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The solution conformation of C-glycosyl analogues of the sialyl-Tn antigen

The solution conformation of C-glycosyl analogues of the sialyl-Tn antigen

Carbohydrate Research 342 (2007) 1974–1982 Note The solution conformation of C-glycosyl analogues of the sialyl-Tn antigen Vı´ctor Garcı´a-Aparicio,...

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Carbohydrate Research 342 (2007) 1974–1982

Note

The solution conformation of C-glycosyl analogues of the sialyl-Tn antigen Vı´ctor Garcı´a-Aparicio,a Adeline Malapelle,b Zouleika Abdallah,b Gilles Doisneau,b Jose´ Ignacio Santos,a Juan Luis Asensio,a Francisco Javier Can˜ada,a Jean Marie Beaub,* and Jesu´s Jime´nez-Barberoa,* a

b

Centro de Investigaciones Biolo´gicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain Laboratoire de Synthe`se de Biomole´cules, Institut de Chimie Mole´culaire et des Mate´riaux associe´ au CNRS, Universite´ Paris Sud 11, 91405 Orsay Cedex, France Received 23 February 2007; received in revised form 9 March 2007; accepted 16 March 2007 Available online 28 March 2007

Abstract—The conformational behavior of two C-glycosyl analogues of the sialyl-Tn antigen has been determined by a combination of NMR methods and molecular mechanics calculations. Both compounds show a major solution conformation that is drastically different from the major one of the natural compound.  2007 Elsevier Ltd. All rights reserved. Keywords: NMR; Glycomimetics; C-Glycosides; Conformational analysis; Molecular mechanics

The sialyl-Tn antigen, which is expressed on membranebound mucin-type glycoproteins, appears in transformed cells by premature sialylation of GalNAc, the first sugar moiety of nascent O-glycan chains in glycoproteins.1 In fact, among the large number of known tumor-associated carbohydrate motifs, the Tn (a-GalNAc-(1!O)-Ser/Thr) and sialyl-Tn (a-Neu5Ac(2!6)-a-GalNAc-(1!O)-Ser/Thr) antigens are the most specific to human epithelial tumor cells (breast, colon, ovarian, lung, and pancreatic cancers).2 These antigens are of interest in the development of synthetic vaccines that induce an anti-cancer immune response in which the carbohydrate domain plays a decisive role in determining immunogenicity.1,2 In particular, anticancer vaccines have been designed based upon the sialyl-Tn epitope. Because the glycosylated antigen is, however, partially deglycosylated during the riming period,3 it is therefore important to access synthetic compounds in which the carbohydrate moiety cannot be detached from the pep-

* Corresponding authors. E-mail: [email protected] 0008-6215/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2007.03.021

tide. In this context, the use of C-glycosyl analogues4 of the Tn antigen might provide stable mimics that may also be useful for the immunodiagnosis of different tumors or for the affinity purification of specific receptors. Indeed, the synthesis of this type of mimic, which can be incorporated into immunogenic glycopeptides, has already been reported.5 A detailed conformational analysis of a number of Cdisaccharides has revealed that the conformational similarity between O- and C-glycosides is not a general phenomenon.4,6 These findings encouraged us to extend the comparison of C- and O-glycosides to other linkages.7 On this basis, we now report the conformational analysis of two C-disaccharide analogues of the sialylTn antigen, with or without a hydroxyl group at the pseudoglycosidic bridge atom (1, 2), in water using NMR spectroscopy in tandem with molecular mechanics calculations.8 The structures of 1 and 2, showing the atomic numbering, are given below. Both C-disaccharides 1 and 2 are expected to be rather flexible around the (2!6) linkage. Moreover, flexibility around the x angle (defined as C7–C6G–C5G–O5G) is also anticipated.

