Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Synthetic Disaccharide Standards Enable Quantitative Analysis of Stored Heparan Sulfate in MPS IIIA Murine Brain Regions Qi Qi He,† Paul J. Trim,‡,∥,∇ Adeline A. Lau,§,∥ Barbara M. King,§ John J. Hopwood,⊥ Kim M. Hemsley,§,∥ Marten F. Snel,‡,∥,∇ and Vito Ferro*,† †
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia Mass Spectrometry Group, Hopwood Centre for Neurobiology, South Australian Health and Medical Research Institute, Adelaide, South Australia 5000, Australia § Childhood Dementia Research Group, Hopwood Centre for Neurobiology, Lifelong Health Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia 5000, Australia ∥ Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, South Australia 5000, Australia ⊥ Hopwood Centre for Neurobiology, South Australian Health and Medical Research Institute, Adelaide, South Australia 5000, Australia ∇ Proteomics, Metabolomics and MS-Imaging Core Facility, South Australian Health and Medical Research Institute, Adelaide, South Australia 5000, Australia
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‡
S Supporting Information *
ABSTRACT: Heparan sulfate (HS) is a complex polysaccharide from the glycosaminoglycan (GAG) family that accumulates in tissues in several neurological lysosomal storage diseases known as mucopolysaccharidosis (MPS) disorders. The quantitation of HS in biological samples is important for studying MPS disorders but is very challenging because of its high molecular weight and heterogeneity. Recently, acid-catalyzed butanolysis followed by LC-MS/MS analysis has emerged as a promising method for the determination of HS. Butanolysis of HS produces fully desulfated disaccharide cleavage products which are detected by LC-MS/MS. Herein we describe the synthesis of butylated HS disaccharide standards and their use for determining the identity of major product peaks in LC-MS chromatograms from butanolysis of HS as well as the related GAGs heparin and heparosan. Furthermore, synthesis of a d9-labeled disaccharide internal standard enabled the development of a quantitative LC-MS/MS assay for HS. The assay was utilized for the analysis of MPS IIIA mouse brain tissues, revealing significant differences in abundance and in the regional accumulation of the various HS disaccharides in affected mice. KEYWORDS: mucopolysaccharidosis, MPS IIIA, heparan sulfate, butanolysis, disaccharide standards, LC-MS/MS
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specifically MPS types I, II, IIIA-D, and VII.4 However, its complex structure, heterogeneity, and high molecular weight renders its quantification extremely challenging. HS is made up of repeating disaccharides comprising a uronic acid (β-Dglucuronic acid (GlcA) or α-L-iduronic acid (IdoA)), (1 → 4)linked to D-glucosamine (GlcN).5,6 The remarkable heterogeneity of HS comes from the variable degrees of sulfation found at the 2-O position of GlcA or IdoA and at the 6-O and
INTRODUCTION
The mucopolysaccharidoses (MPS) are lysosomal storage disorders characterized by genetic defects in the enzymes that degrade glycosaminoglycans (GAG), which are a class of linear, polyanionic polysaccharides. The enzyme dysfunction leads to lysosomal accumulation of undegraded substrate with associated pathology in multiple organs, often including the brain, and ultimately results in reduced life expectancy.1,2 The need to quantify GAG levels in biological samples (e.g., tissues, cerebrospinal fluid, urine, etc.) is thus of great interest for the study of MPS pathology and for determining the effectiveness of new treatments.3 Heparan sulfate (HS) is a GAG that accumulates in a number of neurological MPS disorders, © XXXX American Chemical Society
Received: June 11, 2019 Accepted: July 2, 2019 Published: July 2, 2019 A
DOI: 10.1021/acschemneuro.9b00328 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience
Figure 1. (A) Butanolysis of HS gives disaccharide products predominantly via cleavage of glycosidic bonds of uronic acid residues; (B) MS/MS spectrum of the major product from butanolysis of HS.
standard. We also sought to determine if the butanolysis assay can be applied to the structurally related GAGs heparin and heparosan. Heparin has the same backbone as HS but is more highly sulfated and has a much higher proportion of IdoA.6,19 Heparosan is a nonsulfated GAG of bacterial origin consisting of β-D-GlcA(1 → 4)-α-D-GlcNAc repeating units.20 Finally, we sought to demonstrate the utility of the assay by quantifying HS levels in MPS IIIA mouse brain tissues in order to determine if there are any differences in HS abundance in different regions of the MPS IIIA-affected brain.
