Solution conformations of three small cluster oligosaccharide mimetics (SCOM) from D -glucosamine, D -altrosamine, and (-)-quinic acid

Three small cluster oligosaccharide mimetics were synthesized, and their CDCl 3 solution conformations were determined with NMR experiments. In this paper, we confirmed conformational similarities in rings A and B in all three compounds, and we provide the overall solution geometries. In all cases, an unusual cyclohexane twist-boat conformation with long-range 4 J -(W)-coupling was observed. The origin of the bias towards the twist-boat was attributed primarily to the fused ring system between the cyclohexane ring and the dioxacyclopentane (acetal) including hydrogen bonding. Temperature-annealed molecular dynamics simulations complemented the NMR experiments and supported the overall assignment.


Introduction
Quinic acid (1, Figure 1) 1 and its derivative shikimic acid (2, Figure 1) [2][3][4] (from Illicium japonica and floridanum) are key intermediates during the biochemical synthesis of essential aromatic amino acids, such as L-tryptophan, L-phenylalanine, and L-tyrosine in plants, bacteria, and fungi as well as building blocks for folic acid, alkaloids, and vitamins.The quinic acid/shikimic acid pathway does not exist in mammals, which has led to extensive research activities geared towards the development of suitable antibiotic therapeutics. 5,6Especially the conversion of 3-dehydroquinic acid (DHQ) to 3-dehydroshikimic acid (DHS) has been recognized as an effective target for inhibition in plants.Several commercial herbicides selectively inhibit the DHQ-DHS conversion and prevent growth of weeds.However, disruption of the DHQ-DHS pathway by shikimic acid derivatives (3, Figure 1) has also recently been shown to cause therapeutic inhibition of Mycobacterium tuberculosis. 7(1), shikimic acid (2).Compounds 3, 7 4 (Tamiflu TM , Roche) 8 , and 5 (Relenza TM , Glaxo-Smith) 9,10 show anti-influenza activity.Compound 6 has been previously synthesized in our laboratory. 13rivatives of shikimic acid have been used to inhibit other biochemical pathways for example the infectious pathway of the influenza virus (common flu).Potent anti-influenza A+B effect has been observed for derivatives of shikimic acid 7 inhibiting viral neuraminidase.The widely-prescribed anti-flu medication Tamiflu TM (Oseltamivir, Roche, 4, Figure 1) 8 is not only effective in treating common flu strains, but it has also been used successfully in the treatment of the bird (avian) flu in laboratory animals.Contrary to animal experiments, Tamiflu's TM effectiveness in humans infected with the H5N1 flu strain is still controversial because only a few studies with limited statistical value have been conducted, and the understanding of the drug's action against the virus is only rudimentary.Tamiflu TM is thought to act as influenza virus neuraminidase inhibitor with the possibility of alteration of virus particle aggregation and release.The less well-known Relenza TM (Zanamivir, GlaxoSmithKline, 5, Figure 1) 9,10 is also an effective inhibitor of common flu strains by the same mechanism.The problem of a sufficient supply of these two drugs in case of an influenza pandemic and the necessity for a continuous search for new structural analogs has been discussed in the literature. 11,12herefore, all analogs of shikimic and quinic acid such as compound 6 (Figure 1) 13 and analogs thereof have to be considered potentially valuable bioactive compounds, and their exact structural characterization is of great importance.In this paper, we discuss the NMRspectroscopic properties and solution conformation of the quinic acid derivative 6 and two new structural analogs thereof (16 and 17).

