Protocol to synthesize sequence-controlled glycooligomers for tumor targeting in mice

Summary

Due to the higher and more rapid consumption of carbohydrates by cancer cells compared to normal cells, carbohydrates can be effectively employed as a targeted therapeutic strategy for tumor treatment. Here, we present a protocol for synthesizing sequence-controlled glycooligomers using both solution-phase and solid-phase systems. We outline detailed procedures for evaluating the safety and tumor-targeting properties of the sequence-controlled glycooligomers in vivo.

For complete details on the use and execution of this protocol, please refer to Chen et al.¹

Graphical abstract

Highlights

• •Synthesis of azide, silyl-alkyne, and saccharide glycooligomer blocks
• •Synthesis of sequence-controlled glycooligomers in both solid-phase and solution-phase systems
• •In vivo study of sequence-controlled glycooligomers using the LoVo tumor model

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Due to the higher and more rapid consumption of carbohydrates by cancer cells compared to normal cells, carbohydrates can be effectively employed as a targeted therapeutic strategy for tumor treatment. Here, we present a protocol for synthesizing sequence-controlled glycooligomers using both solution-phase and solid-phase systems. We outline detailed procedures for evaluating the safety and tumor-targeting properties of the sequence-controlled glycooligomers in vivo.

Before you begin

The protocol described here demonstrates the synthesis and in vivo study of sequence-controlled fluorescent glycooligomers. ¹H and ¹³C NMR spectra were recorded using a Bruker AVA400 spectrometer (400 and 100 MHz, respectively) at a temperature of 298 K in deuterated solvents. High-resolution mass spectroscopy was performed on a Thermo Q Exactive Focus mass spectrometer. Analytical high-performance liquid chromatography (HPLC) is performed on an Agilent 1260 Infinity II HPLC system. This system is coupled to a multiwavelength detector and equipped with an Agilent ZORBAX RR Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm), with a flow rate of 0.5 mL/min, eluting initially with 95% water for 5 min, followed by a gradient shift from 95% water to 95% CH₃CN over 20 min, and then maintaining 95% CH₃CN for 5 min (both with 0.1% HCO₂H). Preparative HPLC is performed on an Agilent 1260 Infinity II HPLC system coupled to a multiwavelength detector and equipped with an Agilent ZORBAX StableBond C18 column (9.4 × 250 mm, 5 μm), with a flow rate of 5 mL/min. The elution process star with 95% water for 3 min, followed by a gradient shift from 95% water to 95% CH₃CN over 14 min, and then 95% CH₃CN for 3 min (both with 0.1% HCO₂H). In vivo fluorescent imaging is conducted using an IVIS Spectrum CT system from PerkinElmer.

Institutional permissions

All animal experiments were performed under the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shenzhen Institutes of Advanced Technology (SIAT).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Rink amide (aminomethyl)polystyrene (100–200 mesh)	GL Biochem Ltd. or Sigma-Aldrich	N/A; 8.55130; CAS: 183599-10-2
Anhydrous dichloromethane (DCM)	Energy Chemical or Sigma-Aldrich	W6109425000; 270997; CAS: 75-09-2
Silica gel 60 Å (40–63 micron) high-purity grade	Sigma-Aldrich	60737; CAS: 112926-00-8
Silicone oil (for oil baths, from −50°C to +200°C)	Sigma-Aldrich	85409; CAS: 63148-62-9
Anhydrous N,N-dimethylformamide (DMF)	Energy Chemical or Sigma-Aldrich	W6104935000; 227056; CAS: 68-12-2
Anhydrous tetrahydrofuran (THF)	Energy Chemical or Sigma-Aldrich	W3100755000; 401757; CAS: 109-99-9
Chlorotrimethylsilane (TMSCl)	Tokyo Chemical Industry	C0306; CAS: 75-77-4
Anhydrous sodium sulfate (Na2SO4)	Bidepharm or Sigma-Aldrich	BD31900; 238597; CAS: 7757-82-6
Hexane	Energy Chemical or Sigma-Aldrich	D030155; 270504; CAS: 110-54-3
Ethyl acetate (EtOAc)	Energy Chemical or Sigma-Aldrich	A022002; 319902; CAS: 141-78-6
Ammonium chloride (NH4Cl)	Energy Chemical or Sigma-Aldrich	E010132; 213330; CAS: 12125-02-9
Acetonitrile (MeCN or CH3CN)	Energy Chemical or Sigma-Aldrich	D040604; 34851; CAS: 75-05-8
Methanoic acid (HCO2H)	Aladdin or Sigma-Aldrich	F301957; 5.43804; CAS: 64-18-6
Glycidyl propargyl ether	Bidepharm or Sigma-Aldrich	BD132194; 50062; CAS: 18180-30-8
Sodium azide (NaN3)	LEAPChem Co., Ltd.	C-24251; CAS: 26628-22-8
n-Butyllithium (n-BuLi)	Energy Chemical or Sigma-Aldrich	W420050; 302120; CAS:109-72-8
Dichloromethane (CH2Cl2 or DCM)	Energy Chemical or Sigma-Aldrich	D030158; 34856; CAS: 75-09-2
N,N-dimethylformamide (DMF)	Energy Chemical or Sigma-Aldrich	B020051; 270547; CAS: 68-12-2
N-methyl-2-pyrrolidone (NMP)	Energy Chemical or Sigma-Aldrich	A050168; 443778; CAS: 872-50-4
Succinic anhydride	Bidepharm or Sigma-Aldrich	BD152069; 8.00683; CAS: 108-30-5
4-Dimethylaminopyridine (DMAP)	Bidepharm or Sigma-Aldrich	BD17199; 8.20499; CAS: 1122-58-3
N,N-diisopropylethylamine (DIPEA)	Energy Chemical or Sigma-Aldrich	W320014; 8.00894; CAS: 7087-68-5
Methanol (MeOH)	Energy Chemical or Sigma-Aldrich	A040901; 34860; CAS: 67-56-1
D-glucose	Tokyo Chemical Industry or Sigma-Aldrich	G0048; G5767; CAS: 50-99-7
D-galactose	Tokyo Chemical Industry or Sigma-Aldrich	G0008; G0750; CAS: 59-23-4
D-mannose	Tokyo Chemical Industry or Sigma-Aldrich	M0045; M6020; CAS: 3458-28-4
Aqueous ammonia	Yonghua Chemical Co., Ltd. or Sigma-Aldrich	A104801; 221228; CAS: 1336-21-6
Ammonium carbamate	Aladdin or Sigma-Aldrich	A113918; 292834; CAS: 1111-78-0
Oxalyl chloride	Energy Chemical or Sigma-Aldrich	W8102905000; O8801; CAS: 79-37-8
Methyl-4-hydroxybenzoate	Bidepharm or Sigma-Aldrich	BD35576; 47889; CAS: 99-76-3
Potassium carbonate (K2CO3)	Bidepharm or Sigma-Aldrich	BD148751; 209619; CAS: 584-08-7
Propargyl bromide	Adama or Sigma-Aldrich	13127Y; P51001; CAS: 106-96-7
Anhydrous magnesium sulfate (MgSO4)	Bidepharm or Sigma-Aldrich	BD137048; 208094; CAS: 7487-88-9
Sodium hydroxide (NaOH)	Energy Chemical or Sigma-Aldrich	M0113521; 221465; CAS: 1310-73-2
Hydrochloric acid (HCl)	Yonghua Chemical Co., Ltd. or Sigma-Aldrich	H102901; 258148; CAS: 7647-01-0
Piperidine	Greagent or Sigma-Aldrich	G14242C; 8.22299; CAS: 110-89-4
Hydroxybenzotriazole (HOBT)	Energy Chemical or Sigma-Aldrich	D05010253; CAS: 2592-95-2 Or 54802; CAS: 123333-53-9
N,Nʹ-diisopropylcarbodiimide	Energy Chemical or Sigma-Aldrich	B010023; 8.03649; CAS: 693-13-0
Fluorescein isothiocyanate (FITC)	Bidepharm or Sigma-Aldrich	BD00783262; F3651; CAS: 27072-45-3
11-Azido-3,6,9-trioxaundecan-1-amine	Amatek Scientific or Apollo Scientific Ltd.	B-1526; BIBP1049; CAS: 1162336-72-2
Copper(I) iodide (CuI)	Bidepharm or Sigma-Aldrich	BD122226; 8.18311; CAS: 7681-65-4
Tetrabutylammonium fluoride (TBAF)	Tokyo Chemical Industry or Sigma-Aldrich	T1338; 216143; CAS: 429-41-4
Sodium ascorbate	Bidepharm or Sigma-Aldrich	BD151371; A7631; CAS: 134-03-2
Trifluoroacetic acid (TFA)	Macklin or Sigma-Aldrich	T818778; 302031; CAS: 76-05-1
Triethylamine (TEA)	Energy Chemical or Sigma-Aldrich	B010065; T0886; CAS: 121-44-8
Copper sulfate pentahydrate (CuSO4·5H2O)	Bidepharm or Sigma-Aldrich	BD122189; 209198; CAS: 7758-99-8
Critical commercial assays
CCK-8 assay kit	Boster Biological Technology (China)	Cat#AR1199
Experimental models: Cell lines
LoVo cells	ATCC	CCL-229
Experimental models: Organisms/strains
4- to 6-week-old BALB/c nude female mice (affected gene: forkhead box N1) (immunodeficient)	Guangdong Medical Laboratory Animal Center	CAnN.Cg-Foxn1nu/Crl; RRID:IMSR_CRL:194
6- to 8-week-old ICR female mice (affected gene: colon cancer susceptibility 1) (susceptibility to induced colon cancer)	Guangdong Medical Laboratory Animal Center	ICR/HaJ; RRID: IMSR JAX:009122
Other
Aluminum-back plates coated with silica gel 60 F254	Merck	Cat#1.05554
400 MHz NMR spectrometer	Bruker	Cat#Avance III 400
High-performance liquid chromatography	Agilent	Cat#1260 Infinity II HPLC system
ZORBAX RR Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm)	Agilent	Cat#959963-902
ZORBAX StableBond C18 column (9.4 × 250 mm, 5 μm)	Agilent	Cat#880975-202
High-resolution mass spectrometry (HRMS)	Thermo Scientific	Cat#Thermo Q Exactive Focus mass spectrometer
Mass spectrometry	Bruker	Cat#Ultraflextreme MALDI-TOF/TOF system
IVIS spectrum CT system	Revvity	Cat#CLS158737
Microcentrifuges	Eppendorf	Cat#5424R
Thermostatic oscillation incubator	Yiheng, China	Cat#THZ-98A
Rotary evaporator	Büchi	Cat#Rotavapor R-110
Vacuum pump	Lichen	Cat#2XZ-4
Heating agitator	IKA	Cat#RTC basic
Chromatography column (φ60 mm, 610 mm)	Synthware	Cat#C184605C
Chromatography column (φ32 mm, 457 mm)	Synthware	Cat#C184324C
Round bottom flask (single neck)	Synthware	Cat#F304500 (500 mL); Cat#F301250 (250 mL); Cat#F301200 (200 ML); Cat#F301950 (50mL); Cat#F301925 (25mL)
Round bottom flask (two neck)	Synthware	Cat#F414504 (500 mL); Cat#F411950 (50 mL)
Single neck round bottom reaction flask with glass stopcock	Synthware	Cat#F534250
Hemispherical Dewar flask	Synthware	Cat#F240500 (500 mL)
Cylindrical Dewar flask	Synthware	Cat#F111000 (1000 mL)
Dropping funnel	Synthware	Cat#F652425 (25mL)
Separatory funnel	Synthware	Cat#F787000 (1000 mL); Cat#F474500A (500 mL)
Integrated cold trap (60 × 300 mm)	Synthware	Cat#V236030
Vessel (lower joint and T-bore PTFE plug)	Synthware	Cat#P150050M
Syringe (5 mL, sterile)	Tansoole	Cat#TS069-012
Syringe (10 mL, sterile)	Tansoole	Cat#TS069-002
Syringe (50 mL, sterile)	Tansoole	Cat#TS069-010
Syringe (20 mL, sterile)	Tansoole	Cat#TS069-006
Syringe (1 mL, sterile)	Tansoole	Cat#TS069-004
Syringe needle (φ0.8 × 200 mm)	Tansoole	Cat#02044724
Nitrile gloves	KleenGuard	Cat#54423
Electronic balance	Sartorius	Cat#BSA224S-CW
Autoclave	Zealway	Cat#GR110DP
Glove box	Mikrouna	Cat#Super1220/750
Carbon dioxide (CO2) incubator	Thermo Scientific	Cat#371
Lab-Line water bath	Thermo Scientific	Cat#18802A-1CEQ
Cell culture dish with gripping ring (100 mm)	Wuxi NEST Biotechnology Co., Ltd.	Cat#704202
Dulbecco’s modified Eagle’s medium (DMEM), high glucose	VivaCell Biosciences	Cat#C3113-0500
Trypsin EDTA solution A (0.25% Trypsin)	VivaCell Biosciences	Cat#C3530-0500
Certified fetal bovine serum	VivaCell Biosciences	Cat#C04001-500
Phosphate-buffered saline (PBS)	VivaCell Biosciences	Cat#C3580-0500
1.0 mL pipette tips, sterile	Kirgen	Cat#KG1313
200 μL pipette tips, sterile	Kirgen	Cat#KG1232
10 μL pipette tips, sterile	Kirgen	Cat#KG1031
1.5 mL microcentrifuge tubes, sterile	Wuxi NEST Biotechnology Co., Ltd.	Cat#615601

