THE CONFORMATIONAL BEHAVIOUR OF GLUCOSAMINE I. PEÑA, L. KOLESNIKOVÁ, C. CABEZAS, C. BERMÚDEZ, M. BERDAKIN, A. SIMAO, J.L. ALONSO Grupo de Espectroscopia Molecular. Unidad asociada CSIC Laboratorios de Espectroscopia y Bioespectroscopia Edificio Quifima. Parque Científico Universidad de Valladolid SPAIN

Introduction D-Glucose - 4 C 1 -anomer β - 4 C 1 -anomer Alonso, J. L. et al. Chemical Science 2014, 5, 515. GAS PHASE  The first conformational characterization of isolated D-glucose molecule in gas phase became recently possible due to the latest developments of Fourier transform microwave techniques coupled with laser ablation vaporizations methods (LA-MB-FTMW)

Introduction  Glucosamine is an amino monosaccharide, that differs structurally from the parent D- glucose by replacement of the hydroxyl group on C-2 by an amino group D-Glucosamine D-Glucose  It is an essential precursor of important nitrogen-containing macromolecules like glycoproteins, glycolipids and glycosaminoglycans  D-glucosamine is chemically unstable, only commercially available as D-glucosamine hydrochloride, where it appears in the protonated form  No experimental data on the conformational behavior of its neutral form has been reported hitherto

Aims  Generation of neutral D-glucosamine in gas phase (?) by laser ablation of the crystalline sample D-glucosamine hydrochloride  Study of the conformational behavior of D-glucosamine in isolation conditions of the gas phase (in a supersonic expansion)  Comparison of the conformational behavior of D-glucosamine with that observed in the archetypal D-glucose. How does the replacement of the OH group by the NH 2 group affect the conformational behavior?

Modelling: plausible configurations  -forms β- forms Newman projections of plausible conformations of the hydroxymethyl group around the C 5  C 6 (G , G+, T) and C 6  O 6 (g , g+, t) bonds

Experimental: CP-FTMW + Laser Ablation 6-18 GHz Frequency Range S. Mata I. Peña, et al. J. Mol. Spectr. 280(2012) 91–96 GEM. Valladolid

CP-FTMW spectra: assignment Rotamer I 7 07    6 06 a-type (J + 1) 0 J +1 ← J 0 J and (J + 1) 1 J +1 ← J 1 J and b-type (J + 1) 1 J +1 ← J 0 J and (J + 1) 0 J +1 ← J 1 J R-branch progressions become degenerated with the increasing J 8 08        8 08 unknown (H 2 O) 2 CH 2 CHCHO HC 3 N CH 2 CHCN (H 2 O) 6 unknown

Results ExperimentRotamer IRotamer IIRotamer III A [a] / MHz (23) [e] (29) (18) B / MHz (13) (12) (94) C / MHz (36) (33) (54) a-type transitions [b] observed b-type transitionsobserved  c-type transitions   N [c]  fit [d]  / kHz Theory A [a] BCχ aa χ bb χ cc |μ a ||μ b ||μ c |ΔE [b] ΔG [c]  -G-g+/cc/t   -G+g-/cc/t   -Tg+/cc/t   -G-g+/cl/g   -Tt/cl/g   -Tg-/cl/g  β -G-g+/cc/t d0d 0 β -G+g-/cc/t β -Tg+/cc/t NO CONCLUSIVE IDENTIFICATION!! -forms WHAT ABOUT THE QUADRUPOLE CONSTANTS??

Results The values of  aa,  bb and  cc can discriminate conformers  -G-g+/cc/t  - G+g-/cc/t  - Tg+/cc/t  - G-g+/cl/g-  - Tt/cl/g-  - Tg-/cl/g- cc cl  aa /MHz 2.21  bb /MHz  cc /MHz1.70  aa /MHz 0.66  bb /MHz  cc /MHz1.78  aa /MHz 2.54  bb /MHz  cc /MHz1.79  aa /MHz 2.76  bb /MHz 0.51  cc /MHz-3.26  aa /MHz 2.76  bb /MHz 0.46  cc /MHz-3.22  aa /MHz 2.75  bb /MHz 0.40  cc /MHz N

Experimental: LA-MB-FTMW A high resolution LA-MB-FTMW study is needed FT-MW Spectrometer Fabry-Pérot Resonator Picosecond Laser 3-10 GHz I. Peña et al. JACS 134 (2012) 2305–2312 3232 F’F’’=54 4343 3232 4343 5454 14 N nuclear quadrupole coupling hyperfine structure is completely resolved Rotamer III Rotamer I