Synthesis of quinic amides and NMR analysis
The αand β-anomers of compound 6 (Figure 1) had previously been synthesized and characterized by NMR in our laboratory (Table 1). 13Commercially available D-(-)-quinic acid was concurrently dehydrated and iso-propylidenated in acetone in the presence of p-toluenesulfonic acid to yield (1R,3R,4R,5R)-1,3-dihydroxy-4,5-O-isopropylidene-1-carboxy cyclohexane 1,3-lactone (quinic acid lactone 7). 14The lactone was subsequently opened nucleophilically with benzyl-2-amino-4,6-O-benzylidene-2-deoxy-α/β-D-glucopyranoside to yield 6a and 6b.We have previously shown that the cyclohexane ring in quinamide such as compound 6 (Figure 1) exists to a significant extent in the twist-boat conformation in CDCl 3 .This was concluded from the existence of a long-range 4 J-coupling constant between H2b B and H4 B (W-coupling) as well as small gauche couplings of 2.7 Hz for both H5 B /H6a B and H5 B /H6bB indicating that the C-H5 B single bond was dissecting the H6a B -C-H6b B bond angle.Our assignment of ring A was supported by large coupling constants between H2 A /H3 A , H3 A /H4 A , and H4 A /H5 A consistent with the trans-fused dioxa-decaline system. 13o investigate whether the twist-boat conformation in 6 is the result of the large glycosyl substituent or merely a result of the fusion between the six-membered cyclohexane ring and the five-membered isopropylidene acetal ring, we synthesized compound 8 from quinic lactone 7 through ring opening in methanolic ammonia at 100 ºC for 2 d (Scheme 1).Compound 8 was obtained in 100 % yield with NMR purity after evaporation of the solvent.Scheme 2. Synthesis of compounds 16 and 17.The preparation of 14 and 15 has been reported in the literature. 15e synthesis of 16 and 17 from α-benzyl-3-amino-3-deoxy-4,6-O-benzylidene-Dglucopyranoside 14 and α-benzyl-2-amino-2-deoxy-4,6-O-benzylidene-D-altropyranoside 15, respectively, is shown in Scheme 2. Compounds 14 and 15 were obtained after treatment of D-glucose 9 with benzyl alcohol in the presence of toluenesulfonic acid. 15The benzyl glucoside 10 was 4,6-O-benzylidenated (11) with benzaldehyde in the presence of ZnCl 2 and was subsequently converted into the 2,3-di-O-mesylate 12. Compound 12, upon treatment with NaOMe in CH 2 Cl 2 , yielded the 2,3-anhydro-allopyranoside 13, which was opened under elevated pressure in MeOH saturated with NH 3 .][18] ARKAT USA, Inc.The quinic acid lactone was opened nucleophilically by both 14 and 15 with minimal solvent to yield 16 (65% yield) and 17 (60% yield) as shown in Scheme 3. The mixture of 16 and 17 was observed to crystallize from N,N-dimethylacetamide during the reaction, which was attributed primarily to the high melting point of 16 compared to 17 (218-220 ºC and 105-107 ºC, respectively).