V. Garcı´a-Aparicio et al. / Carbohydrate Research 342 (2007) 1974–1982

1975

OH N8

N9

OH 6G

N6

HO AcHN

COOH N1

OH O

N7 N4

N5

N2

5G

4G

N3

O

R HO

HO

3G

2G AcHN

1 2

1G

OMe

R=H R=OH

The conformational behavior of 1 and 2 was analyzed as previously described,9 by performing molecular mechanics calculations. This was achieved by considering the output of the modeling procedure as determined from NMR parameters (J and NOE) from the population distribution, and by comparing them with the experimental data to validate the theoretical results. Three staggered conformations around the glycosidic bonds are possible.10 Those for the U angle (defined as C1N–C2N–C7–C6G) were previously termed exo-syn (Uexo), exo-anti (Uanti), or non-exo (Unon-exo), by considering their accordance with the exo-anomeric geometry11 and their disposition in a syn- or anti-type arrangement (see Scheme 1a). For the aglyconic bond, W (defined as C5G–C6G–C7–C2N), the three staggered conformations are denoted as W+, W, and Wanti (see Scheme 1b). First, the 1H NMR spectra of 1 and 2 in D2O (Fig. 1) were assigned using a combination of COSY, TOCSY, and HSQC experiments12 (Table 1). The assignment of the prochiral HproR and HproS protons of the methylene bridge was performed as shown below using a similar protocol to that described previously, based on a

combination of J and NOE values.13 The intra-ring vicinal proton–proton coupling constants14 proved that the galactose ring adopts the 4C1 chair conformation, while that of the sialic acid residue is in the 2C5 conformation (see Table 1), as in the natural compound. 1D and 2D NOE-based experiments15 (Figs. 2 and 3) provided a set of short inter-proton distances that could be used to assess the experimental distribution through comparison C1S

C1S

C1S

O5S

Figure 1. Expansions of the key regions of the experimental 500 MHz 1 H NMR spectra of 1 (top) and 2 (bottom) in D2O at 300 K and pH 6.0. N stands for the NeuNAc protons and G for the Gal protons. The large signals at ca. 3.3 ppm correspond to the a-OMe group attached at the Gal anomeric carbon, while that at ca. 1.97 ppm, to the methyl group of the NAc moiety.

C3S

O5S

C6G

C6G

C3S

O5S

C3S

C6G

exo-syn

non-exo

anti Scheme 1a. The three rotamers around U.

C5G

C5G

C5G

C2S H6proS

H6proR

H6proS

H6proR

C2S

H6proR

H6proS

C2S

anti Scheme 1b. The three rotamers around W.

ψ+

ψ−

V. Garcı´a-Aparicio et al. / Carbohydrate Research 342 (2007) 1974–1982

1976 Table 1. 1H and

13

C chemical shifts (d, ppm) and key coupling constants (J, Hz) for 1 and 2 (D2O, pH 7.0) at 500 MHz and 300 K

Proton

1 (CH2 analogue) 1

d H (ppm) H1Gal H2Gal H3Gal H4Gal H5Gal H6proRGal H6proSGal H7proR H7proS H3Neu5NAceq H3Neu5NAcax H4Neu5NAc H5Neu5NAc H6Neu5NAc H7Neu5NAc H8Neu5NAc H9aNeu5NAc H9bNeu5NAc

4.65 4.05 3.78 3.8 3.75 1.48 1.73 1.80 1.80 2.54 1.54 3.61 3.71 3.60 3.47 3.80 3.56 3.80

J (Hz) 3.8 3.8, 10.8

See below See below

4.7, 13.1 11.6, 13.2 4.5, 9.8, 11.5 10.1 10.4 See below See below See below

2 (CHOH analogue) d

13

C

98.0 49.7 67.9 69.8 69.9 35.6 35.6 24.3 24.3 40.2 40.2 73.4 52.1 71.6 68.3 68.3 62.7 62.7

1

d H (ppm) 4.67 4.07 3.78 3.84 3.72 1.77 1.83 — 3.96 2.40 1.87 3.63 3.72 3.74 3.50 3.82 3.80 3.56