Results Theory A [a] BCχ aa χ bb χ cc |μ a ||μ b ||μ c |ΔE [b] ΔG [c]  -G-g+/cc/t   -G+g-/cc/t   -Tg+/cc/t   -G-g+/cl/g   -Tt/cl/g   -Tg-/cl/g  Experiment Rotamer IRotamer IIRotamer III A [a] / MHz (15) [e] (82) (14) B / MHz (26) (14) (13) C / MHz (86) (50) (56) χ aa [b] / MHz2.159 (16)0.637 (5)2.487 (6) χ bb / MHz  (14)  (4)  (5) χ cc / MHz1.567 (14)1.641 (4)1.642 (5) N [c]  fit [d]  / kHz A conclusive identification is achieved!

Conclusions  Neutral D-glucosamine has been succesfully generated in gas phase by laser ablation of the crystalline sample D-glucosamine hydrochloride  Only  -pyranose forms have been observed, thus preserving the  -pyranose form present in the X-ray studies. No interconversion reaction between the  and β forms takes place during the laser ablation process  The most abundant conformers are stabilized by a chain of four cooperative hydrogen bonds (O 4 H  O 3 H  N 2 H  O 1 H  O 5 ) and one non-cooperative (O 6 H  O 5 ). The least abundant exhibits five cooperative H-bonds, O 6 H  O 4 H  O 3 H  N 2 H  O 1 H  O 5.

Conclusions -D-Glucosamine G-g+/cc/tG+g-/cc/tTg-/cc/t O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 6 HO 4 HO 3 HN 2 HO 1 HO 5

Conclusions  Neutral D-glucosamine has been succesfully generated in gas phase by laser ablation of the crystalline sample D-glucosamine hydrochloride  Only  -pyranose forms have been observed, thus preserving the  -pyranose form present in the X-ray studies. No interconversion reaction between the  and β forms takes place during the laser ablation process  The most abundant conformers are stabilized by a chain of four cooperative hydrogen bonds (O 4 H  O 3 H  N 2 H  O 1 H  O 5 ) and one non-cooperative (O 6 H  O 5 ). The least abundant exhibits five cooperative H-bonds, O 6 H  O 4 H  O 3 H  N 2 H  O 1 H  O 5.  The substitution of the hydroxyl group at C-2 by the amino group in  -D-glucosamine does not introduce any changes into the gas phase conformational preferences; the three observed conformers of D- glucosamine correlate with those observed in  -D-glucose.

Conclusions -D-Glucosamine -D-Glucose G-g+/cc/tG+g-/cc/tTg-/cc/t O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 6 HO 4 HO 3 HN 2 HO 1 HO 5 O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 6 HO 4 HO 3 HN 2 HO 1 HO 5

Conclusions  Neutral D-glucosamine has been succesfully generated in gas phase by laser ablation of the crystalline sample D-glucosamine hydrochloride  Only  -pyranose forms have been observed, thus preserving the  -pyranose form present in the X-ray studies. No interconversion reaction between the  and β forms takes place during the laser ablation process  The most abundant conformers are stabilized by a chain of four cooperative hydrogen bonds (O 4 H  O 3 H  N 2 H  O 1 H  O 5 ) and one non-cooperative (O 6 H  O 5 ). The least abundant exhibits five cooperative H-bonds, O 6 H  O 4 H  O 3 H  N 2 H  O 1 H  O 5.  The substitution of the hydroxyl group at C-2 by the amino group in  -D-glucosamine does not introduce any changes into the gas phase conformational preferences; the three observed conformers of D- glucosamine correlate with those observed in  -D-glucose.  The orientation of the NH 2 group within each conformer has been delineated by the values of the nuclear quadrupole constants; adopts the same role than the OH group in the intramolecular hydrogen bonding network.

Conclusions -D-Glucosamine -D-Glucose G-g+/cc/tG+g-/cc/tTg-/cc/t O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 6 HO 4 HO 3 HN 2 HO 1 HO 5 O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 4 HO 3 HN 2 HO 1 HO 5 O 6 HO 5 O 6 HO 4 HO 3 HN 2 HO 1 HO 5

ACKNOWLEDGMENTS Grants CTQ , AYA and AYA CSD Molecular Astrophysics Grants VA070A08 and CIP13/01 Grupo de Espectroscopia Molecular (GEM) Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, UVa,Valladolid, Spain