Overall structure determination and NOE-difference experiments
The cyclohexane ring B of 6 had been shown 13 by analysis of 3 J-values to exist to a significant extent in an unusual twist-boat solution conformation in CDCl 3 in both anomers (Table 1).However, the exact overall solution conformation of the whole molecule had not been determined and only minimal molecular modeling data were discussed.
We determined NOE difference spectra for several proton pairs in 6a.To establish a set of reference buildup curves, we chose the benzylidene CH-resonance with trans-annular spinexchange to H-4 and H-6a in ring A. The benzylidene CH-proton is located in a trans-dioxadecaline system, in which minimal conformational flexibility can be assumed.The mixing time was varied, and the NOE enhancements were integrated relative to the irradiated proton, and the values were plotted against τ mix to generate buildup curves.The NOE buildup followed a standard saturation profile and an initial slope = 0.035*10 -3 s -1 was determined for CHPh/H-6a.This slope served as a reference for all other NOE buildup experiments.
~3.8 (nr) ~3.8 (nr) H4 B 4.12 (ddd~dd) 4 J 4,2b <1 3 J 4,3 =3.3 3 J 4,5 =6.6 4.12 (ddd~dd) 4 J 4,2b <1 3 J 4,3 =3.3 3 J 4,5 =6.3 H5 B 4.54 (dt) 3 J 5,4 =6.3 3 J 5,6a =2.7 3 J 5,6b =2.7 4.53 (dt) 3 J 5,4 =6.9 3 J 5,6a =3.0 3 J 5,6b =3.0 H6a B 2.14 (dd) 3 J 6a,5 =2.1 2 J 6a,6b =15. 3  2.02 (dd) 3 J 6a,5 =3.6 2 J 6a,6b =15.3 H6b B 2.42 (dd) 3 J 6b,5 =2.4 2 J 6b,6a =15.6  2.37 (dd) 3 J 6b,5 =2.7 2 J 6b,6a =15.9 OH1 B 3.53 (s) Not ident.OH3 B 4.39 (d) 3  The cyclohexane ring of 8 showed significant twist-boat conformation (Table 2).This was evident from the two small coupling constants (3.0 Hz) between H5 and H6a/b on the one side of the ring and from the small and medium J-value (2.7 Hz and 5.4 Hz) between H3 and H2a/b on the other side of the ring.In addition, a four-bond coupling of 1.8 Hz was observed between H4 and H2b.Without any substituent attached to the amide group, we concluded that the twist-boat conformation of derivatives such as 8 in CDCl 3 is caused by the fusion between the cyclohexane ring and the five-membered acetal ring.The twist-boat is additionally stabilized by a hydrogen bond between the amide group and OH3, which was inferred from NOE difference experiments (not shown) and temperature annealed molecular dynamics simulations (Scheme 1).Compound 16 with α-D-gluco-configuration displayed consistent coupling constants for a 4 C 1 chair conformation in ring A (Table 2).The NMR signal of the anomeric proton showed a small gauche coupling of 3.6 Hz to H2, which in turn showed trans-diaxial coupling (9.9 Hz) to H3 and the same coupling to the OH-proton.Protons H3, H4, and H5 were characterized by large trans-diaxial coupling constants.Ring B displayed coupling features of a twist-boat conformation very similar to 8. Also here, the four-bond coupling between H2bB and H4B was very distinct with 1.8 Hz as well as the bisecting gauche bond angle of H5 and the two geminal H6 protons (3.3 and 2.4 Hz).The altro-configuration of ring A in compound 17 manifested itself through small or not resolved coupling constants between H1 A , H2 A , and H3 A , followed by a double doublet for H4 A with coupling constants of 3 J 4,3 = 3.0 Hz and 3 J 4,5 = 9.9 Hz (Figure 3, Table 2).Even though ring B represents a rather bulky substituent in axial position, it did not seem to affect the chair conformation in ring A to any significant extent.The coupling constants observed for the protons in ring B were very similar to those observed for both 8 and 16, which implied the same twistboat conformation.Compounds 16 and 17 were analyzed with NOE difference experiments in the same fashion.The fitting of the results to a structure determined from temperature annealed molecular dynamics simulation is described below.

