1. 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

2. Introduction D-Glucose - 4 C 1 -anomer β - 4 C 1 -anomer 1 2 3 4 5 6 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)

3. 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 1 2 3 4 5 6 D-Glucose 1 2 3 4 5 6  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

4. 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?

5. 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

6. 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

7. CP-FTMW spectra: assignment Rotamer I 7 07 6 16 7 07 6 06 7 17 6 16 7 17 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 7 17 9 09 8 18 8 18 7 17 9 19 8 18 8 18 7 07 8 08 7 07 9 09 8 08 9 19 8 08 unknown (H 2 O) 2 CH 2 CHCHO HC 3 N CH 2 CHCN (H 2 O) 6 unknown

8. Results ExperimentRotamer IRotamer IIRotamer III A [a] / MHz1269.4108 (23) [e] 1305.3545 (29)1389.896 (18) B / MHz781.1783 (13)760.1481 (12)738.65091 (94) C / MHz577.43929 (36)531.25706 (33)535.50479 (54) a-type transitions [b] observed b-type transitionsobserved  c-type transitions   N [c] 314221  fit [d]  / kHz23.326.119.2 Theory A [a] BCχ aa χ bb χ cc |μ a ||μ b ||μ c |ΔE [b] ΔG [c]  -G-g+/cc/t 12767845812.21  3.92 1.703.03.80.100  -G+g-/cc/t 13137635340.66  2.44 1.783.03.21.23119  -Tg+/cc/t 13987405382.54  4.33 1.794.11.70.9113205  -G-g+/cl/g- 12967885732.760.51  3.26 1.00.71.2329327  -Tt/cl/g- 1404752544 2.760.46  3.22 2.40.60.3541613  -Tg-/cl/g- 1400748542 2.750.40  3.15 0.10.50.0587672 β -G-g+/cc/t11778185352.34-3.371.032.82.22.50d0d 0 β -G+g-/cc/t11807904950.70-2.381.682.62.01.03716 β -Tg+/cc/t13177354952.40-4.101.713.20.41.0140230 NO CONCLUSIVE IDENTIFICATION!! -forms WHAT ABOUT THE QUADRUPOLE CONSTANTS??

9. 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 -3.92  cc /MHz1.70  aa /MHz 0.66  bb /MHz -2.44  cc /MHz1.78  aa /MHz 2.54  bb /MHz -4.33  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-3.15 14 N

10. 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

11. Results Theory A [a] BCχ aa χ bb χ cc |μ a ||μ b ||μ c |ΔE [b] ΔG [c]  -G-g+/cc/t 12767845812.21  3.92 1.703.03.80.100  -G+g-/cc/t 13137635340.66  2.44 1.783.03.21.23119  -Tg+/cc/t 13987405382.54  4.33 1.794.11.70.9113205  -G-g+/cl/g- 12967885732.760.51  3.26 1.00.71.2329327  -Tt/cl/g- 1404752544 2.760.46  3.22 2.40.60.3541613  -Tg-/cl/g- 1400748542 2.750.40  3.150.10.50.0587672 Experiment Rotamer IRotamer IIRotamer III A [a] / MHz1269.4100 (15) [e] 1305.34810 (82)1390.0011 (14) B / MHz781.18234 (26)760.14999 (14)738.65282 (13) C / MHz577.437380 (86)531.255624 (50)535.499914 (56) χ aa [b] / MHz2.159 (16)0.637 (5)2.487 (6) χ bb / MHz  3.727 (14)  2.278 (4)  4.129 (5) χ cc / MHz1.567 (14)1.641 (4)1.642 (5) N [c] 323018  fit [d]  / kHz 1.3 1.1 A conclusive identification is achieved!

12. 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.

13. 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

14. 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.

15. 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

16. 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.

17. 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

18. ACKNOWLEDGMENTS Grants CTQ 2010- 19008, AYA 2009-07304 and AYA 2012-32032 CSD 2009-00038 Molecular Astrophysics Grants VA070A08 and CIP13/01 Grupo de Espectroscopia Molecular (GEM) Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, UVa,Valladolid, Spain