Metabolic oligosaccharide engineering with N-Acyl functionalized ManNAc analogs: cytotoxicity, metabolic flux, and glycan-display considerations

Abstract

Metabolic oligosaccharide engineering (MOE) is a maturing technology capable of modifying cell surface sugars in living cells and animals through the biosynthetic installation of non-natural monosaccharides into the glycocalyx. A particularly robust area of investigation involves the incorporation of azide functional groups onto the cell surface, which can then be further derivatized using "click chemistry." While considerable effort has gone into optimizing the reagents used for the azide ligation reactions, less optimization of the monosaccharide analogs used in the preceding metabolic incorporation steps has been done. This study fills this void by reporting novel butanoylated ManNAc analogs that are used by cells with greater efficiency and less cytotoxicity than the current "gold standard," which are peracetylated compounds such as Ac₄ ManNAz. In particular, tributanoylated, N-acetyl, N-azido, and N-levulinoyl ManNAc analogs with the high flux 1,3,4-O-hydroxyl pattern of butanoylation were compared with their counterparts having the pro-apoptotic 3,4,6-O-butanoylation pattern. The results reveal that the ketone-bearing N-levulinoyl analog 3,4,6-O-Bu₃ ManNLev is highly apoptotic, and thus is a promising anti-cancer drug candidate. By contrast, the azide-bearing analog 1,3,4-O-Bu₃ ManNAz effectively labeled cellular sialoglycans at concentrations ∼3- to 5-fold lower (e.g., at 12.5-25 µM) than Ac₄ ManNAz (50-150 µM) and exhibited no indications of apoptosis even at concentrations up to 400 µM. In summary, this work extends emerging structure activity relationships that predict the effects of short chain fatty acid modified monosaccharides on mammalian cells and also provides a tangible advance in efforts to make MOE a practical technology for the medical and biotechnology communities.

Figure 1. Use of SCFA-derivatized ManNAc analogues to increase flux into the sialic acid pathway

(a) Under normal cell culture conditions with most mammalian cells type, approximately one million (10⁶) molecules of ManNAc analogue must be added to the culture media to install one analogue-derived sialoside on the cell surface (b). (c) Peracetylation of ManNAc increases the efficiency ∼ 600 fold, such that only ∼1,600 molecules of analogue needs to be added to the media to install one surface sialoside; this gain in efficiency is presumably due to the enhanced membrane permeability of the acylated analog, which was consistent with the even greater efficiency of perbutanoylated analogue (e.g., Bu₄ManNAc, where ∼500 molecules were required (d)). (e) As a final step in increasing efficiency, omission of one ester-linked butyrate increased efficiency yet another ∼25% with “1,3,4” analogues being high flux and “safe” (e.g., with negligible side effects such as the cytotoxicity seen for per-acylated analogues). (f) By contrast, tributanoylated analogues with a “3,4,6” pattern of substitution showed enhanced cytotoxicity and the ability to modulate biological responses (such as signaling pathways) via “whole molecule” mechanisms (as discussed in detail elsewhere (Elmouelhi et al. 2009; Wang et al. 2009)). Examples of “R” groups are shown in Figure 2.

Figure 2. Structures of the ManNAc analogues used in this study

Top row: acetyl- and n-butanoyl-derivatized analogues with the natural ManNAc “core” structure; middle row: analogues with a ketone group (highlighted in blue); bottom row: analogues with an azide group (highlighted in red). In all cases the OH group resulting from the “missing” n-butanoyl of “1,3,4” and “3,4,6” analogues is highlighted in yellow and the four previously unreported compounds are indicated by the boxes.

Figure 3. Cytotoxic properties of SCFA-derivatized N-acyl, N-levulinoyl, and N-azido ManNAc analogues

(a) Comparison of the cytotoxicity of ManNAc and ManLev when incubated with Jurkat cells for five days in the free monosaccharide and peracetylated forms. (b) Comparison of acylation patterns (e.g., peracetylation compared to perbutanoylation or the “1,3,4” vs. the “3,4,6” tributanoylated analogues) of the natural ManNAc “core” with the same information shown for the respecitive ManNLev analogues in (c) and for ManNAz in (d). Panels (e) and (f) show a direct comparison of the three JV-acyl varieties of the “3,4,6” vs. the “1,3,4” tributanoylated analogues, respectively. Error bars shown in (e) and (f) represent SEM from n ≥ 3 replicates (note that in (a)-(d), error bars were omitted for clarity).