J (Hz)

d

3.7 3.7, 11

13

C

98.1 49.7 68 68.6 71.8 31.4 31.4 — 68.4 36 36 68.4 52 73.2 68.5 71.6 62.7 62.7

3.2 See below See below — See below 4.6, 13 11.8, 13 4.7, 9.6, 12 9.5 See below See below See below

Additional coupling constants Proton pair

J (Hz) for 1

J (Hz) for 2

H6Neu5NAc/H7Neu5NAc H7Neu5NAc/H8Neu5NAc H8Neu5NAc/H9aNeu5NAc H9aNeu5NAc/H9bNeu5NAc H7proS/H7proR H6GalproS/H6GalproR H5Gal/H6GalproS H7Gal/H6GalproS H5Gal/H6GalproR H7Gal/H6GalproR

1.6 8.9 6.9 12.5 12.7 Overlap Overlap Overlap Overlap Overlap

1.6 8.9 6.5 12.6 — 14.5 2.8 7.5 10.5 6.5

The Gal protons are noted in the figures and text as G, and the Neu5NAc, as N.

with the short distances estimated from the MM 3* calculations. The analysis of the NMR data was first performed. The x angle for the Gal moiety. Scheme 2 shows the three staggered rotamers around the C5–C6 bond of the Gal moiety. One large and one small value would be expected for the two JH5–H6 couplings in the case of the GT or TG rotamers, while two small values are expected for the GG. For compound 2, the observed coupling constants for the H5G–H6GproS and H5G– H6GproR were 2.8 and 10.5 Hz, indicating the presence of one major rotamer (either GT or TG). The discrimination between these two was obtained through analysis of the NOE data. A strong NOE between the H4 and the H7 protons (Fig. 2a and b) was observed in the 1D-NOESY spectra, thus indicating that the major rotamer in solution was the TG geometry (Scheme 2). Otherwise, a strong NOE would have been observable for the H4/H6proR proton pair and not for the H4/H7 pair. Moreover, molecular mechanics calculations with the MM 3* force field were in agreement with this experimentally based conclusion, and agree that the TG rotamer is the global minimum of both 1 and 2

(Table 2). On this basis, it is straightforward to assign the Gal H6proR and Gal H6proS protons, because the Gal H6proS should be the one with a stronger NOE to Gal H5 (Scheme 2). The comparison between the expected J couplings for the MM 3*-minimized TG conformation (JH5,H6proS 2.6, JH5,H6proR 10.1) of the GalNAc residue and the observed ones (JH5,H6proS 2.8, JH5,H6proR 10.5) is very satisfactory. The stereochemistry at C7 and the conformation around the U angle. To deduce the stereochemistry at the pseudoglycosidic carbon atom, dubbed C7, the two possible isomers were considered and, for 2, molecular mechanics calculations (Table 2) were performed for all the possible staggered rotamers around the sialic U linkage (Scheme 1), Uanti (180), Uexo (60), and Unonexo (+60). In these calculations, the conformation about x was kept as the TG conformer. For both isomers, the major conformer adopts the Unon-exo conformation. This conformer is dominant for 1 (87%) and is the major one for 2 (ca. 67%). Conformers with the Uanti orientation are also present for both compounds (13% for 1 and 33% for 2). According to these MM 3* calculations, the contribution of Uexo conformers is negligible.

V. Garcı´a-Aparicio et al. / Carbohydrate Research 342 (2007) 1974–1982

1977

Figure 2. 1D DPFGNOE NMR spectra (500 MHz, 298 K, 600 ms mixing time), obtained after inversion of the key protons of compound 2. The selectively inverted proton displays negative sign, while the key NOEs are shown as positive signals. N stands for the NeuNAc protons and G for the Gal protons.

The distances between the H3eq and H3ax atoms to the pseudoglycosidic hydrogen, H7, were then computed for the three staggered conformers and compared to the experimental NOEs (Fig. 2a, f, and g). No NOE was observed for either the H3eq/H7 proton pair or the H3ax/H7 one. These observations are only in agreement with either a S configuration having a major anti-U conformation around the sialic acid pseudoglycosidic linkage or R configuration at C7, having a U angle value around +60 (Fig. 4). The trend for compound 1 (with the methylene group at the pseudoglycosidic linkage) was similar. Upon semi-selective inversion of the H7 protons, no NOE to H3eq was observed, while in the latter, the NOE to the H3ax was evident (Fig. 3a and g). This feature was also evident form 2D-ROESY experiments. This trend is in agreement only with a major Unon-exo conformer (Fig. 4c). At this point, there is incomplete evidence that the major conformation in solution around U for 1 may be also applicable to 2. A chemical

