36.2

2.10 2.13 2.16 ∼2.39c nd nd 2.03 2.12 2.15 2.08 2.12 2.05 2.16 nd nd RMSg 0.031 0.041 0.046 0.030

2.05 2.43 2.52 ∼2.39c nd nd 2.49 2.59 2.70 2.74 2.20 2.23 2.25 nd nd

11.90 11.96 12.02 nd nd nd 12.0 12.0 12.1 12.1 12.0 12.0 12.1 nd nd

2.18 2.14 2.39 nd nd nd 2.39 2.53 2.60 2.73 2.12 2.59 2.39 nd nd

3.42 3.70 3.89 nd nd nd 3.42 3.45 3.58 3.73 3.46 3.65 4.00e nd nd

4c 7-deaza

4d 8-Br

PS 6.3f 4ah 4b 4c 4d

Values ± 0.04 Hz. Furanose ring values are from the normal 1D 1H spectrum, J4′5′, J4′5″, and J5′5″ values from the 1H{31P} spectrum. J5′P determined by subtraction of Σ(J4′5′ + J5′5″) from Σ(J4′5′ + J5′5″ + J5′5P), the latter being the width of 5′-signal. J5″P determined analogously. bOnly J1′2′, J2′3′, and J3′4′ are used in PSEUROT. cValue from R4′. dNd = not determined due to sample decomposition. eValue from 1D TOCSY with irradiation at R2′/ R4′. fErrors in PS6.3 output: cADPR (4a) PN ± 0.8°, PS ± 0.3°, ψSm ± 0.3°; 2′-dA cADPR (4b) PN ± 1.2°, PS ± 0.5°, ψSm ± 0.5°; 7-deaza cADPR (4c): PN ± 1.1°, PS ± 0.5°, ψSm ± 0.4°; 8-Br cADPR (4d): PN ± 0.9°, PS ± 0.4°, ψSm ± 0.3°. gRoot-mean-square error of the observed vs calculated J values in Hz. hData for cADPR (4a) from ref 40. iUnderlined values restrained in PSEUROT 6.3. Note the irregularity in the J4′5′ and J5′P values for 4d (bold). a

Figure 5. (a) Five-membered ring furanose geometries and their descriptors. In S-type conformers, note how H1′ and H2′ are anti and H3′ and H4′ are perpendicular, whereas in N-type conformers the opposite is true. (b) Endocyclic bond definitions. (c) The three canonical orientations of the γand β-bonds. (d) Atom, bond, and furanose definitions for cADPR. See ref 53 for details.

through the use of the 1D TOCSY47 experiment, useful especially at the higher temperatures as cADPR/analogues began to decompose. Two notable features of the 298 K spectra were apparent upon inspection. First, as has been noted,22 was the unusually high chemical shift for the A2′ signal in the spectra of 4a, 4c, and 4d (5.4−5.5 ppm vs 4.6−4.8 for adenosine and AMP), characteristic of a syn-oriented base.48 The second feature was

the consistency in the chemical shifts and coupling patterns across the analogue series 4a−4d. The R1′ and all 5′/5″ signals, and to a slightly lesser extent the R2′, R3′, and R4′ signals, were quite similar (see Tables 1 and 2 for coupling constants), suggesting comparable conformations and environments. (The differences in the A-ring chemical shifts can be attributed to the analogue modifications, for example, 2′-deoxyribo vs ribo.) The R5′ and R5″ pair signals were consistently doublets of triplets 2559