Step-by-step method details

The following section details the synthesis of glycooligomer building blocks, alkyne terminated linker and azide functionalized fluorescein isothiocyanate (FITC), coupling protocol of sequence-controlled fluorescent glycooligomers, and in vivo study of its safety and tumor targeting ability.

Part1: Synthesis of glycooligomer building blocks, alkyne terminated linker and azide functionalized FITC

Timing: 19 days

Timing: 1 day (for step 1)

Timing: 2 days (for step 2)

Timing: 2 days (for step 3)

Timing: 4 days (for step 4)

Timing: 2 days (for step 5)

Timing: 2 days (for step 6)

Timing: 2 days (for step 7)

Timing: 1 day (for step 8)

Timing: 1 day (for step 9)

Timing: 2 days (for step 10)

This section describes the synthesis of glycooligomer building blocks B_A, B_B and B_C. Each block contains an azide, a silyl protected alkyne, and a saccharide motif (specifically, glucose, galactose and mannose for B_A, B_B and B_C, respectively). Additionally, it describes the preparation of an alkyne terminated linker for both solid-phase and solution-phase reactions. Furthermore, the synthesis includes azide-functionalized FITC for investigating cellular uptake, as well as assessing the tumor-targeting capabilities and biodistributions of these compounds in vivo.

• 1.Synthesis of TMS-protected glycidyl propargyl ether S1. (Figure 1)
  The following procedure is based on that of Chandra et al.²

  CRITICAL: Remove air/moisture from the flask or other containers and carry out the reaction under a nitrogen atmosphere with an oven-dried apparatus.

  • a.
    Dissolve glycidyl propargyl ether (5.00 g, 44 mmol, 1 equiv.) in anhydrous THF (125 mL) in a two-necked flask (500 mL) connected with a dropping funnel at 25°C or so by stirring.
  • b.
    Cool the mixture to −78°C using the drikold acetone bath method in the hemispherical Dewar flasks (500 mL).
  • c.
    Add n-BuLi (2.5 M in hexanes, 21.4 mL, 1.2 equiv.) dropwise over approximately 30 min using a dropping funnel and stir the mixture at −78°C for 30 min.
  • d.
    Add a solution of TMSCl (8.5 mL, 66 mmol, 1.5 equiv.) in anhydrous THF (15 mL) dropwise over approximately 15 min using a dropping funnel at −78°C.
  • e.
    Warm the reaction to 25°C or so and proceed for 3 h.
  • f.
    Quench the reaction with a cold brine solution (400 mL).
  • g.
    Extract the crude product with EtOAc (3 × 250 mL) with a separatory funnel (1000 mL).
  • h.
    Dry the organic phase over anhydrous Na₂SO₄, filter, and concentrate under reduced pressure using a rotary evaporator.
  • i.
    Purify by silica column chromatography technique (hexane/EtOAc, 20:1) (Figure 2).
  • j.
    Confirm the R_f (0.42, hexane/EtOAc, 10:1) of S1 using a thin layer chromatography (TLC) with the KMnO₄ coloration reaction, collect, and concentrate under vacuum to afford S1 (4.74 g, 79%) as a colorless oil.
  • k.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS and confirm the spectra are in agreement with the literature previously reported.^2,3
    TMS-protected glycidyl propargyl ether S1
    ¹H NMR (400 MHz, CDCl₃) δ/ppm 4.22–4.11 (m, 2H), 3.76 (dd, J = 11.3, 3.1 Hz, 1H), 3.43 (dd, J = 11.3, 5.8 Hz, 1H), 3.13 (dq, J = 7.1, 3.1 Hz, 1H), 2.77 (t, J = 4.6 Hz, 1H), 2.59 (dd, J = 5.0, 2.7 Hz, 1H), 0.13 (s, 9H).
    ¹³C NMR (100 MHz, CDCl₃) δ/ppm 101.0, 91.8, 70.4, 59.3, 50.5, 44.4, −0.2.
    HRMS for C₉H₁₇O₂Si (ESI-TOF) m/z: [M + H]⁺ calc.: 185.0992; found: 185.0994.

    Note: All synthesized products are preferred to be stored at −20°C in the fridge in consideration of more stable preservation.

• 2.Synthesis of 1-Azido-3-(trimethylsilyl propynyl-1-oxy)- 2-propanol S2. (Figure 1)
  The following procedure is based on that of Chandra et al.²

  CRITICAL: Remove air/moisture from the flask or other containers and carry out the reaction under a nitrogen atmosphere with an oven-dried apparatus.

