TWI778596B - Compounds and methods for detecting cholangio-cancerous cells - Google Patents

Abstract

Disclosed herein is a novel compound of formula (I) for detecting circulating cancerous cells, particularly, cholangio-cancerous cells, from a biological sample,

wherein, R₁ and R₂ are independently H, or -SO₃M; and M is a monovalent cation selected from the group consisting of sodium ion, potassium ion, lithium ion or ammonium ion. Also disclosed herein is a method of detecting cholangio-cancerous cells from a biological sample of a subject suspected of having cholangiocarcinoma (CCA). The method includes steps of, contacting the biological sample with a magnetic bead pre-coated with the compound of formula (I); detecting a complex formed between the magnetic bead and the biological sample in an immunoassay; wherein, the presence of the complex is an indication that the subject has CCA.

Description

Compounds and methods for detecting cholangiocarcinoma cells

The present disclosure generally relates to a compound and method for detecting circulating cancer cells in a biological sample.

Cholangiocarcinoma (CCA) is the second most common primary malignant liver cancer, and it often occurs in the bile ducts (the part of the liver that drains bile into the small intestine). It is typically characterized by late diagnosis, poor prognosis, high recurrence rate and frequent tumor metastasis. To date, the main detection tools for CCA include imaging techniques including ultrasonography, MRI and computed tomography, and pathology by forceps biopsy or brush cytology diagnosis, etc. These methods are limited by tumor size, location, and lesions, so that they cannot detect CCA tumors at an early stage. At present, the most commonly used tumor biomarkers for the diagnosis of CCA are carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA), but the sensitivity and specificity of CA19-9 and CEA is far from satisfactory.

Circulating tumor cells (CTCs) have been suggested to play a role in cancer metastasis and spread, so they could be used as markers for the detection of tumors, especially those that metastasize. However, they are not conclusive in the clinical assessment of CCA. The quantification of relatively small numbers of CTCs in the context of millions of blood cells has severely hindered progress in the diagnosis of CCA, and the use of CTC-specific antigens (eg, EpCAM, EphB4, HER2, EGFR, CEA, etc.) Affinity separation techniques have not yet produced results that can be used in clinical applications.

In view of this, there is an urgent need for a novel biomarker in the related fields to facilitate the detection of CTCs in a biological sample, which can then be used to determine the prognosis of CCA.

The present disclosure is based, at least in part, on the unexpected discovery by the inventors of a novel octasaccharide that binds to the surface of cholangiocarcinoma cells, thereby allowing these octasaccharides to be used to detect bile ducts in a biological sample of an individual Cancer cells in which the individual is at risk of developing cholangiocarcinoma (CCA) or suffering from CCA.

其中，R ₁及R ₂個別是H或–SO ₃M；且M是一單價陽離子並選自由鈉離子、鉀離子、鋰離子及銨離子所組成的群組。 Accordingly, the first aspect of the present disclosure relates to a compound of formula (I),

wherein, R ₁ and R ₂ are individually H or -SO ₃ M; and M is a monovalent cation and is selected from the group consisting of sodium ion, potassium ion, lithium ion and ammonium ion.

According to a preferred embodiment of the present disclosure, in the compound of formula (I), R ₁ and R ₂ are individually —SO ₃ M, and M is a sodium ion.

A second aspect of the present disclosure relates to a method for treating and detecting a cholangiocarcinoma cell from a biological sample of an individual suspected of having CCA. The method contains, (a) contacting the biological sample with the compound of formula (I); and (b) in an immunoassay, detecting a complex formed by the compound of formula (I) and the biological sample; Wherein, the presence of the complex means that the individual is CCA positive.

According to a preferred embodiment of the present disclosure, the compound of formula (I) is coupled to streptavidin, and the streptavidin is coupled to the outer surface of a magnetic bead.

According to a preferred embodiment of the present disclosure, in the compound of formula (I), R ₁ and R ₂ are individually —SO ₃ M, and M is a sodium ion.

Exemplary biological samples suitable for use in the methods of the present disclosure include, but are not limited to, blood, plasma, serum, urine, sputum, saliva, tissue samples, biological specimens, and tissue lysates. Preferably, the biological sample is blood from the individual.

According to a preferred embodiment of the present disclosure, the individual suffers from stage III or IV CCA, or metastatic CCA.