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The Journal of Organic Chemistry (dt), indicating JR4′R5′, JR4′5″, JR5′P, and J5″P, are similar in magnitude (∼2−3 Hz). The A5′ (dt) signal was similar in appearance to R5′ and R5″, but A5″ is a ddd, indicating at a minimum a conformation about the A-ring C4′-C5′ bond different than that of the R-ring C4′-C5′ bond.49 These patterns remained largely intact throughout the variable temperature study, although the magnitude of the coupling constants changed. Accurate values for the J4′5′ and J4′5″ couplings were generally extracted from a 1H{31P} experiment (see pages S22, S23, S26, S31, and S33); the J5′P and J5″P couplings were extracted by subtracting the appropriate J4′5′ or J4′5″ and J5′5″ coupling from their corresponding signals.50 As mentioned earlier, cADPR analogues 5−8, 9a, and significantly here 7-deaza analogue 4c show increased resistance to hydrolysis of the R1′-N1 glycosidic bond (compare to cADPR; t1/2 = 2.5 d, pH 7, 37 °C). Pages S23, S27, S28, and S34 show the 1H NMR spectra of 4b−4d at the highest temperature from which coupling data could be extracted. The 2′-dA analogue 4b and 8-Br analogue 4d have completely broken down to their respective ADP-riboses (confirmed by HPLC; data not shown), whereas 7-deaza 4c is largely intact. Conformational Analysis Using PSEUROT. First proposed by Kilpatrick et al. in 1947,51 the pseudorotation concept is the basis for the conformational analysis of five-membered rings such as the furanose ring found in nucleosides and nucleotides. Five-membered rings avoid planarity by displacing one atom (envelope conformation, 2E, 3E, 2E, 3E) or two atoms (twist conformation, 32T , 23T ) from the ring plane defined by the remaining atoms with atoms below (‘exo’) the ring plane indicated by subscripts and those above (‘endo’) by superscripts. (Figure 5a). Nucleos(t)ide furanose rings, lacking symmetry, allow for 20 possible ‘ideal’ or ‘canonical’ conformations, 10 possible envelope forms and 10 possible twist forms, which alternate between E and T forms as a furanose traverses a full pseudorotational itinerary. The Altona−Sundaralingam (AS)52 formalism assigns a numerical value, the ‘phase angle of pseudorotation’ (‘phase angle’, P) to each of the 20 conformers. A second parameter, the maximum puckering amplitude ψm, describes by how much the ring deviates from planarity. (A planar ring would have ψm = 0° and idealized cyclohexane 60°; typical ψm’s in nucleos(t)ides are 25−45°.) The allowed values of P are 0−360°; by analogy to a compass rose, P = 0° (2T3; C2′-exo-C3′-endo) is ‘north’, and P = 180° (2T3; C2′-endo-C3′-exo) is ‘south’. These relationships are shown in Figure 5a. It should be stressed that there is no high-energy barrier between adjacent ‘conformers’ on the pseudorotational wheel and that the notion of 20 ‘stops’ in pseudorotational space is merely a descriptive convenience. A nucleos(t)ide can adopt any value of P. The geometry53 of a furanose ring can also be based on the values of the endocyclic torsion angles ν0−ν4 (Figure 5b); indeed, this is the basis of AS formalism. The relationships are tan P =

ψm =

(ν2 + ν4) − (ν1 + ν3) 2ν0(sin 36° + sin 72°)

υ0 cos P

⎛ 4πi ⎞ ⎟ where i = 0, 1, 2, 3, 4 vi = ψmcos⎜P + ⎝ 5 ⎠

The elegance of the AS formalism resides in eq 1, as it reduces five endocyclic torsion angles to a single parameter, P, that simply describes which atom(s) are puckered out of the ring plane. Any individual endocyclic torsion angles can be recreated with eq 3. For reasons both steric and stereoelectronic,54 not all of the 20 canonical forms are populated. The furanose rings of nucleos(t)ides tend to localize to either a ‘north-type’ (N) or a ‘south-type’ (S) conformation. Under the generally true case of a two-state N ⇆ S equilibrium, five parameters are needed to describe the system: the P of each conformer (PN and PS), the maximum puckering amplitudes of each (ψNm and ψSm), and the mole fraction (of either conformer). For cADPR and its analogues, a complete description of the conformational landscape requires knowledge of the conformation of both furanose rings as well as the A- and R-ring γ- and β-bond conformations (Figure 5c and 5d). These six conformationally flexible subunits, plus the conformation about the glycosidic bonds χA and χR, (Figure 5d) drive the conformation. The aforementioned study22 of cADPR analogues examined only the A-ring conformation, and only to the extent of determining if the A-ring furanose was N-type or S-type. The equilibrium ratios and precise values for PN, PS, ψNm, ψSm and the γ- and βbond conformations of the cADPR analogues 4b−4d can be obtained through a more rigorous analysis of coupling constants, and to our knowledge, this is the first such study to do so in significant detail. The furanose ring coupling constants for cADPR analogues 4b−4d are summarized in Tables 1 and 2. Also included therein are the pseudorotation parameters for 4b−d as determined by PSEUROT as well as the pseudorotation parameters for cADPR itself (4a) determined previously.40 The coupling constants from Tables 1 and 2 were the input for version 6.3 of the PSEUROT program.18 In addition to coupling constants related to the furanose conformation (Aand R-ring J1′2′, J2′3′, and J3′4′ for analogues 4c and 4d plus for analogue 4b J1′2″, and J2″3′), the user provides as input to PSEUROT substituent electronegativities, an initial guess as to the values of the five parameters that describe a two-state N ⇆ S equilibrium (PN, PS, ψNm, PS, ψSm, and the S mole fraction) and a set of parameters to correlate the endocyclic ring atom torsions to the exocyclic 1H−1H torsions (the so-called ‘A’ and ‘B’ parameters). Although the conformation of a furanose ring is described (Figure 5b and eq 1) by the endocyclic ring torsions, the observed 1H−1H coupling constants (Jobs) depend on the exocyclic 1H−1H torsion angles (Figure 5a). Combined with the initial guess of PN and PS, PSEUROT uses the A and B parameters to create exocyclic torsion angles (e.g., H1′-C1′C2′-H2′) by ‘building in’ hydrogen atoms onto the corresponding endocyclic torsions (e.g., ν4 = O4′-C1′-C2′C3′) specified by P. With the exocyclic torsion angles thus specified, a Karplus equation within PSEUROT calculates a predicted coupling constant, Jcalc, which it compares to Jobs. One of the reasons we chose these analogues to study first is that they contain only ribo- and deoxyribofuranose rings for which the A and B values were specifically parametrized.55 (Furanoses different than ribo- and deoxyribo- generally require adjustment of the default A and B values or values specifically derived for the molecule under investigation.) PSEUROT then calculates the expected coupling constants for such a mixture and iteratively adjusts PN, PS, ψNm, ψSm, and the S mole fraction so as to produce the best fit between the calculated and observed coupling constants.