  • a.
    Dissolve S1 (2.50 g, 14 mmol, 1 equiv.) in anhydrous DMF (60 mL) in a single-necked flask (200 mL) at 25°C or so by stirring.
  • b.
    Add NH₄Cl (1.09 g, 20 mmol, 1.5 equiv.) and NaN₃ (5.29 g, 81 mmol, 6.0 equiv.) to above solution.

    Note: Wait until NH₄Cl dissolves before adding NaN₃.

    CRITICAL: Because of the danger and high toxicity of NaN₃, it is highly important to add NaN₃ using a plastic spoon or other nonmetallic container and quench anything contact with NaN₃ in a cold saturated sodium hypochlorite solution.

    Note: There is significant risk of upscaling the reaction, which need to be seriously considered.

  • c.
    Stir the reaction mixture 12–15 h at 65°C using a simple oil bath setup containing methyl silicone oil, equipped with a heating agitator and a temperature probe for monitoring the oil temperature.
  • d.
    Quench the reaction with a cold brine solution (50 mL).
  • e.
    Extract the crude product with EtOAc (3 × 100 mL) with a separatory funnel (500 mL).
  • f.
    Dry the organic phase over anhydrous Na₂SO₄, filter, and concentrate under reduced pressure.
  • g.
    Purify the crude product by silica column chromatography technique (hexane/EtOAc, 20:1).
  • h.
    Confirm the R_f (0.34, hexane/EtOAc, 10:1) of S2 using a TLC with the KMnO₄ coloration reaction.
  • i.
    Collect the eluate and concentrate under vacuum to afford S2 (1.85 g, 60%) as a colorless oil.
  • j.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS and confirm the spectra are in agreement with the literature previously reported.^2,3
    1-Azido-3-(trimethylsilyl propynyl-1-oxy)- 2-propanol S2
    ¹H NMR (400 MHz, CDCl₃) δ/ppm 4.13 (s, 2H), 3.98–3.88 (m, 1H), 3.63–3.45 (m, 3H), 3.38–3.26 (m, 1H), 0.12 (s, 9H).
    ¹³C NMR (100 MHz, CDCl₃) δ/ppm 100.8, 92.1, 71.1, 69.5, 59.4, 53.4, −0.3.
    HRMS for C₉H₁₇N₃O₂SiNa (ESI-TOF) m/z: [M+Na]⁺ calc.: 250.0982; found: 250.0982.
• 3.
  Synthesis of 1-Azido-3-(trimethylsilyl propynyl-1-oxy)- 2-propanyl mono-succinate S3. (Figure 1)

  • a.
    Dissolve S2 (2.30 g, 10 mmol, 1.0 equiv.) in DCM (100 mL) in a single-necked flask (250 mL) at 25°C or so by stirring.

    Note: Use the general solvent unless specifically instructed to use an anhydrous solvent, as is the case with DCM here.

  • b.
    Sequentially add succinic anhydride (2.00 g, 20 mmol, 2.0 equiv.), DMAP (244 mg, 2.0 mmol, 0.2 equiv.) and DIPEA (3.48 mL, 20 mmol, 2.0 equiv.) to the solution above at 25°C or so.

    Note: Wait for it to dissolve fully before adding next one.

  • c.
    Stir the reaction mixture 12–15 h at 25°C or so.
  • d.
    Concentrate the crude product (a burgundy viscous colorless liquid) under vacuum.
  • e.
    Purify by silica column chromatography technique (DCM/MeOH, 20:1).
  • f.
    Confirm the R_f (0.16, DCM/MeOH, 20:1) of S3 using a TLC with the KMnO₄ coloration reaction.
  • g.
    Collect the eluate and concentrate under vacuum to afford S3 (3.30 g, quantitative) as a viscous, colorless liquid.
  • h.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS.
    1-Azido-3-(trimethylsilyl propynyl-1-oxy)- 2-propanyl mono-succinate S3
    ¹H NMR (400 MHz, CDCl₃) δ/ppm 5.22–5.09 (m, 1H), 4.21–4.13 (m, 2H), 3.81–3.60 (m, 3H), 3.50 (d, J = 5.2 Hz, 1H), 2.75–2.63 (m, 4H), 0.17 (s, 9H).
    ¹³C NMR (100 MHz, CDCl₃) δ/ppm 177.9, 171.6, 100.7, 92.4, 71.6, 67.9, 59.5, 50.9, 29.0, 28.9, −0.1.
    HRMS for C₁₃H₂₁N₃O₅SiNa (ESI-TOF) m/z: [M+Na]⁺ calc.: 350.1143; found: 350.1143.
• 4.Synthesis of amino-functionalized carbohydrates (α-D-Glucoxylamine S4_A, α-D-Galactoxylamine S4_B, α-D-Mannoxylamine S4_C). (Figure 3)
  The following procedure is based on that of Tang et al.⁴

  • a.
    Dissolve D-sugars (D-glucose, D-galactose and D-mannose) (4.50 g, 25 mmol, 1.0 equiv.) in aqueous ammonia (100 mL) in a single-necked flask (250 mL) at 25°C or so by stirring.
  • b.
    Add ammonium carbamate (1.98 g, 25 mmol, 1.0 equiv.) in small portions taking about 15 min.
  • c.
    Stir the reaction mixture at 25°C or so for 3 days.
  • d.
    Concentrate under reduced pressure to afford the product (S4_A, S4_B or S4_C) as a white solid (yield: quantitative).
  • e.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS and confirm the spectra agree with the literature previously reported.⁴
    α-D-Glucoxylamine S4_A
    ¹H NMR (400 MHz, D₂O) δ/ppm 4.06 (d, J = 8.8 Hz, 1H), 3.87 (dd, J = 6.9, 2.2 Hz, 1H), 3.67 (dd, J = 5.8, 2.2 Hz, 1H), 3.40–3.36 (m, 1H), 3.34–3.25 (m, 2H), 3.12 (t, J = 9.0 Hz, 1H).
    ¹³C NMR (100 MHz, D₂O) δ/ppm 86.9, 76.7, 74.2, 72.7, 69.8, 60.8.
    HRMS for C₆H₁₂NO₅ (ESI-TOF) m/z: [M-H]^- calc.: 178.0711; found: 178.0721.
    α-D-Galactoxylamine S4_B
    ¹H NMR (400 MHz, D₂O) δ/ppm 4.54 (d, J = 9.1 Hz, 1H), 3.91 (d, J = 8.9 Hz, 1H), 3.88–3.82 (m, 2H), 3.51 (m, 1H), 3.47–3.42 (m, 1H), 3.27 (t, J = 9.2 Hz, 1H).
    ¹³C NMR (100 MHz, D₂O) δ/ppm 87.5, 75.7, 73.4, 70.3, 68.8, 61.1.
    HRMS for C₆H₁₄NO₅ (ESI-TOF) m/z: [M + H]⁺ calc.: 180.0868; found: 180.0867.
    α-D-Mannoxylamine S4_C
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 4.17 (d, J = 8.6 Hz, 1H), 3.72–3.44 (m, 5H), 3.46–3.31 (m, 2H), 3.32–3.14 (m, 3H), 3.04–2.90 (m, 2H).
    ¹³C NMR (100 MHz, DMSO-d₆) δ/ppm 85.4, 78.0, 74.8, 71.6, 67.3, 61.7.
    HRMS for C₆H₁₄NO₅ (ESI-TOF) m/z: [M + H]⁺ calc.: 180.0868; found: 180.0866.

    Note: The product was directly subjected to the next reaction without further purification.

• 5.
  Synthesis of glycooligomer building blocks (Glucose derived building block B_A, Galactose derived building block B_B, Mannose derived building block B_C). (Figure 1)

  CRITICAL: Remove air/moisture from the flask or other containers and carry out the reaction under a nitrogen atmosphere with an oven-dried apparatus.

  • a.
    Dissolve S3 (1.00 g, 4.4 mmol, 1.0 equiv.) in oxalyl chloride (4.6 mL, 66 mmol, 15 equiv.) in a two-necked flask (50 mL) at 25°C or so by stirring.
  • b.
    Stir the reaction mixture 12–15 h at 25°C or so.
  • c.
    Remove the solvent under reduced pressure with a water-containing cold trap placed in the cylindrical Dewar flask with liquid nitrogen to quench superfluous oxalyl chloride.
  • d.
    Redissolve the reaction mixture in anhydrous DMF (20 mL) (Solution-1).
  • e.
    Separately, add amino-functionalized carbohydrates S4_A, S4_B or S4_C (1.00 g, 5.6 mmol, 1.3 equiv.) and TEA (0.9 mL, 6.6 mmol, 1.5 equiv.) in anhydrous DMF (50 mL) in a single neck round bottle reaction flask with glass stopcock (250 mL) (Solution-2).

    Note: Because the hygroscopic property of the amino-functionalized carbohydrates S4_A, S4_B or S4_C, it needs to be dehydrated before being added to this reaction in which water can consume oxalyl chloride and result in a low yield. Dehydration operations include removing water under reduced pressure and weighting the compound under a nitrogen atmosphere with dried apparatus.