Molecular dynamics simulations and geometry refinement with NOE data
After the experimental determination of NOE buildup curves for several proton pairs, we turned our attention to the computational description of both anomers of 6.The molecular dynamics simulation of 6a without dihedral angle restrictions and with temperature annealing from 600 K to 0 K resulted in the plot of selected inter-proton distances in Figure 4a.Uniform convergence of the proton distances to final 0 K values was observed with ring A in chair conformation and ring B in twist-boat conformation consistent with the J-coupling values.All temperatureaveraged distances fall within the limit for reasonably strong NOE-signals (i.e.< 4Å) as experimentally observed.The final lowest energy structure is shown in Figure 4b with selected inter-proton distances and averaged values in parentheses.The averaged distances were calculated from the first 30 ps of the trajectory at thermal equilibrium.Ring A and B were conformationally biased relative to each other through two hydrogen bonds at the amide carbonyl (OH3 A and OH3 B ). Table 3 shows the full list of inter-proton distances.
The temperature-averaged distance between CHPh and H6a A was 2.47 Å and was used as a reference distance for other inter-proton distances.Inter-proton distances can be approximated by their inverse sixth-power relationship to the rate of the experimentally observed NOE buildup (see experimental).Although this relationship is not free from errors, it frequently allows reasonable approximation of inter-proton distances with errors smaller than 10%.This equation can be solved for the distance between any H x /H y proton pair in 6a if their NOE buildup curve can be determined and compared to the slope of the reference pair CHPh/H6a A and the respective distance between CHPh/H6a A from the molecular dynamics simulation.
Several inter-proton distances in 6a relative to the CHPh/H6a A and their respective errors are shown in Table 1.In general, similar values or smaller errors were observed when the temperature-averaged distances from the molecular dynamics trajectory were used to match the NOEDIF data.However, several values deviated significantly more than 10% from the predicted values.For example, the distance determined for the H1 A /ortho-H arom proton pair displayed a 17% error.Rotation of the phenyl ring causes significantly greater time-averaged distance than the 0 K structure suggests.In addition, ring current effects from the aromatic π-system can be expected to affect the efficiency of spatial spin density transfer and increase the error of measurement.The % error was particularly significant in the case for proton pairs that involved hydroxyl protons.Surprisingly, long-range spin density exchange between all hydroxyl protons was observed despite distances greater than 6Å.This observation was explained with partial hydration of the compound and a hydrogen bond network connecting remote OH-groups.In such water-bridged systems, the r -6 -relationship for NOEs does not apply strictly any more and larger distance errors can be expected.To find support for this explanation, the molecule should be computationally modeled in a water-box with molecular dynamics to reveal the existence of such water bridges.Such calculation was not attempted because of lack of computing power, and the structure shown in Figure 4b was assumed to resemble the CDCl 3 solution structure adequately.The investigation of the solution conformation of the β-anomer 6b was carried out in analogous fashion, and inter-proton distances are summarized in Table 1.Temperature-annealed molecular dynamics resulted in a smooth convergence to a 0 K structure and revealed a pair of hydrogen bonds across the amide bond including OH3 A and OH3 B (Figure 5a).The proposed conformation of 6b, as shown in Figure 5b The observed NOE enhancements between both H6b B /NH and H2b B /NH (not shown) were fully consistent with the relative positions of ring A and B. The hydrogen bonding network across the amide bond appears to be only minimally influenced by the anomeric configurations in 6a and 6b and the steric demand of the benzyl group.Unlike 6a, the hydroxyl groups in 6b did not display any long-range NOEs, which implies a stronger intra-molecular H-bond network that is affected less by water present in CDCl 3 .Traces of water were present in both NMR samples at the time of analysis (signal at 1.5 ppm in CDCl 3 ).For 6b, only a weak negative NOE was observed between the hydroxyl resonances at 2.9 and 3.0 ppm. 13These are presumably OH3 A and OH3 B hydrogen-bonded to the amide carbonyl oxygen; however, unequivocal identification was not possible.
The lowest-energy conformation of the quinamide cluster 16 after temperature annealing was fully consistent with the experimental NMR data and is shown in Figure 6a.The equatorial position of OH2 in ring A allowed the formation of a hydrogen bond towards the amide carbonyl oxygen in addition to the hydrogen bond between OH3 B and the same carbonyl.The amide NHgroup was hydrogen bonded to OH1 B , which in turn formed a weak hydrogen bond towards the isopropylidene acetal oxygen at C5 B .The so-obtained twist-boat conformation in ring B was identical to that found in the model compound 8.
Unlike compound 16, the quinic carboxamide 17 displayed the amide linkage at C2 A and the OH-group at C3 A in diaxial positions unsuitable for mutual hydrogen bonding.The conformation of the amide linkage was supported by an experimental NOE at the amide NH-proton after irradiation of H4 A (inset in Figure 6b).between NH, H4 A , and OH1 B .The NOE difference spectrum after irradiation of H4 A is shown as inset.
In this investigation, the solution structures of three quinic amide derivatives in CDCl 3 were solved.For all three carboxamides, NOE signal buildup within rings A/B and across the amide bond was found fully supported by accompanying molecular dynamics calculations in vacuo.Unusually strong spin density exchange between all OH-protons and remote ring protons was interpreted as evidence for hydration in the compounds 6, 16, and 17.In the β-anomer of 6, spin exchange between the hydroxyl groups was less pronounced.The cyclohexane twist-boat conformation was shown to be the result of the cyclic five-membered acetal and trans-annular hydrogen bonding.The twist-boat conformation was found to exist even in the simple unsubstituted quinic carboxamide 8.We are currently synthesizing analogs with different stereochemistries for structural comparison and for evaluation of biological activity.

