GMR Literature Highlights 03-10-2013

Samuele Staderini

Molecular recognition of carbohydrates

• Key role in biological processes • Dedicated classes of biomolecules (lectins,

antibodies…) govern interaction between living cells and other cells or pathogens

• Specific recognition of epitopes of saccharidic chains (usually terminal mono- or oligosaccharides)

Development of articial biomimetic receptors to:1. Mimic the natural ones2. Understand recognition

mechanism3. Develop new therapeutic and

diagnostic toolsThe sugar code: fundamentals of glycosciences (Ed.: H.-J.Gabius), Wiley.VCH, Weinheim, 2009Carbohydrates in chemistry and biology, Part I, Vol.2 (Eds.: B.Ernst, G.W. Hart, P. Sinaÿ), Wiley-VCH, Weinheim, 2000

Mannosides are recognized by several lectins and are involved in infections caused by high-risk pathogenes like:• Yeasts (Candida)• Bacteria (Tubercolosis)• Viruses (HIV and Hepatitis HCV)

Recognition of terminal oligomannosides on pathogen surface enhance potent antiviral activity to:• Lectins (cyanovirin-N, microvirin, actinovirin)• Andibodies (2G12, PGT128)• Natural non-peptidic molecules (benanomicin A,

pradimicin A)

Mono-mannosides and Di-mannosides

Carbohydrates in chemistry and biology, Part II, Vol.4 (Eds.: B.Ernst, G.W. Hart, P. Sinaÿ), Wiley-VCH, Weinheim, 2000, pp. 533-609

Di-mannosides: a relevant example (HIV infection)

Oligomannose glycanOther sugarsTerminal α-

dimannoside motif: the smaller

fragment required for

lectin recognitionThe binding of the DC-SIGN lectin of immune system

dendritic cells to the viral glycoprotein gp120 on the HIV envelope triggers the infection of human cells by the virus

R.A.Dwek, Nature, 2007, 446, 1038-1045

Mannosides synthetic receptors

C.Nativi, O.Francesconi, G.Gabrielli, A.Vacca, S.Roelens, Chem. Eur. J., 2011, 17, 4814-4820

Aminopyrrolic tripodal structures

High affinity and selectivity for

• Polar solvent, but not water• Octyl chain ensure solubility in polar

organic solvents• Different recognition properties by two

enantiomers

Mono-mannosides

Dimannosides: Ditopic structure bridging two units by an appropriate linker

Oct(αMan)αMan

Dimannosides synthesis

C.Nativi, O.Francesconi, G.Gabrielli, A.Vacca, S.Roelens, Chem. Eur. J., 2011, 17, 4814-4820

a) n-octanol, TMSOTf, DCM, 0°C, 15 min, 61%

b) MeONa 1M sol. In MeOH, RT, 45 min, 95%

c) Compound 2, TMSOTf, DCM, 0°C, 50 min, 60%

d) MeONa 1M sol. In MeOH, RT, 1.5h, then H2, [Pd(OH)2]/C, DCM/MeOH 1:1, RT, 16h, 84%

Dimannosides synthesis

Y.Ito, T.Ogawa, Angew. Chem. Int. Ed. Engl., 1994, 33, 1765

IAD: Intramolecular aglycone delivery

a) n-octanol, DDQ, DCM, RT, 1h

b) DTBMP, AgOTf, SnCl2, DCM, RT, 3,5h, 72%

c) Compound 2, TMSOTf, DCM, 0°C, 1h, 63%

d) K2CO3, MeOH, RT, 3h, then H2, [Pd(OH)2]/C, DCM/MeOH 1:1, RT, 16h, 71%

Oct(αMan)βMan

Synthesis of receptors - 1

a) N-BOC-trans-1,2-diaminocycloexane, MeOH,DCM 1:1, 70°C, 7,5h, then NaBH4, RT, 1h, 78%

b) TFA, DCM, 1.5h, 91%c) Pyrrole-2,5-dialdehyde, CHCl3, 70°C, 12h, then

NaBH4, MeOH, RT, 1h, 63%

Synthesis of receptors - 2

The gem-dimethyl group induced a convenient twisting of pyrrolic moieties. On the other hand, the single carbon hinge may impose conformational restrictions