shift-based analysis was conducted on both the H3 axial and equatorial protons in 1 and 2, which showed that H3ax was more strongly deshielded (more than 0.3 ppm) in 2 than in 1 (Fig. 1). This may be adequately explained by the effect of the hydroxyl group at C7. Only when the configuration of C7 is R and the conformer is Unon-exo, is there a clear interaction between H3ax and O7 (Fig. 4a), which conforms with the observed downfield shift with respect to H3ax of 1. For the S configuration with a major Uanti conformation (Fig. 4b), H3eq would be deshielded in 2 with respect to 1, but the opposite trend is observed: it was moderately more shielded in 2 than in 1 (ca. 0.14 ppm). Thus, both compounds display a major Unon-exo conformation around U.16 The conformation around the W angle. The conformation around the W torsion was also explored, keeping the TG geometry for the C5–C6 bond (see above). Indeed, the coupling constants for the Gal H6R,S/H7 proton pairs were 6.5 and 7.5 Hz, thus indicating the

V. Garcı´a-Aparicio et al. / Carbohydrate Research 342 (2007) 1974–1982

1978

Figure 3. 1D DPFGNOE NMR spectra (500 MHz, 298 K, 600 ms mixing time), obtained after inversion of the key protons of compound 1. The selectively inverted proton displays negative sign, while the key NOEs are shown as positive signals. N stands for the NeuNAc protons and G for the Gal protons.

H6proS H5

H5 C7

C7 H4

O5

H4

O5

H6proS H4

O5

OH

OH

OH

TG

H5

C7

H6proS

H6proR

H6proR

H6proR

GT

GG

Scheme 2. The three possible geometries around Gal C5–C6.

presence of conformational averaging around W. These key couplings can be explained by an approximate 50:50 equilibrium between conformers showing W values of Wanti and W (torsion angle around 60), which is in reasonable agreement with those predicted by MM 3* (Table 2, 7:33 for 1, and 69:31 for 2).

The Gal H4–Gal H7 and Gal H5–Gal H7 distances were evaluated for the possible rotamers around W and compared with the experimental NOE data. NOEs were observed (Fig. 2a–c) for both proton pairs, also in agreement with this conformational equilibrium around W. The Wanti conformer is shown

V. Garcı´a-Aparicio et al. / Carbohydrate Research 342 (2007) 1974–1982 Table 2A. Steric energy values (kJ mol1), relative populations (%), and torsion angles () for 1 according to

1979 MM 3*

calculations

Conformer

U, W () DE (kJ mol1) Population (%)

A TG

B TG

C TG

D TG

E TG

54, 72 0 67

160, 72 14 <1

54, 162 3 20

180, 162 4 13

54, 162 16 <1

The TG rotamer is in all cases more stable than the GT one in ca. 4 kJ mol1.

Table 2B. Steric energy values (kJ mol1), relative populations (%) and torsion angles () for 2 (R-isomer) according to

MM 3*

calculations

Conformer

U, W () DE (kJ mol1) Population (%)

