Synthesis and O-Glycosidic Linkage Conformational Analysis of 13C-Labeled Oligosaccharide Fragments of an Antifreeze Glycolipid

Abstract

NMR studies of two ¹³C-labeled disaccharides and a tetrasaccharide were undertaken that comprise the backbone of a novel thermal hysteresis glycolipid containing a linear glycan sequence of alternating [βXyl p-(1→4)-βMan p-(1→4)] _n dimers. Experimental trans-glycoside NMR J-couplings, parameterized equations obtained from density functional theory (DFT) calculations, and an in-house circular statistics package ( MA'AT) were used to derive conformational models of linkage torsion angles ϕ and ψ in solution, which were compared to those obtained from molecular dynamics simulations. Modeling using different probability distribution functions showed that MA'AT models of ϕ in βMan(1→4)βXyl and βXyl(1→4)βMan linkages are very similar in the disaccharide building blocks, whereas MA'AT models of ψ differ. This pattern is conserved in the tetrasaccharide, showing that linkage context does not influence linkage geometry in this linear system. Good agreement was observed between the MA'AT and MD models of ψ with respect to mean values and circular standard deviations. Significant differences were observed for ϕ, indicating that revision of the force-field employed by GLYCAM is probably needed. Incorporation of the experimental models of ϕ and ψ into the backbone of an octasaccharide fragment leads to a helical amphipathic topography that may affect the thermal hysteresis properties of the glycolipid.

Figure 1.

Different statistical models^, of ϕ and ψ in βMan-(1→4)-βXylOCH₃ (2) and βXyl-(1→4)-βManOCH₃ (3). (A) ϕ in 2. (B) ψ in 2. (C) ϕ in 3. (D) ψ in 3. Black, Cartwright’s Power of Cosine. Orange, wrapped normal. Blue, von Mises. Green, wrapped Cauchy. Violet, uniform. The first three models gave similar probability distributions.

Figure 2.

Different statistical models of ψ in tetrasaccharide 4. (A) M₂–X₂ linkage. (B) X₂–M₁ linkage. (C) M₁–X₁ linkage. (See Scheme 1 for residue definitions.) Black, Cartwright’s Power of Cosine. Orange, wrapped normal. Blue, von Mises. Green, wrapped Cauchy. Violet, uniform. The first three models gave similar probability distributions.

Figure 3.

Geometry-optimized structure of octasaccharide 32 obtained from DFT calculations (Gaussian09), showing putative inter-residue H-bonds across the βXyl-(1→4)-βMan and βMan-(1→4)-βXyl linkages and an overall helical shape. Potential intra- and inter-residue H-bonds are also shown. See text for discussion.

Figure 4.

Aqueous 1-μs MD simulation histograms (red) for ϕ and ψ in βMan-(1→4)-βXylOCH₃ (2) (A and B) and βXyl-(1→4)-βManOCH₃ (3) (C and D) superimposed on statistical distributions of ϕ and ψ (von Mises) determined by NMR J-coupling analysis (MA’AT) (blue lines). Mean positions of ϕ (MD) are +162.3° and +161.1° for 2 and 3, respectively. Mean positions of ψ (MD) are +138.9° and +115.8° for 2 and 3, respectively. See Tables 6 and 7 for CSDs.

Figure 5.

Statistical distributions of ψ (von Mises) determined by NMR J-coupling analysis (MA’AT) (blue lines) superimposed on histograms obtained from aqueous 1-μs MD simulations (red) for tetrasaccharide 4. (A) M₂–X₂ linkage. (B) X₂–M₁ linkage. (C) M₁–X₁ linkage. See Scheme 1 for residue definitions.

Scheme 1.. Singly ¹³C-Labeled Disaccharides 2^1′, 2^2′, 3^1′, and 3^2′ and Triply ¹³C-Labeled Tetrasaccharide 4^a

^aSuperscripts denote the labeled carbons in the disaccharides.

Scheme 2.

Synthetic Route To Prepare βMan-(1→4)-βXylOCH₃ (2) Selectively Labeled with ¹³C at C1 of the βMan Residue

Scheme 3.

Synthetic Route To Prepare βXyl-(1→4)-βManOCH₃ (3) Selectively Labeled with ¹³C in the βXyl Residue

Scheme 4.

Synthetic Route To Prepare Tetrasaccharide 4 Containing Selective ¹³C-Labeling at Three Anomeric Carbons

Scheme 5.. J_CH Values (in Hz) in βXylp and βManp Rings Labeled with ¹³C at C1 and C2^a

^aThe signs of ¹J_CH and ³J_CH values are assumed to be positive; the signs of ²J_CH values are shown. A broadened signal is denoted as br (J_CH < 0.5 Hz).

Scheme 6.. J_CC Values (in Hz) in βXylp and βManp Rings Labeled with ¹³C at C1 and C2^a

^aThe signs of ¹J_CC and ³J_CC values are assumed to be positive; the signs of ²J_CC values are shown.

Scheme 7.

NMR Spin-Couplings Sensitive to the ϕ and ψ Torsion Angles Comprising the Internal O-Glycosidic Linkage in 2

Scheme 8.

Model Structures 2^c and 3^c Used in DFT Calculations, Showing Torsion Angle Constraints Applied during Geometry Optimizations and J-Coupling Calculations