    Note: Use the general solvent unless specifically directed to use an anhydrous solvent, as with TEA in this case. For optimal results, open the TEA bottle under a nitrogen atmosphere using equipment that has been dried in an oven.

  • f.
    Heat Solution-2 to 60°C using an oil bath to dissolve the amino-functionalized carbohydrates S4_A, S4_B or S4_C.
  • g.
    Critical step: Add Solution-1 to Solution-2 dropwise using a long needle syringe in 20 min (Figure 4).

    CRITICAL: Avoid contact with air/moisture.

  • h.
    Wait until the mixture cools to 25°C or so.
  • i.
    Stir the reaction mixture at 25°C or so for 12 h.

    Optional: The reaction time can be extended up to 15 h depending on the consumption of S3 and the yield of the desired product, as determined through TLC using the KMnO₄ coloration reaction with the target product serving as a control.

  • j.
    Remove the solvent under vacuum and redissolve the crude product in MeOH.
  • k.
    Critical step: Add silica to obtain a dry sample (Figure 5), then purify it using silica column chromatography (hexane/EtOAc/MeOH, 5:5:1, v/v).
  • l.
    Confirm the R_f (0.13, hexane/EtOAc/MeOH, 4:4:1, v/v) of the product (B_A, B_B or B_C) using a TLC with the KMnO₄ coloration reaction.
  • m.
    Collect the eluate and concentrate under vacuum to afford the glycooligomer building block as a yellowish solid. (yield: B_A = 21%; B_B = 18%; B_C = 22%)

    Note: Due to a series of by-products in this reaction, as indicated by about seven spots on a TLC plate using the KMnO₄ coloration reaction before purification, it is necessary to utilize TLC results to monitor the reaction process closely. This approach aims to achieve the highest possible yield of the target product.

  • n.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS.
    Glucose derived building block B_A
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.42 (d, J = 9.1 Hz, 1H), 5.04–5.01 (m, 1H), 4.99 (d, J = 4.8 Hz, 1H), 4.88 (dd, J = 9.6, 5.1 Hz, 2H), 4.68 (t, J = 9.0 Hz, 1H), 4.50 (t, J = 5.8 Hz, 1H), 4.18 (s, 2H), 3.68–3.43 (m, 6H), 3.21–3.12 (m, 1H), 3.11–2.97 (m, 3H), 2.48–2.35 (m, 4H), 0.15 (s, 9H).
    ¹³C NMR (100 MHz, DMSO-d₆) δ 171.9, 171.0, 102.3, 91.2, 79.6, 78.6, 77.5, 72.6, 70.9, 70.0, 67.9, 60.9, 58.7, 50.4, 29.9, 28.7.
    HRMS for C₁₉H₃₂N₄O₉SiNa (ESI-TOF) m/z: [M+Na]⁺ calc.: 511.1831; found: 511.1834.
    Galactose derived building block B_B
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.44 (d, J = 9.1 Hz, 1H), 5.09–4.99 (m, 1H), 4.82–4.75 (m, 1H), 4.73–4.63 (m, 2H), 4.62–4.55 (m, 1H), 4.44 (d, J = 3.8 Hz, 1H), 4.19 (s, 2H), 3.72–3.66 (m, 1H), 3.63–3.43 (m, 6H), 3.37–3.28 (m, 3H), 2.56–2.40 (m, 4H), 0.16 (s, 9H).
    ¹³C NMR (100 MHz, DMSO-d₆) δ/ppm 171.9, 171.0, 102.3, 91.2, 80.1, 76.7, 74.2, 70.9, 69.8, 68.2, 67.9, 60.5, 58.7, 50.4, 29.9, 28.8.
    HRMS for C₁₉H₃₂N₄O₉SiNa (ESI-TOF) m/z: [M+Na]⁺ calc.: 511.1831; found: 511.1836.
    Mannose derived building block B_C
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.18 (d, J = 9.1 Hz, 1H), 5.15–4.96 (m, 2H), 4.86 (d, J = 4.7 Hz, 1H), 4.79 (s, 1H), 4.74 (d, J = 4.6 Hz, 1H), 4.45 (t, J = 5.9 Hz, 1H), 4.18 (s, 2H), 3.69–3.63 (m, 1H), 3.62–3.37 (m, 8H), 3.09–3.03 (m, 1H), 2.55–2.43 (m, 4H), 0.16 (s, 9H).
    ¹³C NMR (100 MHz, DMSO-d₆) δ/ppm 171.9, 170.5, 102.3, 91.2, 79.1, 77.5, 74.1, 70.9, 70.8, 67.9, 66.8, 61.3, 58.7, 50.4, 29.7, 28.8.
    HRMS for C₁₉H₃₂N₄O₉SiNa (ESI-TOF) m/z: [M+Na]⁺ calc.: 511.1831; found: 511.1830.
• 6.Synthesis of Methyl 4-(prop-2-yn-1-yloxy)benzoate S5. (Figure 6)
  The following procedure is based on that of Zhou et al.⁵

  • a.
    Dissolve methyl-4-hydroxybenzoate (1.52 g, 10 mmol, 1.0 equiv.) and K₂CO₃ (5.60 g, 40 mmol, 4.0 equiv.) in acetonitrile (10 mL) in a single-necked flask (25 mL) under nitrogen atmosphere and cooled to 0°C using ice-water bath method.
  • b.
    Add propargyl bromide (1.43 g, 12 mmol, 1.2 equiv.) and stir the mixture at 35°C using an oil bath method for 24 h.
  • c.
    Remove the solvent under vacuum and redissolve the mixture in EtOAc (200 mL).
  • d.
    Wash with brine solution (3 × 100 mL) using a separatory funnel (500 mL).
  • e.
    Dry the organic phase over anhydrous MgSO₄, filter, and concentrate under reduced pressure to obtain the crude product.
  • f.
    Purify the crude product by silica column chromatography (hexane/EtOAc, 1:1, v/v).
  • g.
    Confirm the R_f (0.19, hexane/EtOAc, 1:1, v/v) of S5 using a TLC under 254 nm UV lamp.
  • h.
    Collect the eluate and concentrate under vacuum to afford S5 (5.94 g, 95%) as a white solid.
  • i.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS and confirm the spectra agree with the literature previously reported.⁵
    Methyl 4-(prop-2-yn-1-yloxy)benzoate S5
    ¹H NMR (400 MHz, CDCl₃) δ/ppm 8.01 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 4.75 (d, J = 2.4 Hz, 2H), 3.89 (s, 3H), 2.55 (t, J = 2.4 Hz, 1H).
    ¹³C NMR (100 MHz, CDCl₃) δ/ppm 166.9, 161.3, 131.7, 123.6, 114.6, 78.0, 76.2, 56.0, 52.1.
    HRMS for C₁₁H₁₁O₃ (ESI-TOF) m/z: [M + H]⁺ calc.: 191.0708; found: 191.0710.
• 7.
  Synthesis of 4-(Prop-2-yn-1-yloxy)benzoic acid S6. (Figure 6)

  • a.
    Dissolve S5 (950 mg, 5 mmol, 1.0 equiv.) in MeOH (250 mL) in a single-necked flask (500 mL) at 25°C or so by stirring.
  • b.
    Add NaOH (800 mg, 20 mmol, 4.0 equiv.) aqueous solution (10 mL) to the solution above.
  • c.
    Stir the mixture at 35°C using an oil bath for 5 h.
  • d.
    Acidify the mixture to a pH of 1 by adding HCl solution (2 M) and monitoring with pH test paper.
  • e.
    Critical step: Remove the MeOH from the mixture under vacuum and concentrate the solution to about 10 mL.
  • f.
    Extract with EtOAc (3 × 100 mL) using a separatory funnel (500 mL).
  • g.
    Dry the organic phase over anhydrous MgSO₄, filter, and concentrate under reduced pressure to obtain the crude product.
  • h.
    Dry the crude product at 40°C under vacuum to obtain S6 (880 mg, quantitative) as a white solid.
  • i.
    Confirm the R_f (0.19, hexane/EtOAc, 1:1) of S6 using a TLC under 254 nm UV lamp.
  • j.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS.

Figure 1.

Synthesis of the glycooligomer building blocks

i) TMSCl, n-BuLi, THF, 3 h; ii) NaN₃, NH₄Cl, DMF, 12 h; iii) Succinic anhydride, DIPEA, DCM, 12 h; iv) (COCl)₂, 12 h; v) S4_A, S4_B or S4_C, TEA, DMF, 12 h.

Figure 2.

Process of the silica column chromatography technique

Figure 3.

Synthesis of α-amino sugars

i) NH₃·H₂O, ammonium carbamate, 3 d.

Figure 4.

Process of adding Solution-1 to Solution-2

Figure 5.

Process of obtaining a dry sample for the silica column chromatography

Figure 6.