(1)

(2)

(3) 2560


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Table 3. Equilibrium Populationsa for the Furanose Rings, γ-Bonds, and β-Bonds in cADPR (4a) and Analogues 4b−4d A-ring 4a cADPRb

R2 d 4b 2′-dA

R2 4c 7-deaza

R2 4d 8-Br

R2

R-ring

T (K)

N⇆S

γt ⇆ γ+

β− ⇆ βt

N⇆S

γt ⇆ γ+

β+ ⇆ βt

277 298 318 338 353

29:71 32:68 35:65 35:65 36:64 0.92 28:72 30:70 33:67 36:64 nd 0.98 25:75 26:74 28:72 30:70 32:68 0.96 33:67 34:66 34:66 35:65 nd 0.90

58:42 57:43 56:44 54:46 53:47 0.97 69:31 68:32 67:33 67:33 nd 0.98 65:35 61:39 59:41 60:40 59:41 0.80 51:49 49:51 47:53 nd nd 0.97

9:91 11:89 12:88 13:87 ndc 0.99 8:92 9:91 11:89 17:83 nd 0.88 nd 10:90 11:89 11:89 13:87 0.92 12:88 13:87 16:84 nd nd 0.95

26:74 27:73 27:73 26:74 26:74 0.04 24:76 24:76 25:75 26:74 nd 0.85 28:72 29:71 29:71 30:70 30:70 0.92 26:74 25:75 26:74 25:75 nd 0.21

4:96 5:95 6:94 7:93 nd 0.98 6:94 10:90 11:89 12:88 nd 0.88 nd 10:90 11:89 13:87 13:87 0.88 8:92 7:93 8:92 nd nd 0.29

2:98 4:96 7:93 8:92 nd 0.94 3:97 4:96 6:94 nd nd 0.99 nd 4:96 5:95 6:94 7:93 0.98 3:97 6:94 7:93 nd nd 0.89

277 298 318 338 353 277 298 318 338 353 277 298 318 338 353

N:S populations from PSEUROT 6.3. A-ring γ-bond population: fγ+ = [13.3 − (J4′5′ + J4′5″)]/9.7 (eq 4).58a R-ring γ-bond: fγ+ = [13.3 − (J4′5′ + J4′5″)]/9.4 (eq 5).58a A-ring β-bond: fβt = [26.4 − (J5′P + J5″P)]/21.4 (eq 6).58a R-ring β-bond: fβt = [25.5 − (J5′P + J5″P)]/20.5 (eq 7).58b Eqs 4 and 6 were used as found in the associated references. Eqs 5 and 7 were modified slightly (see the Supporting Information) to improve the fit between the observed and back-calculated coupling constants;58 the back-calculated coupling constants also allowed for identification of the minor conformer. b Data for cADPR from ref 40. cValue not determined. dCorrelation coefficient from the van’t Hoff plots (Figure 6). a

