Deciphering the conformation of C-linked α -D-mannopyranosides and their application toward the synthesis of low nanomolar E. coli FimH ligands

C -Allyl  –D-mannopyranosides were prepared via a variety of routes to determine an optimal route to the  - anomers. The relative conformational energies of the key intermediate was evaluated by molecular modeling which showed the conventional 4 C 1 chair conformation to be the lowest energy conformer. This finding was also confirmed by NMR and X-ray crystallography. The perbenzoylated C-allyl mannoside was also converted into 1,1’-biphenyl analogues using a palladium-catalyzed Heck reaction. Two of the resulting minor reaction products were co-crystallized with the uropathogenic E. coli FimH. Alternatively, the K D of the major and expected Heck product was in the low nM range as measured by SPR. Crystal data showed that the C-linked derivatives efficiently bind in the FimH binding cavity near the so-called hydrophobic tyrosine gate.


Results and Discussion
In conventional drug design, O-linked glycomimetics should be largely avoided due to their propensity to readily hydrolyze in in vivo settings.To avoid this problem, C-linked α-D-mannopyranosides attached to alkene chains were investigated. 1,11Diversely protected C-allyl α-D-mannopyranosides are useful intermediates for the synthesis of this important family of glycomimetics.Conventional routes for their syntheses are illustrated in Scheme 1.So far, in its most α-stereoselective approach, it was obtained from the known perbenzylated methyl α-D-mannopyranoside 1, 39 which under Sakurai reaction 39,40 (BF 3 •OEt 2 , allyltrimethylsilane) provided 2 in more than 95% of its α-anomer (Scheme 1).Unfortunately, benzyl protecting groups have some drawbacks for their forthcoming palladium-catalyzed Heck coupling and hydrogenolysis deprotection. 11Alternatively, Birch reduction of 2 and acetylation affords the more versatile peracetylated analogue 3α in good overall yield, albeit in three consecutive steps.Disappointingly, application of the Sakurai conditions, directly on peracetylated mannose 6α,β afforded an intractable mixture of 3α and 3β. 41This result prompted us to attempt the reaction on a anomeric mixture of the known perbenzoylated mannopyranose 4α/4β. 42cheme 1. Synthesis of C-allyl α-D-mannopyranosides under Sakurai condition with different protecting groups (see Table 1).
Delightfully, in contrast to the peracetylated analogue 6, 41 the Sakurai products 5α and 5β resulting from the perbenzoylated mixture 4α/4β were separable, but with reduced α-stereoselectivity (7:1) when compared to the perbenzylated derivative 1 (15α:1β).As anticipated, during the course of this transformation, we also observed that 4α reacted faster than its β-anomer 4β.Given that 4α and 4β were also separable, 42 we repeated the reaction on each separated isomer: the 4α anomer was completely converted to 5α/5β (1.7:1) within 2 h while the 4β anomer provided 5α/5β more slowly but with an improved 4:1 anomeric stereoselectivity.The fact that the above three conditions gave different anomeric stereoselectivities was not surprising given the individual propensity of the precursors (α vs β) to react with the Lewis acid competitively.
To simplify access to a suitable and versatile starting material, we attempted the Sakurai reaction directly from the commercially available methyl α-D-mannopyranoside 7 using previously described conditions (BTSFA, AllylTMS, TMSOTf, MeCN, 0 °C,16 h). 43,44Although both the yield and the anomeric diastereoselectivity toward unprotected 8α,β in one single step from 7 was acceptable, the anomeric mixture was not readily separable.The comparative data for the above sets of transformations are illustrated in Table 1.With this information in hand and for practical reasons, it was decided to pursue our goals using the perbenzoylated precursors 4α,β.
ARKAT USA, Inc Table 1.Synthesis and diastereoselectivity of C-allyl α-D-mannopyranosides using different methods a Ratios were obtained from the crude 1 H NMR data.