Synthesis of alkyne terminated linker

i) propargyl bromide, K₂CO₃, acetonitrile; ii) NaOH, MeOH/H₂O; iii) HCl, MeOH/H₂O; iv) Rink amide PS resin, DIC, HOBt, DMF; v) TFA, DCM, 0.5 h.

4-(Prop-2-yn-1-yloxy)benzoic acid S6

¹H NMR (400 MHz, DMSO-d₆) δ/ppm 12.69 (s, 1H), 7.91 (d, J = 8.9 Hz, 2H), 7.07 (d, J = 8.9 Hz, 2H), 4.89 (s, 2H), 3.61 (s, 1H).

¹³C NMR (100 MHz, DMSO-d₆) δ/ppm 167.0, 160.8, 131.3, 123.7, 114.7, 78.8, 78.7, 55.7.

HRMS for C₁₀H₉O₃ (ESI-TOF) m/z: [M + H]⁺ calc.: 177.0552; found: 177.0555.

• 8.
  Synthesis of 4-(Prop-2-yn-1-yloxy)benzylamide bound resin S7. (Figure 6)

  • a.
    Add Fmoc-Rink Amide AM resin (5.00 g, reported loading 0.9 mmol/g, 1.0 equiv.) in a vessel with lower joint and T-bore PTFE plug (50 mL).
  • b.
    Swell the Fmoc-Rink Amide AM resin with DCM (3 × 10 mL, 3 min each time) and drain the solution under reduced pressure.
  • c.
    Add piperidine (15 mL, 20% v/v in DMF) and shake for 2 × 10 min using a thermostatic oscillation incubator.
  • d.
    Remove the solvent and wash the resin with DCM (3 × 10 mL), MeOH (3 × 10 mL), and DMF (3 × 10 mL) to afford the activated Rink Amide AM resin.
    Dissolve S6 (1.74 g, 10 mmol, 2.2 equiv.) and hydroxybenzotriazole (HOBt) (1.88 g, 14 mmol, 3.0 equiv.) in DMF (10 mL) and stir for 5 min.

    Note: To achieve complete transformation of the Fmoc-Rink Amide AM resin, excess S6 and HOBt are used to ensure full reaction, as separating unreacted resin can be challenging.

  • e.
    Add N,Nʹ-diisopropylcarbodiimide (DIC) (1.7 mL, 11 mmol) to the mixture and stir for 1 min.
  • f.
    Add the mixture to the activated Rink Amide AM resin and shake 12 h–15 h at 25°C or so.
  • g.
    Drain the solution and wash the resin with DCM (3 × 10 mL), MeOH (3 × 10 mL) and DMF (3 × 10 mL) to obtain S7.
  • h.
    The resin loading is 0.9 mmol/g according to a quantitative ninhydrin test.

    Note: Wash S7 with MeOH and store at 0°C. S7 remains stable for 3 months at least under these conditions. For extended storage, use diethyl ether instead of MeOH to wash S7.

• 9.
  Synthesis of 4-(Prop-2-yn-1-yloxy)benzylamide S8. (Figure 6)

  • a.
    Add 40 mL of a TFA/DCM solution (1:4, v/v) to S7 (5.0 g, 4.5 mmol) in the vessel with lower joint and T-bore PTFE plug (50 mL) and shake for 1 h.
  • b.
    Collect the filtrate, wash the resin with TFA/DCM (1:4, v/v, 3 × 30 mL), and combine the solution.

    CRITICAL: The color of the resin changes from yellowish to crimson if the reaction succeeds.

  • c.
    Concentrate the filtrate under reduced pressure to obtain the crude product.
  • d.
    Purify the crude product by silica column chromatography (hexane/ EtOAc, 1:1, v/v).
  • e.
    Confirm the R_f (0.25, hexane/EtOAc, 1:1, v/v) of S8 using a TLC under 254 nm UV lamp.
  • f.
    Collect the eluate and concentrate under vacuum to afford S8 (517 mg, 65%) as a white solid.

    Optional: Purify the crude product by preparative HPLC. Begin the elution process with 95% water for the first 3 minutes, then implement a gradient shift from 95% water to 95% CH₃CN over the next 14 minutes, and maintain 95% CH₃CN for an additional 3 minutes, both phases containing 0.1% HCO₂H. (Conditions in before you begin part)

  • e.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS.
    4-(Prop-2-yn-1-yloxy)benzylamide S8
    ¹H NMR (400 MHz, CDCl₃) δ/ppm 7.81 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 4.76 (d, J = 2.5 Hz, 2H), 2.56 (t, J = 2.5 Hz, 1H).
    ¹³C NMR (100 MHz, CDCl₃) δ/ppm 171.0, 161.2, 129.5, 124.7, 115.0, 77.6, 76.3, 55.9.
    HRMS for C₁₀H₁₀NO₂ (ESI-TOF) m/z: [M + H]⁺ calc.: 176.0706; found: 176.0707.
    HPLC (254 nm) t_R = 18.1 min.
• 10.
  Synthesis of azide functionalized FITC S9. (Figure 7)

  • a.
    Dissolve FITC (400 mg, 1.0 mmol, 1.0 equiv.), 11-azido-3,6,9-trioxaundecan-1-amine (262 mg, 1.2 mmol, 1.2 equiv.) and DIPEA (350 μL, 2.0 mmol, 2.0 equiv.) in THF (10 mL) in a single-necked flask (50 mL) at 25°C or so by stirring.
  • b.
    Stir the mixture 12 h–15 h at 25°C or so.
  • c.
    Concentrate to obtain the crude product under vacuum.
  • d.
    Purify the crude product by silica column chromatography (DCM/MeOH, 10:1, v/v).
  • e.
    Confirm the R_f (0.20, EtOAc/MeOH, 10:1, v/v) using a TLC under 254 nm UV lamp, collect and concentrate under vacuum to afford S9 (5.94 g, 95%) as a yellow solid.
  • f.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS.
    Azide functionalized FITC S9
    ¹H NMR (400 MHz, MeOD-d₄) δ/ppm 8.19 (s, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.76–7.70 (m, 1H), 7.65–7.59 (m, 1H), 7.16 (d, J = 8.3 Hz, 1H), 6.74–6.65 (m, 4H), 6.55 (dd, J = 8.7, 2.5 Hz, 2H), 3.75–3.57 (m, 14H), 3.21 (q, J = 7.4 Hz, 2H).
    ¹³C NMR (100 MHz, MeOD-d₄) δ/ppm 184.0, 175.4, 169.9, 162.1, 158.2, 153.0, 141.1, 131.1, 129.07, 128.5, 112.7, 112.7, 112.6, 110.4, 110.4, 110.3, 102.2, 70.2, 70.2, 70.1, 70.0, 69.9, 69.7, 54.4, 51.8, 50.3, 44.1, 42.4.
    HRMS for C₂₉H₂₈N₅O₈S (ESI-TOF) m/z: [M-H]^- calc.: 606.1664; found: 606.1668.

Figure 7.

Synthesis of azide labeled FITC

i) DIPEA, THF, 12 h.

Part2: Solid-phase and liquid-phase synthesis of sequence-controlled fluorescent glycooligomers

Timing: 1 month (variable)

Timing: 2 days (for step 11)

Timing: 4 days (for step 12)

This section demonstrates the procedures for both solid-phase and liquid-phase synthesis of sequence-controlled fluorescent glycooligomers.

• 11.
  Solid-phase synthesis of sequence-controlled fluorescent glycooligomers. (Figure 8)

  • a.
    Coupling protocol.

    • i.
      Add alkyne terminated resin (0.9 mmol/g, 1.0 equiv.), azide-terminated glycooligomer building block (5.0 equiv.) (or azide-terminated FITC, 5.0 equiv.) and DMF to a vessel with lower joint and T-bore PTFE plug (50 mL).
    • ii.
      Add a solution of CuI (1.0 equiv.) and DIPEA (10 equiv.) in DMF to the solution above.

      CRITICAL: The concentration of azide-terminated glycooligomer building block or azide-terminated FITC was 0.15 mol/L in the reaction system.

    • iii.
      Shake the reaction mixture at 25°C or so for 3 h.
    • iv.
      Remove the solution and wash the resin with DCM (3 × 5 mL), MeOH (3 × 5 mL) and DMF (3 × 5 mL).
  • b.
    TMS decapping protocol.

    • i.
      Add 500 μL of a mixture of TBAF (THF, 1.0 mol/L)/DMF/MeOH/H₂O (1/1/0.7/0.14, v/v) to the resin loaded with the glycooligomer structure (50 mg).
    • ii.
      Shake at 25°C or so for 0.5 h to remove the TMS protection group on the glycooligomer moieties.
    • iii.
      Drain the solution and wash the resin with DCM (3 × 5 mL), MeOH (3 × 5 mL) and DMF (3 × 5 mL).
  • c.
    Glycooligomer chain elongation.

    • i.
      Alternate the above-described coupling and decapping reactions until achieving the desired glycooligomer.
  • d.
    Glycooligomer cleavage from resin.