To determine the values of five parameters requires five observables (coupling constants); ribofuranoses, with only J1′2′, J2′3′, and J3′4′, are thus said to be underdetermined, meaning their N ⇆ S equilibria cannot be completely described. Generally being interested in the major conformer, one could restrain the P and ψm of the minor conformer. It has been shown56 that the coupling constants are much less sensitive to changes in ψm than to changes in P. If puckering amplitude information is available from other sources (e.g., a crystal or QM structure), such data can be used to restrain either or both ψNm and ψSm. Regrettably, reliable crystal structure data is not available for either cADPR or analogues 4b−4d. The only crystal structure of cADPR in the Cambridge Structural Database (CSD Entry YINYID) is of the free acid and was deposited without coordinates. There was enough information in the text of the associated paper57a to estimate a PS ∼ 175° for the A-ring and a PS ∼ 208° for the R-ring, but the free acid raises concerns regarding the γ- and β-bond conformations in the crystal. Another study examined the crystal structure of cADPR bound to CD38, but retrieval of the cADPR ligand from the protein structure (PDB ID: 2O3Q) returns an A-ring with the xylo-configuration rather than ribo.57b A third report,57c this time with cADPR bound to the Aplysia cyclase, has a cADPR structure with A-ring 2E (P ∼ 162°)/γ+-bond conformations and R-ring 1E (P ∼ 306°)/γt-bond conformations; the opposite γ-conformations were found here. The option we ultimately chose was to collect NMR spectra over a range of temperatures sufficient to obtain significant changes in the coupling constants. On the basis of our earlier

work38−40 and reluctant to use the crystal structure data, we restrained the ψNm of the A- and R-ring minor conformers to 35° (38° for 2b) and 40°, respectively. All other parameters were freely optimized. Assuming the identities of the two conformers involved in the N ⇆ S equilibrium are temperature independent, a change in the coupling constants reflects a change in their equilibrium ratio (see Thermodynamic Parameters). Each temperature represents not only an additional PSEUROT observable but also the opportunity to perform van’t Hoff analyses from which ΔHo and ΔSo can be determined. Population Analysis. The PSEUROT output is summarized in Tables 1 and 2, and the changes in the north/south ratio as a function of temperature are shown in Table 3. Also included in Tables 1 and 2 are couplings of each 5′- and 5″proton to their respective 4′-protons (related to the γ-bond conformations) and adjacent phosphorus atom (related to the β-bond conformations). The γ- and β-bond conformations and populations were elucidated using the ‘sum rules’58 shown in Table 3. In some cases, these sum rules were slightly modified (see the Supporting Information) so as to provide a better fit between the back-calculated and observed coupling constants. We were concerned that the macrocyclic nature of the cADPR analogues would restrict their conformational freedom, but gratifyingly, the majority of the coupling constants (Tables 1 and 2) showed a regular, albeit small, dependence on temperature. The exception was the R-ring furanose (Table 2), which showed either essentially no variation (J1′2′ and J2′3′ in 4b−4d, and J3′4′ in 4d) or very minor variation (J3′4′ in 4b and 2561


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Figure 6. van’t Hoff plots (ln χS/χN, χγ+/χγt, and χβt/χβ±, as a function of 1000/T) for 2′-dA cADPR (4b, blue ●, blue ○), 7-deaza cADPR (4c, red ■, red □), and 8-Br cADPR (4d, green ▲, green △). Filled and open symbols represent the A- and R-ring portions of each cADPR, respectively. The slope of each line is −ΔHo/R, and the y-intercept is ΔSo/R. For each conformation, the slope, intercept, and correlation coefficient, R2, respectively, are (A) furanose A-rings 4b (blue ●) 0.57, −1.09, 0.98; 4c (red ■) 0.44, −0.48, 0.96; 4d (green ▲) 0.12, 0.26, 0.90. Furanose R-rings: 4b (blue ○) 0.17, 0.56, 0.85; 4c (red □) −0.13, 0.49, 0.92; 4d (green △) −0.05, 1.23, 0.21. (B) A-ring β-bond: 4b (blue ●) 1.26, −1.99, 0.88; 4c (red ■) 0.44, 0.72, 0.92; 4d (green ▲) 0.70, −0.48, 0.95. R-ring β-bond: 4b (blue ○) 1.72, −2.68, 0.99; 4c (red □) 1.17, −0.72, 0.98; 4d (green △) 1.99, −3.75, 0.89. (C) A-ring γ-bond: 4b (blue ●) −0.13, −0.32, 0.98; 4c (red ■) −0.30, 0.54, 0.80; 4d (green ▲) −0.27, 0.95, 0.97. R-ring γ-bond: 4b (blue ○) 1.25, −1.83, 0.88; 4c (red □) 0.65, 0.02, 0.88; 4d (green △) 0.24, 1.71, 0.29. Underlined values indicate a questionable fit.