As stated above, [33][34][35][36][37][38] there are several papers indicating that C-linked α-D-mannopyranosides can exist in conformations other than those observed for the corresponding O-linked derivatives, normally seen as 4 C 1 conformers.Obviously, changing bond lengths, bond angles, torsion angles, and conformations would have a detrimental effect upon binding of antagonists to E. coli FimH.Given the importance of our key precursor, Callyl α-D-mannopyranoside 8α, we measured the energy levels of its various conformations in the gas phase.The geometry optimizations of the 4 C 1 , 1 C 4 , 2 S 0 and 0 S 2 conformers were performed using density functional theory (DFT) with the hybrid functional B3LYP and 6-31G* basis set.For that purpose, the Firefly 45 and GaussSum 46 computer programs were used.Figure 1 clearly showed that the 4 C 1 conformer was more stable than its 1 C 4 conformational isomer by at least 8.10 kcal/mol.In addition, the respective skew boats 2 S 0 and 0 S 2 conformers were 11.72 and 16.42 kcal/mol, respectively higher than that of the 4 C 1 conformer.These results agreed with the solution phase data obtained from high field 1 H NMR (900 MHz) 11 coupling constants and nOe experiments (see below).Solid phase X-ray data also strongly supported these results.2).In addition to the expected correlation between H 1 and H 1'a and H 1'b , we observed a strong nOe between H 1'b and H 3 and H 5 , further confirming that even in the fully protected forms the C-linked mannopyranoside existed in the suitable 4 C 1 conformation.Importantly, the coupling constants for the H 4 signal at δ H 6.02 (dd, 1H, J 3,4 = J 4,5 = 9 Hz, H-4) clearly indicated a trans-diaxial relationship between H 4 and both H 3 and H 5 .Moreover, a crystal structure of 5α showed it existed in the same 4 C 1 conformation.Overall, the data on both fully protected as well as unprotected analogues supported that this family of C-linked mannopyranosides existed in the desired 4 C 1 conformation (Figure 3).Having secured the accurate 4 C 1 conformation of these valuable anomeric precursors (4α and 5α) in the gas phase, solution, and solid-state (X-ray) (Figure 3), we pursued our goals toward potent E. coli FimH antagonists capable of adequately placing hydrophobic pharmacophores within the FimH tyrosine gate (Tyr48/Tyr137).1][12][13][14][15] To this end, we opted for an elongation of the aglycone side chain using palladium-catalyzed Heck cross coupling between allylic 5α and aryl iodide such as 4-iodo-1,1'-biphenyl.
Somewhat surprisingly, when allylic α-D-mannopyranoside 5α was treated under our optimized conditions (4-iodo-1,1'-biphenyl, Pd(OAc) 2 , TBAB, NaHCO 3 , DMF, 85 °C, 12 h), 1,11 compound 9 was obtained as a major product (93%) together with traces amount of stereo-and regio-isomers 11 and 13 (Scheme 2).The mixture of side products (11 and 13) was clearly visible from the 1 H NMR spectra of the crude reaction mixture.Their formation has been previously explained when considering the reaction mechanism of the Heck reaction. 1fter isolation of pure 9 (Scheme 2) and 11/13 mixture, they were separately submitted to de-O-benzoylation (1 M NH 3 , MeOH, r.t., 36 h) to afford pure 10 (Figure 4) and an intractable mixture of 12/14.These two side products could not be readily purified and fully characterized.Nevertheless, their mixture was submitted to co-crystallization with E. coli FimH together with the major product.Even though we could not obtain X-ray data from the complex of FimH and major product 10, both side products were individually and successfully co-crystallized with the FimH 47 in the open and closed tyrosine gate, respectively, thus establishing their exact structures (Figure 5).A high field 1 H NMR spectrum of deprotected 10 (major product) was fully assigned (Figure 4). next undertook affinity measurements between FimH and compound 10 by surface plasmon resonance (SPR). 11Compound 10, having its second phenyl group in the para-position, was a potent ligand (K d 17 nM) but it was less potent than the recently identified ortho-substituted analogue 15 11 having an almost 3fold affinity enhancement (K d 6.9 nM).As opposed to the two side products 12 and 14, both compounds could not be co-crystallized with FimH.However, based on previous work, 11 docking of its lowest energy conformer, obtained through modeling and high field NMR spectra, with the FimH adhesin indicated that the orthosubstituted phenyl ring of 15 was able to interact with an additional amino acid isoleucine-13 (Ile13), located in the clamp loop, thus opening the route for further lead improvement.