Synthesis of receptors - 3

In addition to providing a more flexible linker, the hydrogen-bonding ability of the aminic group may result beneficial to the recognition properties

Synthesis of receptors - 4

Rigid and non-hydrogen bonding moiety served as reference to evaluate the impact of hydrogen-bonding group and the role of size and rigidity of the linker

Synthesis of receptors - 5

Replacing diaminocyclohexane fragment with a simple amine moiety, the narrowest cleft in the receptor set has been obtained

a) NaOAc, DMF, 100°C, 2h, >99%b) K2CO3, MeOH, RT, 70h, >99%c) Pyridinium chlorochromate (PCC), DC, RT, 2h, 95%d) N-BOC-1,2-diaminocyclohexane, DCM, 80°C, 15h,

then NaBH4, MeOH, RT, 3h, >99%e) TFA, DCM, RT, 3h, 63%

Synthesis of receptors - 6

f) Pyrrole-2,5-dicarboaldehyde, CHCl3, RT, 15h, then NaBH4,MeOH, RT, 1,5h, 96%

g) PhP3, H2O, THF, RT, 15h, 87%h) Pyrrole-2,5-dicarboaldehyde, CHCl3, RT, 15h, then

NaBH4,MeOH, RT, 1h, 70%

Recognition studies

Association constants: measured by 1H-NMR spectroscopic titration, simultaneous fit of the complexation induced shifts of all available signals from both receptor and glycoside to the appropriate association model by non-linear regression analysis

Solvent: 30% of [D7]-DMF in CDCl3 (v/v) was found to be an appropriate medium for solubilize both glycosides and receptors. It also has a good polarity value, comparable to CH3CN in terms of competitivity.

Affinity descriptor: Multiple complex species are present in solution, the parameter is necessary to describe quantitatively the overall affinity of a receptor for a ligandA.Vacca, O.Francesconi, S.Roelens, Chem. Rec., 2012, 12, 544-566

1H-NMR spectroscopic titration

Recognition studies: conclusions

• Substantial contribution from pyrrolic groups on the linker

• Twisted bridge of receptor 9 is determinant to the matching to the ligand

• Flexible linker of 12 seems to favor the adaptivity of the receptor at the expenses of selectivity

• Receptor 16 behaves like 12 but with weaker affinity.Altogether, bridging monotopic binding units with a linker of appropriate size and flexibility and endowed with effective binding groups

proved to be a successful strategy

Structural studies and ITC binding studies

• Receptors showing the best affinity results have been tested in more competitive solvent (DMF/CDCl3 40/60): affinity decreases, but follow the same trend.

• Binding affinities were confirmed by isothermal titration calorimetry (ITC) showing generally good agreements with NMR data.

• The complex of receptor (S)-9 with Oct(αMan)βMan was selected as a representative system and its structural features were studied at 50°C in CDCl3 /[D7]DMF 60:40 by NMR techniques (HSQC, DQ-COSY and TOCSY 2D spectra)

• Several intermolecular hydrogen bonds between pyrrolic/aminc NH groups of the receptor and OH groups of the disaccharide could be found in the structure of the complex.

• Both monosaccharidic units are interacting with monotopic subunits of the ditopic receptor.

Structural studies and ITC binding studies

Recognition studies: Results

• All ditopic receptors consistently bound dimannosides more effectively than monomannosides, whereas the opposite was true for the monotopic receptor

• Linker play a crucial role: receptor 15 gave the worst results of the set. On the other hand receptor 9 gave the best affinity of the whole set.

• Receptor 9 shows an outstanding enantioselectivity towards β-dimannoside with the (S) enantiomer more effective than the (R) one by two orders of magnitude

• The best affinity for α-dimannoside is shown by receptor 12, but it shows neither good enantioselectivity neither α/β discrimination

: The intrinsic median binding concentration

The descriptor is defined as the total concentration of receptor necessary for binding 50% of the ligand when the fraction of bound receptor is zero, that is, when forming the first complex molecule. This value coincides with the dissociation constant Kd for 1:1 complexes, whereas it can be viewed as a “global” dissociation constant when more than one complex is present in solution

IAD: Intramolecular Aglycone delivery