According to an optional embodiment of the present disclosure, the method further comprises, before step (a), pre-treating the blood with a lysis buffer to lyse the red blood cells (RBCs) therein .

The details of one or more implementations of the present disclosure are set forth in the detailed description below. Other features and advantages of the present disclosure will become apparent from the detailed description and the scope of the patent application.

It is to be understood that both the foregoing general summary and the following detailed description are exemplary in nature and are intended to provide further explanation of the invention as claimed.

In order to make the description of the present disclosure more detailed and complete, the following provides illustrative descriptions for the embodiments and specific embodiments of the present invention, but this is not the only form of implementing or using the specific embodiments of the present invention.

1. definition

Unless stated otherwise, "patient" or "subject" are used interchangeably in this disclosure and can refer to any animal. The animal may be a human individual, or a non-human individual. The individual can be a human, but can also be a mammal in need of veterinary treatment, eg, livestock or competitive animals, agricultural animals, and laboratory animals (eg, rats, mice, guinea pigs, primates, etc.). Typically the animal is a non-human mammal, such as a non-human primate. Non-human primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (eg, rhesus monkeys or bonobos (Pan)). Domestic and competitive animals include cattle, horses, pigs, sheep, deer, bison, buffalo, mink, felines (eg, domestic cats), canines (eg, dogs, wolves, and foxes), avian species (eg, chickens) , turkey and ostrich), and fish such as trout, catfish and salmon.

As used herein, "contacting" refers to a cell (eg, a cell in a biological sample) and refers to the delivery or "casting" of the cell or the biological sample in any mode ( administration) an agent in which the agent (eg, a compound of the present disclosure or a magnetic bead pre-coated with a compound of the present disclosure) is contacted with one or more cells in sufficient quantity to To achieve the purpose of affinity binding between the two.

Notwithstanding that the numerical ranges and parameters setting forth the broader scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains the standard deviation resulting from individual testing methods. Also, as used herein, the term "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value lies within an acceptable standard error of the mean, as considered by one of ordinary skill in the art to which this invention pertains. In addition to the operating examples, or unless explicitly stated otherwise, all numerical ranges, contents, values and percentages, such as those used in the present disclosure, are used to represent the amount of substances, the length of time, temperature, operating conditions, and content ratios. Equivalent readings should be understood as modified by the word "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and the accompanying claims are approximations that can vary as desired. At the very least, these numerical parameters should be construed to mean the number of significant digits indicated and the numerical values obtained by applying ordinary rounding.

As used in this disclosure, the singular forms "a" (a and an) and "the" (the) include the plural unless the context clearly dictates otherwise.

2. Compounds of the Disclosure

The present disclosure relates to the discovery of a novel octasaccharide as a biomarker that can be used to identify and/or detect cancer cells in a biological sample. The novel octasaccharide will be described in detail herein.

其中，R ₁及R ₂個別是H或–SO ₃M；且M是一單價陽離子並選自由鈉離子、鉀離子、鋰離子及銨離子所組成的群組。 In one aspect, the present disclosure relates to a compound of formula (I):

wherein, R ₁ and R ₂ are individually H or -SO ₃ M; and M is a monovalent cation and is selected from the group consisting of sodium ion, potassium ion, lithium ion and ammonium ion.

In certain embodiments of the present disclosure, in the compound of formula (I), R ₁ and R ₂ are individually —SO ₃ M, and M is a sodium ion.

Compounds of the present disclosure can be prepared according to the procedures described in the working examples. All stereoisomers of the compounds of the present disclosure, such as those isomers that may exist due to the asymmetric carbon atom in the R substituent of the compound of formula (I), including enantiomeric and diastereomeric ), all fall within the expected scope of the present invention. Individual stereoisomers of compounds of the present disclosure may be, for example, substantially free of other isomers, or may be, for example, as racemates or with all others, or other selected stereoisomers 's blend. The chiral center of the present disclosure may be of the S or R configuration as defined by the IUPAC 1974 Recommendations.

3. Instructions

The compounds of formula (I) of the present disclosure can bind to the surface of cancer cells, particularly cancers arising from the bile ducts. Accordingly, the present disclosure encompasses a method for identifying or detecting a cholangiocarcinoma cell from a biological sample of an individual suspected of having cholangiocarcinoma (CCA).