3-O positions of GlcN. The latter can also be sulfated or acetylated at the 2-N position. Several mass spectrometric assays for HS have been reported which are based on depolymerization and desulfation by various means.7−9 For example, methanolysis of HS results in the formation of disaccharides which are amenable to analyis by liquid chromatography/tandem mass spectrometry (LCMS/MS). This simple method was used to analyze HS in various biological samples10−13 and to monitor the effectiveness of enzyme replacement therapy for MPS disorders in animal models of disease. 14−16 Recently, a library of methylated HS disaccharide standards was synthesized to assist in the identification of the major disaccharide peaks in the LC-MS/MS chromatograms from HS methanolysis.17 The results indicated that depolymerization of HS under methanolysis conditions (HCl/MeOH at 65 °C) to give disaccharides occurred predominantly via cleavage of the glycosidic bonds of uronic acid residues. Somewhat surprisingly, the two major disaccharide components identified both contained IdoA, suggesting that IdoA glycosidic bonds are more labile than the corresponding GlcA bonds. Despite the success of the methanolysis depolymerization method, recently it has been shown that butanolysis offers many improvements (Figure 1).18 Butanolysis (3 N HCl/nbutanol at 100 °C) was found to be more sensitive than methanolysis by approximately 70-fold, with maximum disaccharide concentrations observed after only 2 h. In contrast, the methanolysis reaction was incomplete even after 24 h. Another advantage of the butanolysis method is the improved chromatographic behavior of the released butylated disaccharides due to their greater lipophilicity compared with their methylated congeners. The advantages of butanolysis over methanolysis are clear; however, in order to fully realize the potential of this method, disaccharide reference materials are required. This would facilitate peak identification in the LC-MS/MS-chromatograms, including the minor peaks, and could result in identification of potential biomarkers. A suitable isotopelabeled internal standard is also required for the development of a quantitative LC-MS/MS assay with high accuracy and precision. The aims of this study were therefore to (i) synthesize butylated HS disaccharide reference standards for peak identification in the LC-MS/MS chromatograms from the butanolysis assay and (ii) to enable HS quantitation via the butanolysis assay with the addition of a deuterated internal
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RESULTS AND DISCUSSION The structures of the target HS disaccharides (1−8) are shown below (Figure 2). Previous studies13,17 on methanolysis of HS
Figure 2. Structures of the butylated HS disaccharides targeted for synthesis.
demonstrated that the predominant reaction products are the GlcN-(1 → 4)-uronic acid disaccharides (Figure 1A), resulting from cleavage of the more labile β-D- or α-L-glycosidic bonds. Disaccharides resulting from cleavage of α-D-GlcN bonds were not detected in the previous studies, and thus, these were not targeted for synthesis here. Under butanolysis conditions (3N butanolic HCl, 100 °C, 2 h), the newly formed butyl glycosidic bonds were expected to be predominantly α. However, given β-glycosides were detected as major components of the methanolysis reaction,17 their formation during butanolysis could not be excluded, and therefore, both α and β anomers were targeted for synthesis. While previous studies18 also showed that N-acetyl groups are labile to alcoholysis, the formation of minor amounts of N-acetylated disaccharides B
DOI: 10.1021/acschemneuro.9b00328 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience Scheme 1. Synthesis of Butyl GlcN-GlcA Disaccharides 1−4a
a Reagents and conditions: (a) NIS/TMSOTf, DCM, −78 °C → r.t., 20 h, 30−52%; (b) H2, 20% Pd(OH)2/C, n-BuOH, concd HCl, r.t., 24 h, 54− 75%; (c) H2, 20% Pd(OH)2/C, n-BuOH, Ac2O, r.t., 24 h, 14−60%.