    • i.
      Add 5 mL of a solution of TFA/DCM (1:4, v/v) to the resin.
    • ii.
      Shake the reaction mixture at 25°C or so for 0.5 h.
    • iii.
      Collect the filtrate and wash the resin with TFA/DCM (1:4, v/v, 3 × 2 mL).
    • iv.
      Combine the solutions and evaporate in vacuo.
    • v.
      Purify the crude sequence-controlled glycooligomers by preparative HPLC (Conditions in before you begin part).
      Expected peaks for HPLC
      FITC-S_A: t_R = 17.3 min.
      FITC-S_AA: t_R = 16.6 min.
      FITC-S_AAA, FITC-S_BAA, FITC-S_BBA, FITC-S_CAA, FITC-S_CCA: t_R = 16.1 min.
      FITC-S_AAAA: t_R = 15.8 min.
      FITC-S_SS: t_R = 18.64 min.
      FITC-S_SSS: t_R = 18.55 min.
      Side-products
      During the coupling reaction, the TMS group may be removed from the FITC-labeled glycooligomer. For example, FITC-S_A without TMS, FITC-S_AA without TMS, FITC-S_AAA without TMS, FITC-S_AAAA without TMS and so on. The FITC-labeled glycooligomer without TMS is expected to have a shorter t_R than the corresponding desired products. However, this TMS-protected free by-product may subsequently react with the glycooligomer building blocks, resulting in a longer glycooligomer, which can then be collected as another desired product.
  • e.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR and HRMS.
    FITC labeled glycooligomer FITC-S_A
    ¹H NMR (400 MHz, MeOD-d₄) δ/ppm 8.17–8.08 (m, 2H), 8.05 (s, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.3 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.07 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 2.4 Hz, 2H), 6.58 (dd, J = 8.8, 2.4 Hz, 2H), 5.38-5.28 (m, 2H), 5.23 (s, 2H), 4.76–4.51 (m, 6H), 3.92–3.51 (m, 22H), 2.57–2.42 (m, 4H).
    ¹³C NMR (100 MHz, MeOD-d₄) δ/ppm 182.3, 175.3, 175.3, 173.5, 173.4, 172.0, 171.9, 162.4, 155.6, 145.1, 144.5, 144.5, 131.1, 130.9, 130.7, 127.2, 126.9, 126.9, 126.2, 115.7, 112.9, 103.7, 81.0, 79.3, 79.32, 78.6, 73.6, 72.2, 72.2, 71.3, 71.2, 71.2, 71.1, 70.2, 69.4, 65.0, 62.4, 62.2, 51.4, 45.4, 31.2, 29.9.
    HRMS for C₅₅H₆₃N₁₀O₁₉S (ESI-TOF) m/z: [M + H]⁺ calc.: 1199.3986; found: 1199.3987.
    HPLC: t_R = 17.3 min.
    FITC labeled glycooligomer FITC-S_AA
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.48–8.20 (m, 5H), 8.14–8.03 (m, 3H), 7.88-7.70 (m, 4H), 7.30–7.15 (m, 2H), 7.07 (d, J = 8.6 Hz, 2H), 6.69–6.53 (m, 6H), 5.30–5.11 (m, 4H), 5.03–4.84 (m, 6H), 4.78–4.42 (m, 14H), 3.88–2.90 (m, 30H), 2.48–2.29 (m, 8H).
    ¹³C NMR (100 MHz, DMSO-d₆) δ/ppm 171.6, 171.1, 168.5, 167.4, 160.3, 159.5, 151.9, 143.5, 141.3, 129.4, 129.1, 126.8, 125.8, 125.2, 124.5, 114.2, 112.6, 109.7, 102.2, 83.0, 79.6, 78.6, 77.5, 72.5, 70.7, 69.9, 69.8, 69.6, 69.6, 68.7, 68.2, 63.9, 61.1, 60.9, 49.7, 49.4, 43.8, 29.8, 28.6.
    HRMS for C₇₁H₈₇N₁₄O₂₈S (ESI-TOF) m/z: [M + H]⁺ calc.: 1615.5529; found: 1615.5531.
    HPLC: t_R = 16.6 min.
    FITC labeled glycooligomer FITC-S_AAA
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.55–8.20 (m, 6H), 8.11 (s, 4H), 7.90–7.72 (m, 4H), 7.26–7.15 (m, 2H), 7.08 (d, J = 8.5 Hz, 2H), 6.78–6.50 (m, 6H), 5.36–5.15 (m, 6H), 5.08–4.87 (m, 8H), 4.77–4.47 (m, 20H), 3.72–2.95 (m, 38H), 2.46–2.31 (m, 12H).
    ¹³C NMR (100 MHz, D₂O) δ/ppm 183.4, 175.4, 175.0, 173.7, 173.0, 171.9, 163.4, 160.7, 152.7, 152.6, 152.4, 152.3, 145.1, 143.7, 142.3, 130.4, 130.1, 127.7, 127.5, 125.8, 125.3, 114.8, 114.6, 111.4, 111.3, 104.3, 79.3, 78.8, 77.4, 76.5, 71.8, 69.5, 69.4, 69.2, 63.5, 60.5, 50.1, 48.8, 33.9, 28.0.
    HRMS for C₈₇H₁₁₁N₁₈O₃₇SNa (ESI-TOF) m/z: [M + H + Na]²⁺ calc.: 1027.3482; found: 1027.3488.
    HPLC: t_R = 16.1 min.
    FITC labeled glycooligomer FITC-S_AAAA
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.65–8.17 (m, 7H), 8.11 (s, 5H), 7.86-7.83 (m, 4H), 7.28–7.12 (m, 2H), 7.08 (d, J = 8.4 Hz, 2H), 6.72–6.33 (m, 6H), 5.29–5.12 (m, 8H), 5.09–4.88 (m, 10H), 4.79–4.42 (m, 26H), 3.90–2.93 (m, 46H), 2.49–2.29 (m, 16H).
    ¹³C NMR (100 MHz, D₂O) δ/ppm 185.7, 175.6, 175.1, 175.1, 175.0, 173.0, 170.6, 169.8, 168.8, 161.2, 155.2, 143.6, 143.4, 143.2, 130.3, 129.5, 126.6, 125.8, 118.0, 114.7, 114.6, 110.7, 103.6, 79.2, 77.4, 76.4, 71.7, 70.9, 69.1, 63.2, 60.4, 49.9, 44.8, 29.7, 28.6.
    HRMS for C₁₀₃H₁₃₅N₂₂O₄₆SNa (ESI-TOF) m/z: [M + H + Na]²⁺ calc.: 1235.9271; found: 1235.9276.
    HPLC: t_R = 15.8 min.
    FITC labeled glycooligomer FITC-S_BAA
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.52–8.26 (m, 3H), 8.22 (s, 1H), 8.14–8.07 (m, 4H), 7.85-7.72 (m, 4H), 7.28–7.15 (m, 2H), 7.08 (d, J = 8.5 Hz, 2H), 6.74–6.54 (m, 6H), 5.28–5.17 (m, 6H), 5.05–4.74 (m, 8H), 4.70–4.41 (m, 20H), 3.89–3.22 (m, 38H), 2.47–2.26 (m, 12H).
    ¹³C NMR (100 MHz, D₂O) δ/ppm 185.0, 175.3, 175.0, 174.2, 173.1, 170.9, 163.8, 160.6, 159.3, 157.9, 157.2, 143.7, 129.5, 125.9, 125.2, 114.6, 79.3, 77.7, 77.5, 76.5, 73.4, 71.8, 71.1, 69.3, 66.4, 63.5, 60.8, 58.0, 49.9, 29.8, 28.6.
    HRMS for C₈₇H₁₁₁N₁₈O₃₇SNa (ESI-TOF) m/z: [M + H + Na]²⁺ calc.: 1027.8499; found: 1027.8496.
    HPLC: t_R = 16.1 _min.
    FITC labeled glycooligomer FITC-S_BBA
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.83–8.25 (m, 5H), 8.23(s, 1H), 8.19–8.02 (m, 4H), 7.82–7.85 (m, 4H), 7.26–7.12 (m, 2H), 7.07 (d, J = 8.6 Hz, 2H), 6.69–6.44 (m, 6H), 5.30–5.15 (m, 6H), 5.12–4.80 (m, 8H), 4.74–4.42 (m, 20H), 3.72–3.22 (m, 38H), 2.47–2.33 (m, 12H) .
    ¹³C NMR (100 MHz, D₂O) δ/ppm 182.4, 177.4, 175.6, 175.2, 173.9, 173.4, 173.0, 164.8, 163.3, 161.6, 157.3, 146.1, 145.2, 143.3, 122.1, 121.1, 116.8, 116.0, 114.1, 104.5, 79.4, 78.5, 75.0, 73.3, 72.6, 69.3, 69.3, 69.3, 60.9, 60.8, 52.1, 50.6, 34.5, 29.8.
    HRMS for C₈₇H₁₁₂N₁₈O₃₇S (ESI-TOF) m/z: [M+2H]²⁺ calc.: 1016.8590; found: 1016.8590.
    HPLC: t_R = 16.1 min.
    FITC labeled glycooligomer FITC-S_CAA
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.57–8.16 (m, 6H), 8.10 (s, 4H), 7.85-7.75 (m, 4H), 7.24–7.12 (m, 2H), 7.07 (d, J = 8.4 Hz, 2H), 6.69–6.47 (m, 6H), 5.25–5.18 (m, 6H), 5.05–4.88 (m, 8H), 4.73–4.43 (m, 20H), 3.73–3.01 (m, 38H), 2.46–2.30 (m, 12H).
    ¹³C NMR (100 MHz, D₂O) δ/ppm 182.5, 174.7, 173.0, 172.5, 171.6, 160.6, 155.5, 145.9, 143.6, 139.8, 129.6, 129.4, 125.7, 117.0, 114.8, 114.4, 111.4, 103.3, 79.2, 77.4, 76.4, 73.3, 71.8, 70.8, 69.6, 69.2, 64.4, 63.8, 63.5, 63.3, 60.7, 48.8, 45.6, 31.7, 29.8.
    HRMS for C₈₇H₁₁₁N₁₈O₃₇SNa (ESI-TOF) m/z: [M + H + Na]²⁺ calc.: 1027.8499; found: 1027.8502.
    HPLC: t_R = 16.1 min.
    FITC labeled glycooligomer FITC-S_CCA
    ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.56–8.13 (m, 6H), 8.10 (s, 4H), 7.85-7.72 (m, 4H), 7.25–7.14 (m, 2H), 7.07 (d, J = 8.5 Hz, 2H), 6.70–6.48 (m, 6H), 5.30–5.14 (m, 6H), 5.07–4.87 (m, 8H), 4.66–4.43 (m, 20H), 3.78–2.93 (m, 38H), 2.45–2.34 (m, 12H).
    ¹³C NMR (100 MHz, D₂O) δ/ppm 183.4, 175.3, 173.7, 172.5, 171.4, 171.0, 164.9, 160.6, 156.9, 146.1, 144.0, 134.2, 129.6, 127.6, 125.9, 125.8, 125.8, 113.2, 103.6, 79.0, 78.0, 77.1, 73.6, 71.8, 71.2, 70.3, 70.0, 69.1, 64.4, 64.0, 63.4, 50.5, 44.7, 31.3, 29.4.
    HRMS for C₈₇H₁₁₁N₁₈O₃₇SNa (ESI-TOF) m/z: [M + H + Na]²⁺ calc.: 1027.8499; found: 1027.8496.
    HPLC: t_R = 16.1 min.
    FITC labeled glycooligomer FITC-S_SS
    ¹H NMR (400 MHz, MeOD-d₄) δ/ppm 8.15 (d, J = 2.1 Hz, 1H), 8.06 (s, 1H), 8.02 (s, 1H), 7.98 (s, 1H), 7.84 (d, J = 8.9 Hz, 2H), 7.76 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 8.9 Hz, 2H), 6.70–6.62 (m, 4H), 6.54 (d, J = 2.4 Hz, 1H), 6.52 (d, J = 2.4 Hz, 1H), 5.33 (dq, J = 7.7, 4.9 Hz, 2H), 4.71–4.52 (m, 12H), 3.90–3.53 (m, 18H), 2.56–2.47 (m, 8H).
    ¹³C NMR (100 MHz, MeOD-d₄) δ/ppm 175.8, 173.2, 171.1, 162.6, 161.3, 154.1, 145.5, 145.3, 144.6, 130.7, 130.3, 127.5, 126.7, 126.4, 126.1, 115.7, 113.6, 111.4, 103.6, 72.2, 71.4, 70.3, 69.6, 69.3, 65.2, 65.1, 62.4, 51.4, 45.5, 30.0, 29.6.
    HRMS for C₅₉H₆₃N₁₂O₂₀S (ESI-TOF) m/z: [M-H]^- calc.: 1291.4008; found: 1291.3988.
    HPLC: t_R = 18.64 min.
    FITC labeled glycooligomer FITC-S_SSS
    ¹H NMR (400 MHz, MeOD-d₄) δ/ppm 8.17–8.14 (m, 2H), 8.09 (s, 1H), 8.03 (s, 1H), 8.00 (s, 1H),7.84 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.07 (d, J = 8.9 Hz,2H), 6.70–6.64 (m, 4H), 6.58–6.51 (m, 2H), 5.37–5.31 (m, 3H), 4.69–4.56 (m, 16H), 3.90–3.54 (m, 20H), 2.55–2.47 (m, 12H).
    ¹³C NMR (100 MHz, MeOD-d₄) δ/ppm 175.8, 173.2, 171.9, 171.1, 162.6, 161.3, 154.1, 145.3, 144.6, 130.8, 130.7, 130.3, 127.5, 126.7, 126.1, 115.7, 113.6, 111.4, 103.6, 72.2, 72.1, 71.5, 71.4, 70.3, 69.6, 69.3, 65.2, 65.1, 62.4, 51.4, 49.6, 45.5, 30.0, 29.6.
    HRMS for C₆₉H₇₈N₁₅O₂₅S (ESI-TOF) m/z: [M + H]⁺ calc.: 1548.5009; found: 1548.5015.
    HPLC: t_R = 18.55 min.
• 12.
  Liquid-phase synthesis of sequence-controlled fluorescent glycooligomers. (Figure 9)

