Synthesis, separation, and characterization of amphiphilic sulfated oligosaccharides enabled by reversed-phase ion pairing LC and LC-MS methods

Abstract

Synthesis of amphiphilic oligosaccharides is problematic because traditional methods for separating and purifying oligosaccharides, including sulfated oligosaccharides, are generally not applicable to working with amphiphilic sugars. We report here RPIP-LC and LC-MS methods that enable the synthesis, separation, and characterization of amphiphilic N-arylacyl O-sulfonated aminoglycosides, which are being pursued as small-molecule glycosaminoglycan mimics. The methods described in this work for separating and characterizing these amphiphilic saccharides are further applied to a number of uses: monitoring the progression of sulfonation reactions with analytical RP-HPLC, characterizing sulfate content for individual molecules with ESI-MS, determining the degree of sulfation for products having mixed degrees of sulfation with HPLC and LC-MS, and purifying products with benchtop C18 column chromatography. We believe that the methods described here will be broadly applicable to enabling the synthesis, separation, and characterization of amphiphilic, sulfated, and phosphorylated oligosaccharides and other types of molecules substituted to varying degrees with both anionic and hydrophobic groups.

Figure 1

Sulfonation of N-cbz aminoglycosides (kanamycin, apramycin, and neomycin) with Pyr·SO₃ or ClSO₃H resulted in products with varied degrees of sulfation (DS). RPIP-HPLC methods developed here were used to separate reaction products based on degree and position of sulfate groups. Sulfonation of N-cbz neomycin was complete, giving persulfated product, nz7. Sulfonation of N-cbz kanamycin and N-cbz apramycin produced per sulfated derivatives kz7 and az6, respectively, as well as under-sulfated kanamycin derivatives (kz5, kz6) and under-sulfated apramycin derivatives (az4, az5), where the number indicates the number of sulfate groups attached.

Figure 2

Representative comparison of analytical HPLC chromatograms from various RPIP conditions eluting persulfated N-cbz neomycin. Each chromatogram was generated from a 20 min. gradient increase in percent ACN, and the ending percent ACN was then maintained isocratic for an additional 22 min. (A) 10–80% ACN; 10 mM ammonium acetate, 10 mM TMAI, pH = 7.3. (B) 10–80% ACN; 10 mM ammonium acetate, 0.8 mM TMAI, pH = 5.0. (C) 10–80% ACN; 10 mM ammonium acetate, 0.8 mM TMAI, pH = 7.4. (D) 10–80% ACN; 10 mM ammonium acetate, 0.8 mM TMAI, pH = 8.0. (E) 10–70% ACN; 10 mM ammonium acetate, 10 mM TBAI, pH = 7.3. (F) 10–30% ACN; 10 mM ammonium acetate, 0.8 mM TEA, pH = 8.3.

Figure 3

Tuning of the ESI-ion trap mass spectrometer is critical for decreasing sulfate fragmentation and detecting the many ionic states of highly sulfated products. . Panel A: Before tuning, direct inject ESI of persulfated N-cbz neomycin, nz7, detects only desulfated ions m/z = 788.51 [M-5SO₃ + 5H]⁻² and m/z = 828.12 [M-4SO₃ + 4H]². Panel B: After tuning, higher molecular weight ions are more abundant and persulfated nz7 is observed in various adducts with ion pairing reagents (denoted in Table 1).

Figure 4

Separation of N-cbz O-sulfonated kanamycin derivatives that differ by degree of sulfation and the position of sulfate groups using RPIP-HPLC interfaced with an ESI- ion trap mass analyzer. Peaks for all sulfonation products are shown in the total ion chromatogram (TIC, A). The extracted ion chromatograms (B–D) were extracted based on m/z ratios that fall within the range predicted for a degree of sulfation (5, 6, or 7 sulfate groups); see Table 1 for an example of predicted m/z ratios. Ion chromatograms were extracted from the TIC for the indicated degrees of sulfation (DS): (B) persulfated, DS = 7, kz7; (C) DS = 6, kz6; (D) DS =5, kz5.

Figure 5

RPIP-HPLC-MS total ion chromatograms (TICs) of reaction products from the sulfonation of N-cbz aminoglycosides. Shown are product mixtures from the sulfonation of kanamycin and apramycin with Pyr·SO₃ (A and B, respectively) and the sulfonation of neomycin and apramycin with ClSO₃H (C and D, respectively). A and B demonstrate separation of different sulfated states of N-cbz kanamycin (DS = 5–7) and N-cbz O-sulfonated apramycin (DS = 4–6), respectively. O-sulfonation with ClSO₃H afforded persulfated products: (C) nz7 and (D) az6.

Figure 6

RPIP-HPLC to follow reaction progression over time for O-sulfonation of N-cbz neomycin. Following addition of ClSO₃H, reaction aliquots were analyzed at: (A) 5 min, (B) 15 min, (C) 20 min, (D) 50 min, (E) 275 min. Percent of reaction product that is persulfated product (nz7) is indicated next to the nz7 peak at 28 minutes.

Figure 7

Adaptation of RPIP-HPLC method developed here to separate sulfonated products from reaction mixtures by gravity flow benchtop C18 resin. N-cbz O-sulfonated neomycin was eluted in 10 mM ammonium acetate, initially in 100% water, and then with addition of 30% ACN as indicated by the dashed arrow. 1 mL fractions were collected and analyzed for UV absorbance at 258 nm. UV-absorbing fractions were pooled, freeze-dried and injected on analytical HPLC, detected at 258 nm.

Scheme 1

Representative synthesis showing concept for making N-aryl O-sulfonated aminoglycosides; shown is kanamycin. a) R-NHS, NaHCO₃, H₂O, 23 °C, 12–16 hr, 30–85%; b) i: Pyr·SO₃, DMF, anhydrous pyr, 66 °C, 5–7 hr ii: H₂O, 10 mM NaOH, 4 °C, 45 65%; c) i: ClSO₃H, pyr, 57 °C, 4–6 hr, ii: H₂O, NaHCO₃, 35–95%.