Scheme 2. Synthesis of Butyl GlcN-IdoA Disaccharides 5−8a
a
Reagents and conditions: (a) NIS/TMSOTf, DCM, r.t., 2−3.5 h, 56−81%; (b) i. n-BuONa/n-BuOH, 2 h; ii. Amberlite IR-120 H+; iii. n-BuI, KHCO3, DMF, o/n, 31−61% (c) 20% Pd(OH)2/C, MeOH, concd HCl, r.t., 24 h, 65−71%; (d) 20% Pd(OH)2/C, MeOH, Ac2O, r.t., 24 h, 97%.
disaccharides 1 and 2 in moderate yields. Hydrogenolysis of disaccharides 12 and 14 in the presence of Ac2O directly converted the azide to the acetamide to give the N-acetate disaccharides 3 and 4 in low to moderate yields. The GlcN-IdoA disaccharides 5−8 were similarly prepared as shown in Scheme 2. The IdoA acceptors 15 and 16 or 17, prepared from 4′-methyphenyl 2-O-benzoyl-3-O-benzyl-4,6-Oisopropylidene-1-thio-α-L-idopyranoside25 (see Supporting Information) were glycosylated with donor 9 using NIS/ TMSOTf as promoter which resulted in the exclusive formation of the desired α-linked disaccharides, generally in improved yields (up to 81%). Deprotection, with or without N-acetylation, then furnished the target disaccharides 5−8. Interestingly, in the case of protected disaccharide 20, the 1 H NMR spectrum indicated that the IdoA residue was in equilibrium between the 1C4 and 2SO conformations,26,27 with larger coupling constants for J2,3 = J3,4 = 6.7 Hz, while J1,2 = 2.5 Hz and J4,5 = 4.2 Hz (Table 1).28 The conformation of the IdoA residue in the deprotected target disaccharides 7 and 8 reverted to 1C4 as indicated by 1H NMR spectroscopy (Table 1). Following preliminary LC-MS/MS analyses of the disaccharides (vide infra) which confirmed 1 as the major disaccharide present following butanolysis of HS, it was decided to prepare an isotope-labeled derivative of 1 for use as an internal standard. The butyl ester of protected disaccharide
could not be excluded, and these were also targeted for synthesis. Synthesis of Butylated HS Disaccharides. The synthetic methodology for the preparation of HS oligosaccharides has advanced significantly in the past two decades (for recent reviews, see refs 21−23). However, it remains a nontrivial undertaking because of the need for suitably protected monosaccharide building blocks, especially for IdoA which is not commercially available. Building on earlier work on the synthesis of disaccharides from methanolysis of HS,17 a similar synthetic strategy was followed, which enabled the synthesis of both free amino and N-acetylated disaccharides from the same intermediates. The target butyl GlcN-GlcA disaccharides were prepared via the glycosylation of acceptors 10 and 11, prepared from D-glucose (see Supporting Information), with the thioglycoside donor17 9. Compound 9 contains a nonparticipating azido group at C-2 to enable formation of the desired α- (1,2-cis) glycosidic linkage and subsequent conversion into a 2-amino or 2-acetamido group. Glycosylation of acceptor 10 with donor 9 and NIS/TMSOTf as promoter24 gave a 3.3:1 mixture of α/β-linked disaccharides (12 and 13) in 52% yield from which the desired 12 was carefully separated by flash chromatography (Scheme 1). Similar glycosylation of acceptor 11 gave only the α-linked disaccharide product 14 (J1′,2′ = 3.7 Hz). Global deprotection by hydrogenolysis with Pearlman’s catalyst afforded the target butyl GlcN-GlcA C
DOI: 10.1021/acschemneuro.9b00328 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience Table 1. Coupling Constants (J, Hz) Measured from 1H NMR Spectra (CDCl3 or CD3OD) for the L-Iduronic Acid Rings of Selected Compounds, with Predominant Conformation in Parentheses J1,2 J2,3 J3,4 J4,5
7 (1C4)
8 (1C4)
20 (1C4/2S0)
1.4 3.1 3.1 1.9
1.4 3.0 3.4 2.1
2.5 6.7 6.7 4.2