  • a.
    Coupling protocol.

    • i.
      Dissolve the alkyne-terminated glycooligomers (1.0 equiv.) and azide-terminated saccharide monomer (1.2 equiv.) (or azide-terminated FITC) in MeOH/H₂O (1:1, v/v).

      CRITICAL: For the coupling of the first azide-terminated saccharide monomer, dissolve 4-(Prop-2-yn-1-yloxy)benzylamide completely using a solvent mixture of MeOH/H₂O (3:1, v/v).

    • ii.
      Dissolve CuSO₄·5H₂O (0.2 equiv.) and sodium ascorbate (0.4 equiv.) separately in water, then combine and add to the reaction mixture.

      CRITICAL: Add the mixture of CuSO₄·5H₂O and sodium ascorbate to the reaction system when its color changes from black to yellow. The reagent concentration was 0.15 mol/L in MeOH/H₂O (1:1, v/v) solution.

      Note: For FITC labeling, use CuSO₄·5H₂O (4 equiv.) and sodium ascorbate (8 equiv.).

    • iii.
      Stir the reaction mixture at 25°C or so for 1 h.
    • iv.
      Filter the reaction mixture and purify by preparative HPLC.

      Note: Monitor the reaction process by the analytic HPLC. Part of the product may have a TMS-decapping during coupling.

  • b.
    TMS decapping protocol.

    • i.
      The obtained glycooligomer iterative product (1.0 equiv.) from the step above was dissolved in MeOH/H₂O (1:1, v/v) and cool to 0°C.
    • ii.
      Add TBAF (20 equiv.) dropwise and stir at 0°C for 0.5 h.
    • iii.
      Warm to 25°C or so and stir at 25°C or so for 1.5 h.
    • iv.
      Filter the reaction mixture and purify by preparative HPLC.
  • c.
    Critical Step: Analyze the product by ¹H NMR, ¹³C NMR, HRMS.

Figure 8.

Solid-phase synthesis of FITC labeled sequence-controlled glycooligomers

i) B_A, B_B or B_C, CuI, DIPEA, DMF, 3 h; ii) TBAF, THF/DMF/MeOH/H₂O, 0.5 h; iii) S9, CuI, DIPEA, DMF, 3 h; iv) TFA, DCM, 0.5 h.

Figure 9.

Liquid-phase synthesis of FITC labeled sequence-controlled glycooligomers and skeleton dimer and trimer without saccharide moieties

i) B_A, B_B, B_C or S3, CuSO₄·5H₂O, sodium ascorbate, MeOH/H₂O, 1 h; ii) TBAF, MeOH/H₂O, 2 h; iii) S9, CuSO₄·5H₂O, sodium ascorbate, MeOH/H₂O, 1 h.

Part3: In vivo studies of sequence-controlled glycooligomers

Timing: 1 month

This section outlines the method for examining the biodistribution and tumor targeting capability of sequence-controlled glycooligomers in vivo, utilizing the LoVo tumor model.

• 13.
  Biodistribution evaluation.

  • a.
    Inject LoVo cells (logarithmic growth stage) suspended in Matrigel into the right flank subcutaneous tissues of the 4–6 weeks old, female BALB/c nude mice (1 × 10⁶ cells/mouse, 100 μL).
  • b.
    When the tumor size reached 500 mm³, randomly divide the mice into 5 groups (n = 3) and treated with FITC-S_AA and FITC-S_SS (8.0 and 6.5 mg/kg, respectively, with the same molar concentration) by intravenous injections.
  • c.
    Sacrifice the mice 1, 2, 4, 8 and 12 h after injection to collect main organs (heart, liver, lung, kidney spleen and tumor) (n = 3).
  • d.
    Measure the FITC fluorescence intensity of the collected organs and tumors using an in vivo imaging system (IVIS Spectrum CT system, λ_ex/em = 488/512 nm) to obtain the biodistribution of FITC-S_AA and FITC-S_SS in the treated mice. Use Living Image 4.4 software to calculate the average fluorescence intensities of the major organs and tumors for quantitative analyses.
• 14.
  Pharmacokinetic study.