The methods of the present disclosure include contacting a biological sample of the individual with a compound of formula (I), wherein a binder is formed between the compound of formula (I) and the biological sample, indicating the presence of cholangiocarcinoma in the biological sample cell. Alternatively, to better identify or detect cholangiocarcinoma cells in a blood sample, prior to starting the methods of the present disclosure (ie, prior to contacting the blood with the magnetic beads), use A lysis buffer pretreats the blood sample to lyse red blood cells in the blood sample. Exemplary biological samples suitable for use in the methods of the present disclosure include, but are not limited to, blood, plasma, serum, urine, sputum, saliva, tissue samples, biological specimens, and tissue lysates. In a preferred embodiment, the biological sample is blood. Accordingly, the method comprises admixing a blood sample from the individual with the compounds of the present disclosure. During mixing, if the blood sample does contain cholangiocarcinoma cells, they will automatically bind to the compound of formula (I) and form a complex that can be detected by any suitable method, For example, enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining, etc. are used; and the presence of the complex indicates that the individual is CCA positive. On the contrary, if the blood sample does not contain cholangiocarcinoma cells, the blood sample will not form binding with the compound of formula (I), and the absence of such binding indicates that the individual is CCA negative. Preferably, the compound of formula (I) is coated on the surface of a magnetic bead and coupled via streptavidin pre-bonded to the outer surface of the magnetic bead. Therefore, when mixed with the biological sample, the magnetic beads coated with the compound of formula (I) can capture the cholangiocarcinoma cells present in the biological sample, and the cholangiocarcinoma cells on which the cholangiocarcinoma cells have been captured Magnetic beads can be formulated and collected using magnets.

After the disease state of the individual is confirmed, particularly when the individual is positive for CCA, appropriate treatment (eg, radiation therapy, chemotherapy, etc.) can then be administered to the individual to improve symptoms associated with CCA. In certain embodiments, the methods of the present disclosure further comprise administering to the individual an effective amount of a chemotherapeutic agent. Examples of chemotherapeutic agents suitable for use in the methods of the present disclosure include, but are not limited to, S-1, leucovorin, oxaliplatin, gemcitabine, cisplatin, and combinations thereof . In certain embodiments, an effective amount of S-1 is administered to the individual to ameliorate symptoms associated with CCA, wherein the S-1 is tegafur, 5-chloro-2,4 - 5-chloro-2,4-dihydropyrimidine (CDHP), and potassium oxonate (potassium oxonate) were administered as a combination. In optional embodiments, a radiation therapy is administered to the individual prior to, concurrently with, or after administration of the S-1. In other embodiments, a composition comprising gemcitabine and cisplatin is administered to the individual to improve symptoms associated with CCA. In certain further embodiments, a composition comprising S-1, chrysanthemum folic acid, oxaliplatin, and gemcitabine is administered to the individual to ameliorate symptoms associated with CCA.

According to a preferred embodiment of the present disclosure, the individual suffers from stage III or IV CCA, or metastatic CCA.

The present invention will now be described in more detail with reference to the following embodiments, which are intended to illustrate the invention and should not be construed as limiting the scope of the invention. Although they are generally routine method steps, other steps, methods or techniques well known to those skilled in the art may also be used.

Example

Coupling compounds of the present disclosure to magnetic beads

Compound 20 (100 × ^10-6 volume molar) was mixed with Dynabeads MyOne Streptavidin C1 magnetic beads (≈7–10 × ¹⁰ magnetic beads ml ^-1 , Ø = 1 μm, Invitrogen, USA) in 1 The reaction was carried out at a volume/volume ratio of : 10 and placed on a RM-2L INTELLI mixer (ELMI Ltd., Latvia) at 25 revolutions per minute (rpm) for 30 minutes at room temperature (C2 mode). Magnetic beads were then collected using a magnetic particle concentrator (MPC, Dynabeads MPC-1, Life Technologies) for 2 minutes. The supernatant was removed and the magnetic beads were washed three times with 1 mL of deionized water (DI water). These magnetic beads coupled to MB-SCH45 were suspended in the same volume of DI water as the initial volume of Dynabeads.