A TG

B TG

C TG

D TG

E TG

54, 72 0 68

160, 72 12 <1

160, 60 11 1

180, 162 11 1

54, 162 2 30

in Figure 4a, while the W conformer is depicted in Figure 4b. Unfortunately, the severe overlap existing in the NMR spectrum (Fig. 1) of compound 1, with two contiguous methylene groups at Gal C6 and C7, precluded the measurement of the key couplings and, thus no unambiguous assignment of the prochiral protons could be obtained. Nevertheless, because a H4/H7 NOE was observed with a strong-medium intensity (Fig. 3g), the presence of the TG rotamer was assumed (see above for 2). In addition, because the behavior around U was similar for both 1 and 2, it seems reasonable to assume that a similar tendency also is present in 1. Previous reports17 have indicated that for Gal (1!6) linked disaccharides, Gal H6proS is deshielded versus Gal H6proR. Although merely speculative, a tentative assignment was based on this fact. Nevertheless, regarding the NOEs that could validate such an assignment, it can be observed that Gal H4 and H5 (Fig. 5) give small to medium sized NOEs to both Gal H6proR and H6proS (Fig. 3c, h, e) indicating the presence of a conformational equilibrium for x (Scheme 2). The conformation of the sialic acid lateral chain. The conformation of the lateral chain of the sialic acid moiety was also evaluated, by probing two torsion angles, namely v (H6N–C6N–C7N–C8N) and h (C6N–C7N– C8N–C9N). The conformational map obtained (Fig. 6) is in agreement with an exclusive population around v, while h may have the three staggered conformations, although a 180 value is preferred. The expected couplings for the three conformers were compared to the experimental J values, namely JH6N–H7N 1.6 Hz and JH7N–H8N 8.9 Hz. The major conformer is indeed that which shows a better agreement with the experimental data. The expected couplings are JH6N–H7N 1.9 Hz and JH7N–H8N 9.9 Hz, indicating that this conformer is the major one present in solution, indicated in Figures 4 and 5, with minor contributions from the other two.

Discussion. The conformational analysis of 1 and 2, glycomimetics of the sialyl-Tn antigen, has been performed by using NMR and molecular mechanics. The analysis of the data indicates that, for both compounds, there is a conformational equilibrium that primarily affects the W angle, which populates the Wanti and W torsion angles. There is a major conformer for the U angle (the ‘non-natural’ Unon-exo conformer),18 which is essentially absent in natural sialyl-a-(2!6)-lactose,19 as well as for x angle (the TG rotamer, one of the naturally existing ones, although only about 20% in a-NeuNAc(2!6)-Gal linkages).19,20 There is one crystal structure available in the Protein Data Bank for a a-NeuAc-(2!6)-Gal-containing molecule, which is this disaccharide bound to the pertussis toxin (code 1PTO).21 In this crystal structure, the saccharide adopts several conformations around the U (Uexo and Unon-exo), W (Wanti and W), and x (the three staggered rotamers) torsion angles, confirming the great flexibility of this a-(2!6)-linkage and the relatively low energy barriers required for conformer inter-conversions. There is also a model of the a-NeuAc-(2!6)-aGalNAc disaccharide docked to Maackia amurensis hemagglutinin (MAH).22 Here, the a-NeuAc-(2!6)-aGalNAc glycosidic linkage displays Uexo and Wanti values, which correspond to the most abundant conformer in solution.19 Regarding the lateral chain of the sialic acid moiety, the data indicate that the major conformer is that which exists in naturally occurring sialic acid derivatives.23 Conclusions. The conformational behavior of 1 and 2 is drastically different from the major solution conformer of the natural O-glycosyl compound, especially around U and x. Nevertheless, the intrinsic low energy barriers for conformer inter-conversion might easily access other conformers allowing them to be bound by natural receptors without major entropy penalties. The existence of non-natural conformations for C-glycosyl compounds has also been reported for other analogues,24

1980

V. Garcı´a-Aparicio et al. / Carbohydrate Research 342 (2007) 1974–1982

Figure 5. The alternative W geometry of compound 2. The coupling constants for the pseudoglycosidic H7 indicate that this geometry is also present in solution. The same geometry also takes place in the conformational equilibrium of 1 (without the pseudoglycosidic hydroxyl group).