Figure 4. Comparison of the N-acyl group and time dependence of ManNAc analogue cytotoxicity

(a) The relative number of Jurkat cells incubated with 3,4,6-O-Bu₃ManNAc for various lengths of time is shown. (b) The long term (15 day) impact of SCFA-derivatized ManNAz analogue treatment (comparison with short term effects can be made with the data shown in Fig. 3d or panel (c)). (c) Cell counts based on the starting, rather than the final, number of cells.

Figure 5. Caspase activation – a surrogate measure for apoptosis

Caspase activity displayed for analogues at 20 μM, 50 μM, 100 μM, and 200 μM showing time and dose-dependent onset of apoptosis. Panels (a) and (c) represent ManNLev-based analogues and panels (b) and (d) shows the equivalent data for ManNAz-based compounds; also, panels (a) and (b) depict caspase 3/7 activity and panels (c) and (d) show caspase 9 activity. In all cases, data for 5 and 24 hour time points are shown. Error bars represent the SEM for n ≥ 3 replicates.

Figure 6. Cell cycle arrest and apoptosis analysis of analogue-treated Jurkat cells

Cells were incubated for 3 days with (a) 3,4,6-O -Bu₃ManNLev, (b) 3,4,6-O-Bu₃ManNAz, (c) Bu₄ManNAz, and (d) 1,3,4-O-Bu₃ManNAz after which the DNA content was measured by PI/RNase A staining.

Figure 7. Comparison of Azido-ManNAc flux in Jurkat cells

Intracellular sialic acid levels in cells incubated with peracetylated and three varieties of butanoylated ManNAz analogues (panel a) or ManNAc analogues (panel b). Error bars represent the SEM for n ≥ 3 replicates.

Figure 8. Comparison of glycoconjugate labeling with Ac₄ManNAz and 1,3,4-O-Bu₃ManNAz by bioorthogonal glycan blots

(a) Jurkat, (b) SW 1990, (c) MDA-MB-231, (d) CHO, or (e) PANC-1 cells were metabolically labeled with either analogue for 48 h and cell surface azido groups were conjugated with biotin-alkyne, lysed, and equal quantities of protein were loaded for each sample, resolved by SDS-PAGE, and visualized by incubation with streptavidin-HRP. In all cases, samples were treated with (from left to right) 12.5, 25, 50, 100, 150 μM Ac₄ManNAz or 1,3,4-o-Bu₃ManNAz. Lanes indicated with (-) represent control samples from cells treated with neither analogue. (f) The intensity of the labeled glycoconjugates in each lane was measured using NIH ImageJ software and samples from a particular cell line were compared to the signal from cells incubated with 12.5 μM Ac₄ManNAz. Error bars represent the SEM for n ≥ 3 replicates.

Figure 9. Comparison of surface labeling with 1,3,4-O-Bu₃ManNAz and Ac₄ManNAz by flow cytometry

(a) PANC-1, SW1990, Jurkat and CHO cells treated with (or without) 1,3,4-O-Bu₃ManNAz (black bars) or Ac₄ManNAz (outline bars) for two days, detached and incubated for 1.0 h with and click-labeled with Alexa Fluor®488 alkyne as described in experimental procedures; these data are obtained from n ≥ 3 flow cytometry replicates, for which representative histograms are shown in panel (b) for PANC-1 and SW1990 cells. (c) Summary of the geometric means of samples treated with 1,3,4-O- Bu₃ManNAz divided by the geometric means of their Ac₄ManNAz counterparts. Error bars represent the SEM for n ≥ 3 replicates.

Figure 10. Comparison of surface labeling with Ac₄ManNAz and 1,3,4-O-Bu₃ManNAz by fluorescence microscopy

Representative images of PANC-1, SW1990, and CHO cells after treatment with 100 μM Ac₄ManNAz or 1,3,4-O-Bu₃ManNAz followed by labeling with Alexa488-Alkyne to detect surface azide groups and DAPI to stain nuclei (the procedure for labeling of cells is described in detail in the text). The fluorescence intensity landscape is shown for dotted the line indicated on each image. Scale bar = 25 μm. Representative images of analogue-treated cells are given in the Supporting Information.