Supporting Information Figure S1 for an overlay of the three chromatograms). Using the retention time (RT) of the internal standard as a reference, the synthesized butyl amino disaccharide standards were analyzed using the same method (Figure 4). The MRM transition m/z 510.240−204.080 was also monitored for the analysis of any GlcNAc-GlcUA disaccharides (Figure S2). It was found that α-butyl GlcNGlcA disaccharide 1 and α-butyl GlcN-IdoA disaccharide 5 had nearly identical retention times, 1.60 and 1.61 min, respectively, and these match the major peak observed in HS, heparin, and heparosan. There are small amounts of disaccharides 3 and 7 in different ratios between the GAGs measured. The β-butyl GlcN-GlcA disaccharide 3 matched well with a minor peak in the LC-MS/MS of HS, heparosan, and heparin (Figures 3 and 4). There were only two peaks observed in the LC-MS/MS for butanolysis products of heparosan, and on the basis of the structure of heparosan, the major amino reaction product for heparosan was found to be the disaccharide 1, while the minor product for heparosan was the disaccharide 3. Taken together, α-butyl GlcN-GlcA disaccharide 1 gave one peak in the LC-MS/MS chromatogram, corresponding to the main peak in the chromatograms for butanolysis of HS and heparosan, confirming that disaccharide 1 is the major product for butanolysis of these GAGs. Much smaller than the amino disaccharides shown in Figure 3, the N-acetylated signals were barely observed for the GAGs, especially heparin (Figure S2). The butanolysis of heparosan had the largest detectable amount of these disaccharides, as would be expected for a fully N-acetylated GAG. The Nacetylated reaction products obtained from the butanolysis of heparosan were found to be butyl GlcNAc-GlcA disaccharides 2 and 6 (primary peaks) and disaccharides 4 and 8 (minor products). Regional Biodistribution of HS Disaccharides in the MPS IIIA Mouse Brain. To validate this HS quantitation method, we analyzed the HS disaccharide content in a naturally occurring murine model of MPS IIIA. Brains from 21-week-old wild-type control and MPS IIIA mice were microdissected to explore the hypothesis that there are regional
12 was first saponified with aq NaOH in MeOH-chloroform (Scheme 3). The crude carboxylate was then esterified under Scheme 3. Synthesis of Deuterium-Labelled Disaccharide Standard 22a
a Reagents and conditions: (a) i. 5M NaOH/MeOH/CHCl3/H2O, 1:7:2:1, r.t., 48 h; ii. n-BuI-d9, KHCO3, DMF, r.t. 24 h, 86% (2 steps); (b) 20% Pd(OH)2/C, MeOH, r.t., 24 h, 57%.
basic conditions with commercially available 1-iodobutane-d9 to give the disaccharide 21 in excellent overall yield (86%). Hydrogenolysis then gave the deuterated disaccharide 22 in an acceptable 57% yield. LC-MS/MS Analyses. Identification of the major disaccharide products from the butanolysis of HS, heparin, and heparosan were identified by comparing the retention times of each disaccharide to the internal standard d9 deuterated disaccharide 22. Figure 3 shows the chromatograms for each GAG after butanolysis digestion by monitoring the MRM (multiple reaction monitoring) transition m/z 468.245− 162.077 for the disaccharide GlcN-GlcUA18 (see also
Figure 3. Comparison of the chromatograms using the MRM transition m/z 468.245−162.077 obtained from the butanolic digestion of (a) HS (red line), (b) heparin (purple line), and (c) heparosan (green line) compared to the deuterated internal standard 22 (black dotted line, m/z 477.300−162.077, RT 1.57). All three GAGs show a dominant peak at RT 1.60 or 1.61 min, heparin had a secondary peak at RT 1.70 min, and heparosan had a secondary peak at RT 1.68 min, whereas HS had secondary peak at RT 1.69 and an additional peak at 1.52 min. D
DOI: 10.1021/acschemneuro.9b00328 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Figure 4. LC-MS/MS analysis of the 8 synthesized disaccharides standards (solid trace) compared to the deuterated internal standard 22 (dotted line, m/z 477.300−162.077, RT 1.57). Retention times for each disaccharide are marked on the chromatograms. MRM transitions monitored were m/z 468.245−162.077 and m/z 510.240−204.080 for odd and even numbered disaccharides, respectively.