Collect blood samples

Informed consent was obtained from all 65 patients with advanced or metastatic CCA. Three healthy blood samples used in this study were collected from Tainan Blood Bank, Taiwan. Peripheral blood (10 mL) was drawn from each donor into K2EDTA-coated BD vacuum tubes (BD Vacutainer spray coated K2EDTA tubes, BD Diagnostics, USA). All samples were stored and shipped at 4°C and processed within 30 hours of sampling. To avoid bias, the samples were blindly processed, that is, the patients were not informed about the disease state, cancer stage, or tumor metastasis in advance.

Experimental setup

The experimental protocol consists of four steps: (1) lysis of RBCs and separation of the pellet, (2) removal of white blood cells (WBCs) by negative depletion, (3) positive enrichment using a magnet, and (4) immunofluorescence staining.

(1) dissolve RBC and separate the precipitate

9 mL of 1× RBC lysis buffer (Biolegend, USA) was added to 1 mL of peripheral blood, and the reaction was gently mixed with a rotary shaker (TKS ROCKER-S-01, Lab666, Taiwan) at room temperature. for 5 minutes to dissolve the RBCs. The sample was then dispensed into the uppermost PDMS layer of the wafer via the inlet provided by the microfluidic wafer (FIG. 1A). The microfluidic wafer was placed on a PMMA plate with grooves so that the microfluidic wafer could remain inside the PMMA plate, and then the microfluidic wafer was rotated in the manner described above. The device was then placed on a spin-coater (spin-coater; M and R Nanotechnology Co., Taoyuan, Taiwan), and under a negative gauge pressure of -67 kilopascals (kPa) at 1,200 Spinning at revolutions per minute (rpm) for 2 minutes, then the cell pellet was moved into the blood processing unit of the liquid gate layer of the wafer by the vacuum force generated by the microvalve. The blood waste containing dissolved RBCs was drained through the waste collection outlet (marked as (iii) in Figure 1A ) by vacuum suction. The remaining 5 ml of lysed blood was then dispensed into the uppermost layer of the microfluidic wafer and the above steps were repeated. The pellet was transported to a micromixer, and the micromixer was activated for 2 minutes to suspend the pellet in 180 microliters of Ix Phosphate Buffered Saline (PBS). The resulting cell suspension is mainly composed of nucleated cells, and the suspension is then negatively depleted to remove leukocytes.

(2) Negative consumption and positive enrichment using magnets

A negative depletion strategy of leukocyte depletion by binding of leukocytes to anti-CD45 antibodies on magnetic beads (Dynabeads CD45, Thermo-Fisher Scientific) was employed on the wafer. The cell-magnetic bead complex is then attracted to a magnet to facilitate removal of bound leukocytes from the lysed blood sample. After four rounds of negative depletion, the remaining cells (mainly CTCs) in the collected supernatant were treated with either MB-SCH45 (magnetic beads coupled to compound 20 above) or MB-anti-EpCAM (EpiEnrich, 4 × 10 ⁸ magnetic beads ml ^-1 , Ø = 4.5 μm, Thermo-Fisher Scientific) mixed. The magnetic bead-CTC complexes were then collected, washed, and transferred to the immunofluorescence staining unit of the wafer for further analysis.

(3) Immunofluorescence staining

For immunofluorescence staining, primary anti-CK17 (1:500 dilution; 100 μl, 0.6 mg ml ⁻¹ , GTX103765, rabbit antibody, GeneTex, USA) was used in this experiment. The secondary antibodies were goat anti-rabbit IgG DyLight 488 (GeneTex) diluted 1:500; and anti-CD45-PE (RRID: AB_10375163, HUCD45 PE, Life Technologies). It should be noted here that the anti-CD45 antibody used has a PE tag, so no additional primary antibody is required. Nuclear staining was then performed using DAPI (1 mg ml ^-1 , Thermo Fischer, US). Finally, after collection using a magnet, the magnetic bead-cell complexes were suspended in 10 μl of 1×PBS and collected through the waste collection chamber. This was transferred to numbered microscope slides and the cell-magnetic bead complexes were embedded using ProLong Gold Antifade Reagent (ProLong Gold Antifade Reagent; Invitrogen).