Table 4. Thermodynamic Parametersa at 298 K for the Furanose Rings, γ-Bonds, and β-Bonds in cADPR A-ring ΔH° 4a 4b 4c 4d Adoc Ado•H+c 4a 4b 4c 4d 4a 4b 4c 4d

N⇆S N⇆S N⇆S N⇆S N⇆S N⇆S γt ⇆ γ+ γt ⇆ γ+ γt ⇆ γ+ γt ⇆ γ+ β∓ ⇆ βt β∓ ⇆ βt β∓ ⇆ βt β∓ ⇆ βt

−0.81 −1.13 −0.88 −0.24 −1.05 −0.05 +0.52 +0.25 +0.60 +0.54 −1.03 −2.50 −0.88 −1.39

ΔS° −1.20 −2.17 −0.95 +0.52

+1.23 −0.64 +1.07 +1.89 +0.80 −3.96 +1.44 −0.96

TΔS° −0.36 −0.65 −0.28 +0.15 −0.62 +0.10 +0.37 −0.19 +0.32 +0.56 +0.24 −1.18 +0.43 −0.29

R-ring ΔG° −0.45 −0.49 −0.60 −0.40 −0.43 −0.15 +0.16 +0.44 +0.29 −0.02 −1.27 −1.32 −1.31 −1.10

pop.b 32:68 31:69 27:73 34:66 33:67 44:56 57:43 68:32 62:38 51:49 10:90 10:90 10:90 13:87

ΔH°

ΔS°

ΔG°

TΔS°

pop.b

+0.04 −0.33 −0.25 +0.10

+2.15 +1.12 +0.97 +2.45

+0.64 +0.33 +0.29 +0.73

−0.61 −0.67 −0.54 −0.63

26:74 24:76 29:71 26:74

−1.81 −2.49 −1.30 −0.47 −4.51 −3.42 −2.33 −3.96

−0.24 −3.64 +0.04 +3.39 −8.72 −5.32 +1.43 −7.46

−0.07 −1.08 +0.01 +1.01d −2.60 −1.58 −0.43 −2.22

−1.74 −1.41 −1.31 −1.48 −1.91 −1.84 −1.91 −1.74

5:95 8:92 10:90 8:92 4:96 4:96 4:96 5:95

ΔHo, TΔSo, and ΔGo in kcal/mol; ΔS in cal/(mol K). Underlined values denote the major contributor to ΔGo. bPopulations recalculated based on the calculated ΔGo; compare to Table 3. Values in bold are discussed in the A Model for cADPR section. cData from ref 60b and c. Ado = adenosine, Ado•H+ = N1-protonated adenosine. dPoor fit in the van’t Hoff plot; R2 = 0.29. a

ring PS ∼ 175°, β = −138°, R-ring PS ∼ 208°, β = +160°), though both γ-bonds in the crystal structure were trans, as well as to our recent preliminary MD simulation of cADPR.40 Despite these surface similarities in cADPR 4a and cADPR analogues 4b−4d, analysis of the thermodynamic parameters reveals some important distinctions relevant to the SAR of the cADPR system (next section). Thermodynamic Parameters. Data for cADPR 4a is from ref 40. Inspired in part by the work of the Chattopadhyaya group, who have performed extensive conformational analysis and thermodynamic studies on monomeric nucleosides and nucleotides as well as DNA and RNA,54a,60 we wished to establish if the same methodology could be applied to cADPR and its analogues. Having determined the conformer populations in 4b−4d, we next sought to determine the thermodynamics (ΔG°, ΔH°, and ΔS°) for each of the conformational equilibria. Qualitatively, the expression ΔG° = ΔH° − TΔS° predicts the conformer whose population