with 1−5% (disaccharides 1 and 5) and 11−12% variability (disaccharides 3/7; n = 3). For the remaining HS disaccharides, both the intra- and interbatch coefficients of variation exceeded 15%, presumably due to low survival of these acetylated HS species during the digestion process. While accumulation of HS in MPS IIIA mouse brain has previously been demonstrated using mass spectrometric methods (e.g., refs 13,18,32), this is the first quantitative LC-MS/MS assay for HS measurement. Other laboratories have characterized HS disaccharide structures using enzymatic digestion methods and have demonstrated that HS GAGs are abnormally sulfated in MPS I, IIIA, and IIIB mouse tissues in their relative quantitation methods.33,34 The sulfation status of the HS cannot be determined under the conditions employed here due to the removal of sulfate groups in the acid butanolysis reaction. Comparison of the HS disaccharide content across the various brain regions in MPS IIIA mice revealed statistically significant regional differences for HS disaccharides 1, 2, 3, 4, 5, and 7 (p < 0.05). Of note, the olfactory bulb consistently contained significantly more HS disaccharides 1/5 and 3/7
differences in HS accumulation that correlate with the functional changes in affected mice (i.e., locomotor and cognitive impairments).29−31 HS disaccharides 1, 3, 5, and 7 were quantifiable in all regions of wild-type mouse brain. Significant genotype effects were found for all eight HS disaccharides, with elevated HS evident in all brain regions from MPS IIIA mice (p < 0.05; Figure 5). The overall amount of each HS disaccharide varied, with 1 α-butyl GlcN-GlcA and 5 α-butyl GlcN-IdoA the most abundant HS species measured. MPS IIIA brains were also enriched for HS disaccharides 3 and 7. However, the levels were approximately half that of disaccharides 1 and 5. The N-acetylated reaction products for HS disaccharides 2, 4, 6, and 8 were detected at low levels in both the control and MPS IIIA mouse brains. The intrabatch and interbatch coefficients of variation in the assay were also determined using one representative MPS IIIA mouse whole brain homogenate. The intrabatch coefficient of variation values from four replicates assessed in three independent experiments were 6−7% and 11−16% for disaccharides 1/5 and 3/7, respectively. The interbatch coefficient of variation was also low for these HS species E
DOI: 10.1021/acschemneuro.9b00328 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Figure 5. Concentration of HS disaccharide in 21-week-old wild-type control and MPS IIIA mouse brain homogenates (n = 5 male mice per genotype and area). HS is expressed as the counts of each specific HS disaccharide versus the d9 labeled disaccharide internal standard (IS) counts. The brain regions depicted in the schematic diagram include the olfactory bulb (OB), cortex (Ctx), striatum (Str), hippocampus (Hp), thalamus (Th), cerebellum (Cb), and brainstem (Bs). Data are mean + 1 SEM.