Figure 4. There is only one possible U conformer for each possible isomer at C7 of compound 2 that explains the lack of NOEs between the pseudoglycosidic H7 atom and H3ax, H3eq at the sialic acid moiety (see Fig. 2a, f and g for the experimental data): (a) the Unon-exo conformer of the C7-(R)isomer or (b) the Uanti conformer of the C7(S)-epimer. In both cases, H7 is far from both Neu5NAc H3 protons. (c) For compound 1, only the Unon-exo conformer can explain the absence of the H7/H3eq NOE and the presence of the H7/H3eq NOE (see Fig. 3a and g for the experimental data). Thus, the conformers around U and x depicted in (a) and (c) are the major ones for compounds 2 and 1, respectively. These conformations also may explain the observed chemical shifts differences for H3ax (Fig. 1 and Table 1) between both compounds (see text). In all cases, the anti W geometry is shown.

especially in C-mannosyl compounds.16,25 The lack of the exo-anomeric effect as well as the lack of destabilizing interactions between one equatorially oriented hydroxyl group (Glc- or Gal-type glycosyl compounds) with the aglycon moiety are at the heart of the existence of non-exo-anomeric conformers.16 Recently, other sialic acid glycomimetics (with a S-glycosyl linkage) have been employed as possible rotavirus inhibitors.26 Thus, the use of all these types of mimetics may open new avenues for potential drugs robust to metabolic or chemical degradation or for probes in exploring molecular interactions.

Figure 6. Steric energy map (MM 3*) for the lateral chain of the sialic acid moiety. v/h are defined in the text. Energy contours are given every kJ mol1.

1. Experimental 1.1. Materials Compounds 1 and 2 were synthesized using a two-step deprotection procedure (Malapelle, A.; Doisneau, G.; Beau, J.-M.; unpublished results) on the corresponding fully protected derivatives previously described.5b 1.2. Molecular mechanics calculations First, all the possible 27 staggered rotamers around /, w, x were built and minimized using the MM 3* force field27 (e = 80) as integrated in MAESTRO package. The obtained energies were used to compute a possible Boltzmann-based distribution at 300 K. The geometries were compared against the NMR parameters as described in the text.

V. Garcı´a-Aparicio et al. / Carbohydrate Research 342 (2007) 1974–1982

For the sialic acid lateral chain, v, h potential energy maps for the sialic acid moiety of compound 1 were generated by systematic rotation around both linkages using a grid step of 18. Optimization of the geometry at every point using conjugate gradients iterations was carried out until the rms derivative was smaller than ˚ 1 via energy calculations with the same 0.05 kJ mol1 A force field. v is defined as H6N–C6N–C7N–C8N and h as C6N–C7N–C8N–C9N. The probability distribution was calculated for each point according to a Boltzmann function at 300 K. 1.3. J and NOE calculations The vicinal coupling constants were calculated for each /, w, x conformation using the Karplus–Altona equation. Ensemble average values were P calculated from the distribution according to: J = P/wJi/w. The inter-proton average distances were calculated using the following expression: X hr6 ikl ¼ P /w r6 klð/wÞ : NOE intensities were determined according to the complete relaxation matrix as previously described using home-made software available from the authors upon request.28 Isotropic motion and external relaxation of 0.1 s1 were assumed. A correlation time of 90 ps was used. 1.4. NMR spectroscopy NMR spectra were recorded on a Bruker Avance 500 instrument at 25 C. A concentration of ca. 2 mM of 1 and 2 was used. Chemical shifts were referenced to external DSS in D2O. 1D spectra were acquired using 32K data points, which were zero-filled to 64K data points prior to Fourier transformation. Absolute value COSY, and phase-sensitive HSQC spectra and ROESY (mixing times of 300 and 500 ms) were acquired using standard techniques. Acquisition data matrices were defined by 2K · 256 points and zero-filled to 2K · 512 matrices prior to Fourier transformation. Baseline correction was applied in both dimensions. 1D-selective NOE spectra were acquired using the double echo sequence proposed by Shaka and co-workers29 at three different mixing times (300, 500, and 600 ms). Spectra were processed using the Bruker XWIN-NMR program on a Silicon-Graphics computer.

Acknowledgments Financial support by the Ministry of Education and Science of Spain is gratefully acknowledged (CTQ2006-10874-C02-01), as is a EC Marie Curie Research Training Network grant (Contract No. MRTN-CT-

1981

2006-035546). We also gratefully acknowledge the Ministe`re de la Recherche in France for a Ph.D. grant to A.M. and Z.A. The CAI-NMR facility at the University Complutense of Madrid is thanked for the access to the 500 MHz spectrometer.

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