Experimental Section
General Procedures.NOEDIF experiments.Approximately 10 mg of the compound was dissolved in CDCl 3 and was subjected to three freeze-thaw cycles in liquid nitrogen with evacuation/N 2 -flushing on a Schlenk line while frozen.NOEDIF spectra were recorded nonspinning for 512 transients at room temperature on a 300 MHz Varian Mercury system.An array of eight different mixing times between 0.5*10 -3 s and 3*10 -3 s yielded spectra whose NOE signals were integrated relative to the negative irradiated proton signal, and the resulting integrated NOE signal intensity values were plotted against the mixing time.With the first three or four data points, a linear buildup curve was constructed for the respective proton pair.The slope from the benzylidene/H6a A proton pair (slope = 0.035*10 -3 s -1 and 0.023*10 -3 s -1 for 6a and 6b, respectively) in the rigid ring A was used as a reference slope.All other experimental buildup slopes were brought in relation to the reference slope and the respective inter-proton distances from molecular modeling by equation (1).

Molecular dynamics
The molecule was constructed in HyperChem 5.1.Ring B was constructed in the twist-boat conformation, and two dihedral angles were restrained to maintain the twist-boat conformation during the dynamics simulation.The compound was initially constructed with protons oriented to match NOE information.The structure was energy minimized with molecular mechanics (MM+ force field) followed by the semi-empirical AM1-method.The molecule was subsequently modeled with molecular dynamics (T init = 280 K, T simul = 600 K, T final = 0 K, temperature steps = 10 K, heat time = 0.5 ps, run time = 100 ps, cool time = 0.5 ps, step size = 0.1 fs).Data from dihedral angles or inter-proton distances were collected in a snapshot file at every 100 time steps, i.e. every 10 fs.

Figure 2 .
Figure 2. a) Selected NOEDIF spectrum of 6a after irradiation of H1 A at room temperature; b) NOEDIF buildup curves from arrayed mixing times.

Figure 4 .
Figure 4. (a) Selected inter-proton distances from the 180 ps trajectory of 6a during temperature annealing from 600 K to 0 K; (b) Lowest energy structure for 6a with selected 0 K distances.High-temperature average distances from the first 30 ps of the trajectory are shown in parentheses.

Figure 5 .
Figure 5. (a) Inter-proton distance for OH3 A and OH3 B hydrogen-bonded, respectively, to the amide carbonyl from the 150 ps trajectory of 6b during temperature annealing from 600 K to 0 K; (b) Lowest energy structure for 6b with selected 0 K distances.High-temperature average distances from the first 30 ps of the trajectory are shown in parentheses.

Figure 6 .
Figure 6.Temperature annealed lowest energy structures in vacuo.a) Compound 16.The conformation of the amide linkage was confirmed through an NOE between NH, H3 A and OH1 B ; b) 17.The conformation of the amide linkage in 17 was confirmed through a strong NOE

7 8 Scheme 1. Synthesis of compound 8. The most stable conformation in vacuo for 8 is
shown which was consistent with all observed J-values and NOEs (temperature annealed molecular dynamics simulations, AM1, 600 K → 0 K).