  • a.
    Randomly divide ICR female tumor-free mice at 6–8 weeks into 2 groups (n = 3).
  • b.
    Inject the mice intravenously within each group with FITC-S_AA and FITC-S_SS (dosage: 8.0 mg/kg) individually.
  • c.
    Collect blood through eye socket bleeding using capillary tubes at 1 h, 2 h, 4 h, and 8 h, and deposit it into 1.5 mL centrifuge tubes containing heparin anticoagulant.
  • d.
    Centrifuge the blood samples at 4°C, 1,503 x g for 30 s.
  • e.
    Take 10 μL of plasma samples and add them to the 96-well plates (10 μL of blank plasma for a blank control and an internal standard blank control), followed by the addition of 100 μL of internal standard solution (100 ng/mL Tolbutamide in 50% MeOH/CH₃CN). (A double blank control consists solely of 100 μL of internal standard solution).
  • f.
    Take each sample in a 0.5 mL centrifuge tube, vortex the samples for 5 min, and then centrifuge the samples at 4°C, 18,407 x g for 5 min.
  • g.
    Collect 50 μL of the supernatant, add 50 μL of ultrapure water, and mix thoroughly to prepare the final sample for analysis in the LC/MS machine.
  • h.
    Determine the amount of compounds by analytical HPLC equipped with a Synergi 4 μm Fusion-RP 80 Å Luna C18 column (2∗50 mm), eluting initially with 85% water for 0.2 min with a flow rate of 0.8 mL/min, followed by a gradient shift from 85% water to 95% CH₃CN over 1.6 min and a change of the flow rate to 0.6 mL/min, and then maintaining 95% CH₃CN for 0.3 min with a flow rate of 0.8 mL/min (Water with 0.1% HCO₂H).
• 15.
  Hemolytic test.

  • a.
    Collect blood samples (1 mL) from ICR female mice (6–8 weeks) and add into 1.5 mL centrifuge tubes containing heparin anticoagulant.
  • b.
    Centrifuge the blood samples at 4°C, 845 x g for 5 min, and discard the supernatant.
  • c.
    Add erythrocyte stock dispersion (50 μL) to saline containing different concentrations of FITC-S_AA (950 μL, 1, 10 and 50 mM), maintaining the erythrocyte concentration at 2% (w/v).
  • d.
    Employ saline solution (0.9% NaCl) as a negative control (0% lysis) and distilled water as a positive control (100% lysis).
  • e.
    Incubate the resulting suspension at 37°C for 30 min.
  • f.
    Centrifuge the samples at 25°C or so, 845 x g for 5 min.
  • g.
    Measure the absorbance of the resulting supernatant at 540 nm.
  • h.
    Calculate the hemolysis ratio according to the following equation:

$$\text{Hemolysis}\%=\frac{{\mathrm{A}}_{\text{sample}}-{\mathrm{A}}_{\text{negative}}}{{\mathrm{A}}_{\text{positive}}-{\mathrm{A}}_{\text{negative}}}\times 100\%$$

A_sample, A_negative and A_positive represent the absorbance of the samples, the negative and the positive control, respectively.

Expected outcomes

The products are expected to be purified as shown in Table 1 (Figure 10). For in vivo tumor targeting properties, compared to FITC-S_SS, FITC-S_AA is expected to rapidly concentrate in the tumor region, exhibiting significantly higher fluorescence intensities than any other normal tissue (Figure 11). Moreover, FITC-S_AA probably has higher plasma concentration and longer circulation time than FITC-S_SS in pharmacokinetic study (Figure 12), along with better biocompatibility in a hemolytic test (Figure 13).

Table 1.

Compound synthetic yields and appearance

Compound#	Percent of yield	Appearance
S1	79	Colorless oil
S2	60	Colorless oil
S3	Quantitative	Viscous colorless liquid
S4A	Quantitative	White solid
S4B	Quantitative	White solid
S4C	Quantitative	White solid
BA	21	Yellowish solid
BB	18	Yellowish solid
BC	22	Yellowish solid
S5	95	White solid
S6	Quantitative	White solid
S8	65	White solid
S9	95	Yellow solid
FITC-SA	75 (solid-phase synthesis); 50 (liquid-phase synthesis)	Yellow solid
FITC-SAA	69 (solid-phase synthesis); 28 (liquid-phase synthesis)	Yellow solid
FITC-SAAA	57 (solid-phase synthesis); 16 (liquid-phase synthesis)	Yellow solid
FITC-SAAAA	49 (solid-phase synthesis); 6.9 (liquid-phase synthesis)	Yellow solid
FITC-SBAA	49 (solid-phase synthesis); 8.3 (liquid-phase synthesis)	Yellow solid
FITC-SBBA	43 (solid-phase synthesis); 7.4 (liquid-phase synthesis)	Yellow solid
FITC-SCAA	53 (solid-phase synthesis); 13 (liquid-phase synthesis)	Yellow solid
FITC-SCCA	55 (solid-phase synthesis); 17 (liquid-phase synthesis)	Yellow solid
FITC-SSS	34 (liquid-phase synthesis)	Yellow solid
FITC-SSSS	11 (liquid-phase synthesis)	Yellow solid

Figure 10.

HPLC traces of purified homo-glycooligomers S_A, S_AA, S_AAA and S_AAAA

Figure 11.

In vivo tumor targeting properties of FITC-S_AA and FITC-S_SS

(A and B) Ex vivo fluorescence images of major organs and tumors collected 1, 2, 4, 8 and 12 h after intravenous injection of FITC-S_AA (A) or FITC-S_SS (B).

(C and D) The average fluorescence intensities of the major organs and tumors from the mice treated with FITC-S_AA or FITC-S_SS were quantified and concluded in (C) and (D), respectively. (n = 3/group) Data are presented as mean ± SD.

Figure 12.

Pharmacokinetics of FITC-S_AA and FITC-S_SS in healthy BALB/c mice

Compound levels were determined by HPLC.

Figure 13.

Hemolytic test carried out for FITC-S_AA

Saline solution (0.9% NaCl) was employed as a negative control (0% lysis) and distilled water as a positive control (100% lysis). (n = 3/group) Data are presented as mean ± SD.

Quantification and statistical analysis

Data were presented as mean ± SD. Dunnett’s t-tests were used to determine whether the variance between two groups is similar. One-way analysis of variance (ANOVA) was applied for comparison of multiple groups. Statistical analysis was performed using GraphPad Prism. A “P” value < 0.05 was considered statistically significance.

Limitations

The protocol involves multiple steps that are time-intensive. Some steps in the synthesis have low yields, which could affect the overall efficiency of the protocol and increase the cost of the process.

Troubleshooting

Problem 1

Step 5, Synthesis of glycooligomer building blocks (B_A, B_B, B_C): The yield of the reaction is low.

Potential solution

Use a freshly opened oxalyl chloride for improper storage may causes oxalyl chloride to react with water in the air.

Ensure that the reaction proceed under a nitrogen atmosphere without moisture.

Avoid contact with air/moisture when mix Solution-1 with Solution-2.

Ensure that adding Solution-1 to Solution-2 dropwise is taking at least 10 min or a longer time.

Shorten the reaction time in Substep i of Step 5 to reduce the appearance of by-products.

Problem 2

Step 7, Synthesis of 4-(Prop-2-yn-1-yloxy)benzoic acid S6: Uncomplete reaction of S5 result in a lower yield of the reaction than that expected.

Potential solution

Use extra amount of NaOH (a double dosage) in Substep b of Step 7 to consume superfluous propargyl bromide if the crude product of S5 is directly used for step 7 without purification.

Problem 3

Step 13, Liquid-phase synthesis of sequence-controlled fluorescent glycooligomers: The yield of the reaction is low or the reaction fail to proceed.

Potential solution

Replace MeOH with MeCN in Substep a of Step 13 to ensure that the alkyne-terminated glycooligomers and azide-terminated saccharide monomer (or azide-terminated FITC) are dissolved completely.

Increase the amounts of CuSO₄·5H₂O and sodium ascorbate in Substep ii of Substep a of Step 13.

Problem 4

Step 13, Liquid-phase synthesis of sequence-controlled fluorescent glycooligomers: The yield of the reaction is low due to the ease with which the TMS (trimethylsilyl) protecting group is removed from the glycooligomer moieties in the CuAAC system.

Potential solution

Use a more stable silyl protective group for alkynyl, such as triisopropylsilyl (TIPS) and tert-butyldiphenylsilyl (TBDPS).

Optimize the catalytic system or other conditions, such as temperature, time and so on.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jin Geng (jin.geng@siat.ac.cn).

Technical contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the technical contact, Jin Geng (jin.geng@siat.ac.cn).

Materials availability

All materials generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

• •Additional data relevant to this work are available from the lead contact upon reasonable request.
• •This paper does not report original code.