4c) in the couplings. All A-ring furanoses in 4b−4d favored south conformations, generally by a factor of 2:1 with PS = 166° ± 7° and ψSm= 29° ± 2° (somewhat flatter than their free nucleoside counterparts). As we found for cADPR, the A-ring γ-bonds favored the trans conformation (percent γt at 298 K: 4a, 57%; 4b, 68% ;4c, 61%; 4d, only 49%), and all β-bonds were heavily biased (83−92%) toward the βt conformer. For the R-ring furanoses, also similar to that of cADPR, the favored conformers were all south-type (N:S ∼ 1:3) with PS = 214° ± 2°, ψSm= 35° ± 1°, a highly populated (∼90% or more) βt state, but now an almost exclusively (>87−98%) populated γ+ state. The favored conformers of the triethylammonium salts59 of 4a−4d all show a remarkable degree of similarity in both identity and population with an S-type furanose, γt bond, and βt bond for the A-ring and an S-type furanose, γ+ bond, and βt bond for the R-ring. Many of the solution conformations determined herein compare quite well to those of the published57a crystal structure of the free acid of cADPR (A2562


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attributed to differing strengths of the AE in the neutral (weaker) and protonated (stronger) forms. The N6-amino group of cADPR/analogues is protonated at physiological pH,28,61 and yet their A-ring N ⇆ S ΔH° values are hardly diminished relative to those of neutral Ado. It is curious that the A-ring furanoses in 4a−4c ‘resist’ the AE and seem to be thermodynamically more similar to neutral Ado. The ΔH° for the A-ring N ⇆ S equilibria in 8-Br 4d, on the other hand, is significantly diminished (−0.24 kcal/mol) compared to those of 4a−4c, suggesting perhaps a steric interaction between the 8-bromo substituent and the A-ring furanose in the S-form. Interestingly, the 8-Br 4d N ⇆ S equilibria is the only A-ring furanose with a positive TΔS° term, so although the −ΔH° contribution to ΔG° has been reduced, a more disordered S-type conformer apparently compensates. R-Ring Furanoses. The N ⇆ S equilibria in the R-rings of 4a, 4c, and 4d are characterized by small values of ΔH° and positive TΔS° terms that favor S-type conformers, demonstrating that indeed the R-ring N ⇆ S equilibria have a significant entropy component. These R-rings are inherently more disordered than their A-ring counterparts. This is somewhat surprising; in a comprehensive study of nine dideoxy-, deoxy-, and ribo nucleosides, Plavec60a et al. found only one (deoxycytidine) whose ΔHo value did not make a significant contribution to its ΔGo, but now the comparison to Ado•H+ is more appropriate. The positive charge in Ado•H+ led to a 0.72 kcal/mol diminishment of the TΔS° term that originally favored N-type neutral Ado (TΔS° = −0.62 kcal/mol in Ado and +0.10 kcal/mol in Ado•H+). The positive charge in cADPR/analogues is closer to the R-ring than to the A-ring and thus reasonably exerts more influence on the R-ring TΔS° term. Whether the ΔH° value for the N ⇆ S equilibria in the 2′-deoxy-4b and 7-deaza-4c is truly negative, though, will require further study on additional analogues over greater temperature ranges. A van’t Hoff plot lacking a significant slope is extremely sensitive to additional data points, but it is safe to say, based on the general lack of effect of temperature on the Rring N:S ratios, that the ΔH° term is small and that the TΔS° term dominates the N ⇆ S equilibria. Although thermodynamically the R-rings resemble Ado•H+ (comparable ΔH° and TΔS° terms), neither the A- nor R-ring populations shift much from their ∼1:3 N:S ratios, suggesting again some resistance to conformational change. γ-Bonds. In contrast to all the other conformationally mobile rotatable bonds where a distinct preference for one conformer is observed, the A-ring γ-bonds exist as a mixture of the γt and γ+ rotamers with a slight preference for the trans rotamer. The ΔH° and TΔS° terms are more evenly matched with cADPR (1a), 7-deaza analogue 4c, and 8-bromo analogue 4d possessing positive ΔH° values that disfavor the γ+rotamer but positive TΔS° values that favor it. The TΔS° term for 4b, however, is negative, and the γ+ state is disfavored both enthalpically and entropically. Of all the analogues, 4b has the highest populated A-γt state at ∼69%. The situation is different for the R-ring γ-bonds, most of which show a strong (>90%) ΔH°-driven preference for the γ+ rotamer. (Although the Rring γ-bond in cADPR analogue 4d exists almost exclusively as the γ+ rotamer, the poor fit in the van’t Hoff plot (R2 = 0.29) makes it impossible to conclude that it too is favored for enthalpic reasons.) The crystal structure of the free acid of cADPR showed both γ-bonds as trans. Our recent MD simulation40 of cADPR found both γ-bonds to be γ+ and a north-favoring A-ring; though once the A-γ bond was restrained

