Synthesis and characterization of biotin derivatives as multifunctional oligosaccharide tags

The synthesis of novel multifunctional oligosaccharide tags with amino, azido, and alkyne termini is described. Tags with an amino terminus can be introduced into the carbohydrate at the reducing end through reductive amination, while 1,3-dipolar cycloaddition (“click chemistry”) can be used to label azido sugars with oligosaccharide tags having an alkyne terminus and vice versa.


Introduction
Glycosylation of proteins represents the most structurally elaborate form of protein posttranslational modification.Such a process can affect folding, stability, and biological activity of proteins; further it can also influence their interaction with other biomolecules.These glycans have been found to participate in molecular recognition, inter-and intracellular signaling, embryonic development, fertilization, immune defense, inflammation, cell adhesion and division processes, viral replication and parasitic infections. 1 The analysis of carbohydrate oligomers is crucial for complete characterization of polysaccharides and glycoconjugates.The analysis of biologically important oligosaccharides is complicated by structural complexity (stereochemistry, linkage, and anomericity), by poor detectability in chromatography, and by limitation in sample amount. 2,30][11][12][13] The label is most commonly introduced by reductive amination. 14,15ur group has previously synthesized a prototypical multifunctional small-molecule tag (Figure 1) for covalent introduction into oligosaccharides which helps to increase the UV sensitivity, separation and isolation of the labeled oligosaccharides. 16The structure includes a primary amine for reductive amination, a UV-active portion, and biotin functionality.The tag is different in that many other small-molecule tags described in the literature have an aromatic amine functionality with lower nucleophilicity. 2The primary amine group of the new tag was successfully used in aminomethyl-C-glycoside formation through in situ reduction of the intermediate imine by sodium cyanoborohydride (NaBH 3 CN).Low molecular weight carbohydrates and linear oligosaccharides could be labeled with high efficiency.However, subsequent experiments with higher molecular weight oligosaccharides resulted in greatly reduced labeling efficiency with concurrent aldehyde reduction (alditol formation) in the competing reaction channel.Here, we report modifications in the structure of 1.The impact that length and polarity of the tether between the primary amino group and the chromophore had on the derivatization reaction was systematically investigated.We also introduced structural changes to the UV core.
As an alternative to reductive amination, oligosaccharide azides or alkynes can also be labeled through Huisgen 1,3-dipolar cycloaddition with alkyne tags and azide tags, respectively, to afford triazoles.8][19][20][21][22][23] The copper-catalyzed version of this reaction has proven very successful which was first investigated independently by Sharpless 24 and Meldal 25 groups.It has become a mild, efficient, and widely-used method to synthesize five-membered ring 1,2,3-triazoles and has high compatibility with functional groups (alcohols, carboxylic acids, amines) in different solvent systems, including water.In the field of carbohydrate chemistry, click chemistry has been used for the synthesis of glycoconjugates 26,27 and carbohydrate macrocycles 28,29 in which a sugar possessing an azido function is grafted onto a saccharide, 30 a peptide, 31 or a polymeric chain. 32Here we report the synthesis of oligosaccharide tags bearing azido and alkyne termini which can be used to label oligosaccharides with alkyne or azido termini, respectively.We have applied this method to the covalent labeling of carbohydrate azides with new small-molecule alkyne-tags.Labeling of sugar azides with alkyne-tags or alternatively sugar alkynes with azidetags under Huisgen cycloaddition conditions is possible with excellent yields and regioselectivity.

Results and Discussion
We had previously observed that the efficiency of labeling oligosaccharides with compound 1 decreased with increasing molecular mass and branching of the oligosaccharides; a fact that was attributed to insufficient length of the spacer between biotin and the amino group.The first modification we pursued was to increase the length of the tether by incorporating propane-1,3diamine and hexane-1,6-diamine.D-Biotin was activated with 1,1'-carbonyldiimidazole in dry freshly-distilled dimethylformamide (DMF) to give compound 2 (2.05 g, 6.96 mmol, 85%) (Table 1).Moisture and amines in DMF are deleterious as they lead to hydrolysis and aminolysis, respectively, of the reactive intermediate 2. Hence the use of dry, fresh DMF is crucial to the success of the reaction.The activated biotin 2 can be isolated and is stable under dry, inert (under nitrogen) conditions.Compound 2 was used for coupling with 4aminomethylbenzoic acid to give 3 (2.78g, 7.36 mmol, 90%) in excellent yield.In turn, compound 3 was activated with 1,1'-carbonyldiimidazole and was immediately added to a vigorously stirred solution of excess diamine (propane-1,3-diamine and hexane-1,6-diamine) to give 3a (98 mg, 0.23 mmol, 85%) and 3b (117 mg, 0.25 mmol, 64%) in good to very good yields.Use of excess solution of the diamine helps to avoid addition of activated 2 on both amino groups of the diamine.Surprisingly, the solubility of 3a and 3b was found to be better in polar solvents such as water and methanol in comparison to the original compound 1.This led us to the additional hypothesis that the observed decrease in labeling efficiency may be connected to the solubility of the tag and the oligosaccharide in methanol or water.Consequently, to further improve the solubility of the tag, we incorporated 2,2'-[ethane-1,2-diylbis(oxy)]diethanamine as a spacer to obtain 3c (105 mg, 0.21 mmol, 56%) which showed very good solubility in polar solvents and much improved labeling efficiency as discussed further below.For the investigation of 1,3-dipolar cycloaddition, we prepared compounds 3d and 3e.Compound 3d requires a carbohydrate azide for coupling while compound 3e requires a carbohydrate alkyne.Compounds 3d (176 mg, 0.42 mmol, 80%) and 3e (295 mg, 0.66 mmol, 50 %) were obtained from 3 after 1,1'-carbonyldiimidazole activation with an equimolar ratio of prop-2-yn-1-amine and 2azidoethanamine (2.35 g, 27.30 mmol, 56%) 33 , respectively.In contrast to this result, the primary amine group of 4-ethynylbenzenamine failed to couple with 2 in DMF during the preparation of 5.This was attributed to lower nucleophilicity of the amino group due to resonance with the aromatic ring.However, coupling promoted by N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) [34][35][36][37] gave 5 (202 mg, 0.59 mmol, 72%).Subsequent reaction of 5 with 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine gave 5a (73 mg, 0.13 mmol, 56%) in moderate yield.