Conclusions
Several approaches to an important key intermediate for the synthesis of families of C-linked α-Dmannopyranosides have been presented.Although some were more α-stereoselective than others, we opted for the use of perbenzoylated precursors for practical reasons.Using optimized Sakurai allylation, good yields of the C-allylated glycosyl derivatives were obtained.We demonstrated that these existed in the required 4 C 1 conformation, in the gas phase, solutions, and in the solid-state, as requested for efficient binding interactions with one of the essential E.coli virulence factor FimH.We then explored the formation of the typical, as well as of side products, obtained during the course of our palladium-catalyzed Heck cross-coupling reaction with a key aryl iodide (4-iodo-1,1'-biphenyl).Even though, an X-ray complex between the FimH and the major and expected Heck product could not be acquired, data were obtained from the two minor side products.The findings reveal new opportunities for the design of drug candidates against FimH and offer previously unexplored binding interactions within the active site of the protein.

Experimental Section
General.Reactions were carried out under Nitrogen using commercially available ACS grade solvents which were stored over 4 Ǻ molecular sieves.Solutions in organic solvents were dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure.Reagents were obtained from Sigma Aldrich Canada LTD and used without further purification.Compounds 1 and 6 are commercially available, 2, 39 and 8 43 were prepared according to published data and their physical data were in agreement with the proposed structures.Melting points were measured on a Fisher Jones apparatus and are uncorrected.Optical rotations were measured with a JASCO P-1010 polarimeter.Reactions were monitored by thin-layer chromatography using silica gel 60 F 254 coated plates (E.Merck). 1 H and 13 C NMR spectra were recorded at 300 or 600 MHz and 75 or 150 MHz, respectively, with Varian Gemini 2000 (300 MHz) and Varian Inova (600 MHz) spectrometers.All NMR spectra were measured at 25 °C in indicated deuterated solvents.Proton and carbon chemical shifts (δ) are reported in ppm relative to the chemical shift of residual CHCl 3 , which was set at 7.28 ppm ( 1 H) and 77.16 ppm ( 13 C).Coupling constants (J) are reported in Hertz (Hz), and the following abbreviations are used for peak multiplicities: singlet (s), doublet (d), doublet of doublets (dd), doublet of doublet with equal coupling constants (t ap ), triplet (t), multiplet (m).Assignments were made using COSY (COrrelated SpectroscopY) and HSQC (Heteronuclear Single Quantum Coherence) experiments.High-resolution mass spectra (HRMS) were measured with a LC-MS-TOF (Liquid Chromatography Mass Spectrometry Time of Flight) instrument (Agilent Technologies) in positive and/or negative electrospray mode by the analytical platform of UQAM.Compounds 11-14 were only obtained in trace amounts that could not be fully characterized except through the X-ray data of their unprotected FimH complexes (12, 14) (Figure 5).The X-ray data were deposited as PDB accession no.5AAL and 5AAP, respectively.

Figure 6 .
Figure 6.Comparison between para-substituted biphenyl 10 (K d 17 nM) and ortho-substituted biphenyl 15 (K d 6.9 nM); docking of the lowest energy conformer of 15 in the active site of FimH indicated additional hydrophobic interactions with Ile13 (centroid with the ortho-phenyl at 4.8 Å).