increases with increasing temperature is the more disordered conformer. As was found for cADPR (1a) itself,40 it was always the minor conformer whose population increased with temperature; thus, the minor conformers (N-type furanoses, β+ or β− bonds, A-ring γ+ bond, R-ring γt or γ−) are more disordered. It thus follows that, as was the case in cADPR,40 the major conformers are favored because of enthalpy. The exception to this trend was the R-ring furanoses in 4a−4d whose N:S ratios were either essentially unchanged, or changed marginally, with increasing temperature. The van’t Hoff equation (vide infra) predicts a temperature-insensitive population such as the R-ring furanoses in 4a−4d to have a zero or small ΔH° for their N ⇆ S equilibrium and thus that the R-ring major conformer (here, S-type) is favored by entropy. The population data from Table 3 formed the basis of a more thorough analysis using van’t Hoff plots (ln K as a function of 1000/T, Figure 6), revealing the precise values of ΔH° and ΔS° for each equilibria; this data is shown in Table 4. The majority (15 of 18) of the plots in Figure 6 showed excellent linearity as reflected in correlation coefficients (R2) of ∼0.90 or higher (Table 3). The moderately good fit (R2 = 0.80) for the A-ring γ-bond in 7-deaza analogue 4c can likely be attributed to the sum rule used to derive the populations, as the coupling constants changed with temperature in a consistent manner. The R-ring furanose and γ-bond in 8-bromo analogue 4d displayed the smallest (and somewhat inconsistent) temperature-dependent variations in coupling constants, resulting in essentially unchanging populations and a flat van’t Hoff plot of poorer fit (R2 = 0.21 and 0.29, respectively). Thus, other than (1) the exceptional R-ring N ⇆ S equilibria, which is indeed driven largely (in 4a and 4d) or significantly (in 4b and 4c) by an entropy term favoring the S-conformer, and (2) the A-ring γt ⇆ γ+ transition, where γt is only slightly favored over γ+ and ΔH° and ΔS° are roughly comparable, the remaining conformational equilibria are generally characterized by a negative ΔH° term that outweighs the TΔS° component by a factor of approximately two. This conf irms that in the majority of cases the higher populated conformer is favored for reasons of enthalpy. Exceptions are noted in the subsequent discussion. A-Ring Furanoses. The N ⇆ S equilibria in the in the Aring furanoses of cADPR (4a), 2′-deoxy 4b, and 7-deaza 4c are characterized by a larger negative ΔH° value (−0.97 ± 0.16 kcal/mol) and a smaller negative TΔS° value (−0.46 ± 0.18 kcal/mol), somewhat smaller than that of adenosine (Ado; ΔH°, TΔS° −1.05, −0.62 kcal/mol). The Chattopadhyaya60a group found that Ado, over the same temperature range as our study, experienced a broader change in equilibrium composition (10% decrease in S-type) with increasing temperature compared to A-ring of cADPR (7% decrease in S-type). One might expect that cADPR, being cyclic, has less conformational freedom than its acyclic adenosine counterpart. One stereoelectronic effect that drives the N ⇆ S equilibrium is the anomeric effect (AE). The AE (donation of an O4′ lone pair into the C1′-N9 antibonding σ* orbital) increases with increasing electron demand of the nucleobase, or with protonation of the nucleobase. The AE is generally more favorable in N-type conformations.60a−d In two studies60b,c of the effect of pH on the N ⇆ S equilibrium, the same group found a dramatic decrease in the amount of S-type (or increase in N-type) conformer at pH values at or below the pKa of the adenine N1 atom. The N ⇆ S ΔH° for protonated adenosine (Ado•H+) had dropped to a mere −0.05 kcal/mol, which they 2563


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Figure 7. A model for cADPR. (a) Schematic representation of 4a−4d based on our NMR data and the cADPR crystal structure/MD simulation values for the glycosidic bonds A-χ and R-χ (b) and (c) HyperChem (HC) minimized structures (Amber99). The A-ring is in the γt (b) and γ+ (c) conformation. The A-χ bond was defined using standard accepted rules, but the R-χ bond was defined as O4′R−C1′−N1−C2 so that it will have the same magnitude and sign as the A-ring.