than other brain regions (except cerebellum; p < 0.05; Figure 5). In contrast, the cerebral cortex contained the lowest amount of HS disaccharides 1, 3, 5, and 7 compared with other brain regions, though this was still >22-fold that of wild-type levels. Regional expression of HS has been examined in MPS IIIA mouse brain using immunohistochemical methods, with HS expression semiquantitated on the basis of the staining intensity and the relative amount of positive staining area.35
HS expression was dependent on cortical layer as well as brain region (piriform and retrosplenial cortex, amygdala, hippocampus). The olfactory system has not been a focus in this or any other studies in either animal models or in patients and its role in MPS IIIA disease pathogenesis requires further examination. In infantile neuronal ceroid lipofuscinosis mice, the rate of accumulation of autofluorescent storage material in the thalamus and cerebral cortex was found to be similar.36 F
DOI: 10.1021/acschemneuro.9b00328 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Children’s Health Network Animal Care Facility. Genotyping was conducted using published methods.38 All institutional and national guidelines for the care and use of laboratory animals were followed. Male mice with advanced disease (21 weeks old) were humanely killed with carbon dioxide gas and perfused with ice-cold PBS to remove blood. The brain was removed, divided along the midline, and microdissected on a Petri dish chilled on ice to obtain the olfactory bulb, cerebral cortex, striatum, hippocampus, thalamaus, cerebellum, and brainstem. Tissues were snap frozen in liquid nitrogen and stored at −80 °C. Brain regions were homogenized in 500 μL of 20 mM Tris, 500 mM NaCl, pH 7.4 in Lysing Matrix D tubes (MP Biomedicals) using a Precellys 24 Tissue Homogenizer (2 cycles of 6500 rpm of 30 s each at 4 °C). Total protein was quantitated using a MicroBCA kit (Pierce). Homogenates (20 μg protein) were prepared to 50 μL of total volume in 20 mM Tris, 500 mM NaCl, pH 7.4 in glass tubes and freeze-dried overnight. Butanolysis, data acquisition, and analysis was then conducted as described in “LC-MS/MS analyses”. Statistics. Data was analyzed using Graph Pad Prism (v7.04) using a two-way ANOVA with genotype and brain region as the main effects followed by a Bonferroni posthoc multiple comparisons test. p ≤ 0.05 was considered statistically significant. n-Butyl (2-Azido-3,4,6,-tri-O-benzyl-2-deoxy-D-glucopyranosyl)-(1 → 4)-n-butyl 2,3-di-O-benzyl-α-D-glucopyranosiduronate (12, 13). A solution of thioglycoside donor 917 (22 mg, 0.04 mmol) and acceptor 10 (15 mg, 0.03 mmol) and freshly dried AW300 mol. sieves (40 mg) in anhydrous DCM (3 mL) was stirred at r.t. for 30 min under N2. NIS (9.7 mg, 0.04 mmol) was added, and the solution was stirred at −78 °C for 10 min. TMSOTf (1.1 μL, 0.006 mmol) was then added, and the resulting solution was stirred at −78 °C and slowly allowed to warm up to r.t. for 20 h under N2. The reaction was quenched by addition of Et3N (2 mL), sat. aq NaHCO3 solution (5 mL) and 10% aq Na2S2O3 solution (5 mL) to pH 8. The mixture was filtered through Celite and the filter cake was washed with DCM. The combined filtrate and washings were then washed with 10% aq Na2S2O3 solution (5 mL), dried (MgSO4), filtered, and concentrated. The residue was then purified by flash chromatography (EtOAc/n-hexane, 1:9) to give α-isomer 12 as a colorless oil (10 mg), β-isomer 13 as a colorless oil (3 mg), and a mixed fraction (12, 13) as a colorless oil (2 mg). In total, it was a 3.3:1 α/β mixture in 52% yield. 