Scheme 1. Synthesis of 4a and 5a.
In an effort to increase the UV sensitivity of the label, we introduced an aromatic nitro moiety in the tags.Compound 6 (0.96 g, 2.35 mmol, 79%) (Scheme 2) was synthesized by coupling biotin with 4-amino-2-nitrobenzoic acid with the help of HATU.Compound 6 serves as intermediate for the synthesis of two tags, 6a (85 mg, 0.19 mmol, 52%) and 6c (105 mg, 0.21 mmol, 56%) downstream.The UV activity of 6a and 6c is not enhanced significantly as compared to original tag 1.This was attributed to the electron withdrawing nature of all three functional groups on the aromatic ring in compound 6a and 6c including the nitro group.However, the nitro group on the aromatic ring can be selectively reduced SnCl 2 38 in anhydrous ethanol without affecting the other functional groups [39][40][41][42] to produce fluorescent compounds 6b (20 mg, 0.05 mmol, 71%) and 6d (quantitative yield).

Scheme 2. Synthesis of 6a-d.
To evaluate the labeling efficiency of the tag (3c), we labeled standard oligosaccharides (Nacetylglucosamine, maltose, maltotriose, lacto-N-fucopentaose-II (LNFP-II) (human milk oligosaccharide) and lacto-N-difucohexaose-II (LNDFH-II) (human milk oligosaccharide)) with 3c using a previously established procedure 16 .Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) analysis (Figure 2) of the labeling reaction showed that compound 3c can be covalently attached to oligosaccharides without any side reaction such as alditol formation to give quantitative conversion of oligosaccharides to their respective labeled derivatives.

Conclusions
Novel oligosaccharide tags were synthesized and characterized.During the synthesis, we observed that the intermediate, activated biotin can be isolated and is stable under dry and inert conditions.The compounds containing a primary amine group can be used to label oligosaccharides through reductive amination.In addition, compounds containing an azide functional group can be used to label oligosaccharide with an alkyne terminus and vice versa.

Experimental Section
General Procedures.Thin layer chromatography (TLC) was performed on plates coated with 0.25 mm of silica gel with UV-indicator (254 nm).Column chromatography was performed on silica gel (Premium Rf Grade, porosity 60Å, particle size 40-75µm, 200x400 mesh).

2-Azidoethanamine.
The compound was prepared according to the method reported by Foster and Newman. 33In brief, 2-bromoethylamine hydrochloride (10 g, 48.81 mmol) and sodium azide (6.35 g, 97.68 mmol) dissolved in water (20 mL) in a round bottom flask (50 mL) attached to a water condenser.The solution was heated to 70 °C for 5 hours.After cooling to room temperature, potassium hydroxide (20 g) was added, and 2-azidoethyl amine was separated by steam distillation.Addition of potassium hydroxide (20 g) to the distillate caused phaseseparation of the free amine from the aqueous phase.The free amine was extracted from the aqueous phase with Et 2 O. Solvent removal at 0 °C under reduced pressure gave the crude product (3.9 g).Distillation of the crude product from potassium hydroxide pellets gave the product as colorless oil (2.35 g, 27.30 mmol, 56%); 1 H-NMR (CD 3 OD), δ 2.76 (t, J =6.0 Hz, 2H), 3.38 (t, J =6.0 Hz, 2H). 13

Table 1 . Synthesis of compound 3a-e
The samples were coprecipitated with 2,5-dihydroxybenzoic acid (DHB, 5 mg/100 µL in acetonitrile:water (1:1) and were irradiated by a N 2 -laser (λ = 335 nm) unless stated otherwise.Melting point ranges were determined using a Thomas Hoover Capillary Melting Point Apparatus and are uncorrected.During purification of labeled oligosaccharides, a heated CentriVap Concentrator (<?xml:namespace prefix = st1 ns = "urn:schemas-microsoftcom:office:smarttags" />Labconco, Kansas City, MO) was used to remove solvent and concentrate the samples.Aliquots of sample were desalted using the Porous Graphitized Carbon (PGC) cartridges (Supplier-Thermo Electron Hypersil Keystone, UK).The cartridges were washed with 60% acetonitrile and deionized water (H 2 O) prior to use.Micro liter volumes of solvent were removed in a heated (40 °C) CentriVap Concentrator with spinning at reduced pressure (12 mTorr).
13-NMR,13C-NMR spectra were recorded on a Varian Mercury 300 MHz NMR spectrometer with specified deuterated solvents.Exact mass measurements were performed on the AccuTOF DART (Direct Analysis in Real Time, Time-of-Flight Mass Spectrometer) (JEOL Ltd.,Tokyo, Japan). Marix-Assisted Laser Desorption/Ionization (MALDI) Time Of Flight (TOF) mass spectra were recorded on a Shimadzu/Kratos (Columbia, MD) AXIMA CFR mass spectrometer in reflectron mode.