factor. The TΔS° term for the A-ring β-bonds is somewhat variable in sign, but consistently negative for the R-ring βbonds. A Model for cADPR. On the basis of the above discussion, we propose the following model (Figure 7) for cADPR that accommodates our data, potentially explaining some of the SAR for cADPR and providing direction for future efforts. What parts of the story should the model include? It is clear from the discussion above that the conformations of cADPR 4a and analogues 4b−4d are overall quite similar, meaning activity cannot be explained on the basis of divergent structures. Changes to the preferred conformations of the A-ring β-bond, (A-β), R-β, and R-γ bonds will incur a substantial enthalpy penalty. In a sense, these subunits could be considered to be thermodynamically ‘restrained’. Establishing that these conformers are kinetically restrained due to high barriers to rotation would require either line shape analysis or substantial modeling efforts well beyond the scope of the present study. The furanose rings are certainly flexible, but there is no NMRbased evidence that the structural modifications (2′-deoxy-, 8bromo, 7-deaza) had any impact on the preferred furanose conformations (A-ring PS ∼ 168°; R-ring PS ∼ 215°). If cADPR is indeed ‘conformationally agnostic’ as to the orientation of its furanose rings, this would seem to leave the A-ring γ-bond and the two N-glycosidic bonds, A-χ and R-χ (defined in the legend to Figure 7), as the only sites capable of variability. In our model (Figure 7), the nucleobase is positioned so that A-χ is ∼60° and R-χ is ∼10°, approximating the values from the crystal structure57a and our MD simulation,40 with the adenine C2−N3−C4 edge pointing toward the area between A2′ and A5″ hydrogens. The phosphate backbone is behind the plane of the page. One attractive feature of this model is that it immediately provides a rationale for the ‘unusually high’ 1H chemical shift of A2′, as it is edge on to the adenine and deshielded by the aromatic ring current of the nucleobase.

to trans, a south A-ring was favored. We suspect this may be due to the bsc0 refinement of the AMBER force field, which was designed to minimize the γt state.62 Thus, three different experiments, this NMR study (R-γ+ and a mixture of A-γt and A-γ+), our recent MD simulation (R-γ+ and A-γ+ when unrestrained), and the crystal structure (of the free acid; R-γt and A-γt), give three different outcomes for the γ-bonds. Clearly the A-γ bond, finely balanced between its two observed orientations, is sensitive to its environment. Analogue 4b is also unusual in that its R-γ bond shows much larger favorable −ΔH° and much larger unfavorable −TΔS° values for the γ+ state compared to those of the other three compounds, though the ΔG° values are comparable. The removal of the A-ring 2′OH somehow greatly enthalpically favors the remote R-γ+ state while simultaneously penalizing it entropically. It is to be expected that there will be differences in the conformational preferences of the γ-bonds in cADPR compared to those of the free nucleosides. In the Chattopadhyaya60a group study, they noted that without exception the population of the γ+ state decreased with increasing temperature regardless of whether the γ+ state was the favored rotamer. The conclusion to be drawn is that in free nucleosides the γt state is the more disordered rotamer. Only in the R-ring γ-bonds of 4a−4d was this trend observed; the A-γ+ state became more populated with increasing temperature. Generally in 4a−4d we can say the more disordered rotamers are the A-γ+ and R-γt. The adenine base is necessarily in the syn orientation in cADPR and the analogues. Given that in free nucleosides a syn conformation generally decreases the γ+ population,48,58a it is remarkable that the A-γ+ state in cADPR is populated to the extent that it is. β-Bonds. The βt is favored for enthalpy reasons and is highly populated in both rings of cADPR 4a and analogues 4b− 4d. The TΔS° term is smaller than the ΔH° term by an approximate factor of 2, and the favorable ΔH° for the R-ring β-bonds is greater than that of the A-ring β-bonds by the same 2564


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equipotent with cADPR, perhaps because the nucleobase, free of its R-ring, is not forced to adopt the χ angles that promote antagonism. Of course, the conformational behavior of a ligand in solution, even if correctly modeled, is never guaranteed to translate to the ligand in the bound state. The penalty cADPR or an analogue would have to pay to bind in, for example, the (solution-state) less-favored A-γ+ conformation (

Variable-Temperature NMR Spectroscopy, Conformational Analysis

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