12: Rf = 0.35 (EtOAc/ n-hexane, 1:4); [α]24 D +27.85 (c 1.24, CHCl3); 1 H NMR (500 MHz, CDCl3): δ 7.34−7.24 (m, 23H, Ph), 7.11−7.09 (m, 2H, Ph), 5.64 (d, 1H, J1′,2′ = 3.7 Hz, H-1′), 5.05, 4.85 (ABq, 2H, JA,B = 10.6 Hz, CH2Ph), 4.84 (s, 2H, CH2Ph), 4.75, 4.48 (ABq, 2H, JA,B = 10.9 Hz, CH2Ph), 4.74, 4.62 (ABq, 2H, JA,B = 11.2 Hz, CH2Ph), 4.71 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.59, 4.42 (ABq, 2H, JA,B = 12.1 Hz, CH2Ph), 4.20 (d, 1H, J4,5 = 9.4 Hz, H-5), 4.10 (dt, 1H, J = 6.9, 10.8 Hz, CO2CH2−a), 4.06 (dd, 1H, J2,3 = 9.2 Hz, J3,4 = 8.8 Hz, H-3), 4.02 (dd, 1H, H-4), 3.97 (dt, 1H, J = 6.8, 10.8 Hz, CO2CH2−b), 3.85 (dd, 1H, J2′,3′ = 10.3 Hz, J3′,4′ = 9.0 Hz, H-3′), 3.77−3.73 (m, 2H, H-4′, H6a′), 3.65 (dt, 1H, J = 6.8, 9.9 Hz, OCH2−a), 3.61−3.57 (m, 2H, H-2, H-6′b), 3.45−3.41 (m, 2H, H-5′, OCH2−b), 3.29 (dd, 1H, H-2′), 1.68−1.53 (m, 4H, 2 × CH2), 1.45−1.39 (m, 2H, CH2), 1.30−1.24 (m, 2H, CH2), 0.94 (t, 3H, J = 7.4 Hz, CH3), 0.82 (t, 3H, J = 7.4 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ 169.9 (C = O), 138.4, 138.2, 137.9, 137.8, 128.4, 128.4, 128.3, 128.2, 128.1, 127.99, 127.9, 127.8, 127.6, 127.6, 127.5 (Ph), 97.7 (C-1′), 97.2 (C-1), 81.3 (C-3), 80.0 (C-3′), 79.8 (C-2), 77.8 (C-4′), 75.3, 75.3, 74.7, 73.5, 73.2 (5 × CH2Ph), 74.6 (C-4), 70.9 (C-5′), 70.1 (C-5), 68.5 (OCH2), 67.6 (C6′), 65.7 (CO2CH2), 63.1 (C-2′), 31.42 (CH2), 30.30 (CH2), 19.28 (CH2), 18.98 (CH2), 13.80 (CH3), 13.67 (CH3); 13: Rf = 0.31 (EtOAc/n-hexane, 1:4); 1H NMR (500 MHz, CDCl3): δ 7.34−7.11 (m, 25H, Ph), 5.03, 4.77 (ABq, 2H, JAB = 11.4 Hz, PhCH2), 4.81, 4.75 (ABq, 2H, JAB = 10.8 Hz, PhCH2), 4.73, 4.51 (ABq, 2H, JAB = 10.9 Hz, PhCH2), 4.72, 4.54 (ABq, 2H, JAB = 12.1 Hz, PhCH2), 4.67 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.38 (d, 1H, J1′,2′ = 7.7 Hz, H-1′), 4.35, 4.30 (ABq, 2H, JAB = 12.1 Hz, PhCH2), 4.23 (dt, 1H, J = 6.6, 10.8 Hz, CO2CH2−a), 4.22 (d, 1H, J4,5 = 9.8 Hz, H-5), 4.12 (dt, 1H, J = 6.6, 10.8 Hz, CO2CH2−b), 4.06 (dd, 1H, J3,4 = 8.8 Hz, J4,5 = 9.8 Hz, H-4), 3.91 (dd, 1H, J2,3 = 9.6 Hz, H-3), 3.65 (dt, 1H, J = 6.7, 9.7 Hz,
However, quantitation of neuron loss within these regions demonstrated that it occurred first within the thalamus and only subsequently impacted the cortex. This potentially indicates that factors downstream of substrate accumulation mediate neuron toxicity or that there are cell-specific vulnerabilities to the effects of substrate accumulation. The survival of N-acetyl groups under the reaction conditions leading to the formation of N-acetylated disaccharides as minor products was assessed by comparing the pre- and postdigestion chromatograms for each of the synthesized disaccharides. It was expected that the N-acetyl group would not survive the digestion process. The post digestion chromatograms of disaccharides 2, 4, 6, and 8 had