Numbered microscope slides were fabricated using a SU-8 standard photolithography process for CTC counting. Briefly, 30 μm thick SU-8 3035 (MicroChem, USA) was spin-coated on glass microscope slides. A 10-minute soft-baking process was performed, and the SU-8 microstructures were patterned with a UV irradiation dose of 147 mJ/cm2. Standard SU-8 development procedures were followed after a post-exposure bake for 1 min at 65°C and 5 min at 95°C. The microstructured glass slides are thus formed and then analyzed under a microscope using a DS-Qi1Mc camera equipped with a Peltier cooling device and a programmable gain amplifier (Nikon), which is equipped with a digital control The inverted microscope of the module is coupled. The software used is NIS-Elements basic research software (Br, version 4.20.00, 64 bit, Nikon). If cells are: (1) expressing CK, an internal structural protein that is mostly associated with epithelial cells, (2) having a high nuclear:cytoplasmic ratio (after DAPI staining), and (3) ) do not express CD45, the cells are considered CTCs.

Assess for metastatic CCA patients before and after chemotherapy CTC

The role of CTC in reflecting the assessment of treatment efficacy was investigated by analyzing blood samples (1 mL) from 5 patients with advanced or metastatic CCA before and after chemotherapy over a six-month period. The presence and number of CTCs in the blood was assessed using the previously mentioned MB-SCH45 and MB-anti-EpCAM, as well as immunofluorescence staining and conventional FACS analysis, and CT scans were used to measure tumor size. In the section measured by the CT scan, solid tumors were measured in at least one dimension of the longest diameter according to RECIST guidelines. A baseline study was performed on CTC counts from blood and radiology analysis within an average of 3 days prior to initiation of chemotherapy prior to initiation of chemotherapy. Blood samples were obtained from five CCA patients every 4-12 weeks, and the same type of imaging study was performed within an average of 4 days between angiography and blood draw.

Constructing Microfluidics wafer

To effectively utilize magnetic beads to capture cancer cells, an integrated microfluidic chip was constructed and used in this study. The microfluidic wafer is composed of 4 layers of polydimethylsiloxane (PDMS) and a layer of double-sided tape (Fig. 1). The PDMS on the top layer of the wafer is used to collect cell pellets from blood, and the second and third layers are the air control layer and the liquid channel layer, respectively. The fourth layer (the bottom layer) of the wafer is the thinnest and provides enough support to keep the liquid channels in it intact. All layers were bonded together by oxygen plasma treatment at 90 watts for 1 min (FC-12064, FEMTO Science, USA), in which polar functional silanol (SiOH) groups were introduced to change the surface properties of PDMS from Hydrophobicity changes to hydrophilicity. It was then copolymerized with a triblock containing Pluronic P123 (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) Compound; prepared in 99% ethanol aqueous solution (weight ratio)) solution treatment wafer to increase the hydrophilicity of PDMS. P123 was dispensed to the uppermost layer of the wafer and allowed to react with PDMS for 5 minutes. Then the microvalve was activated to make The P123 solution is delivered to the liquid channel layer of the wafer and finally through the waste collection chamber. The wafer is capable of performing various fluid functions (eg, mixing, transport, etc.) using two types of air: compressed air and suction pressure (i.e. vacuum). The The flow of compressed air or the generation of suction pressure is controlled by an electromagnetic valve (EMV). The entire device for operating the wafer is composed of an air compressor, a vacuum pump and a computer-controlled EMV functional unit.

A microfluidic wafer as shown in Figure 1A has two enclosed micromixer/micropump/transport units. As shown in Figure 1A, the dissolved blood is dispensed into the uppermost PDMS layer through the inlet (i) of the wafer. The channel marked (iii) acts as a drain for any air bubbles trapped during dispensing of the dissolved blood to the inlet, and it also serves as a waste collection unit for the dissolved blood after sedimentation. The _C1 chamber is designed in a way that ensures no loss of sediment after the collection and transfer of the above substances directly to the micromixer I of the liquid channel layer (L2). The second and third PDMS layers (L2 and L3) are used to perform positive enrichment and immunostaining steps by active mechanical displacement of two microvalves and an actuation chamber ) to operate pneumatically. The air control layer of the wafer is divided into two parts: a first and a second micro-mixer. The first micromixer (I in Figure 1A) serves as a blood processing unit, where negative depletion (using anti-CD45 (MB-CD45) coated magnetic beads to remove white blood cells (WBC)), and using a coating Positive enrichment was performed on magnetic beads of the compound of the invention (MB-SCH45) or anti-EpCAM antibody (MB-anti-EpCAM). The second micromixer (II in Figure 1A) was used as an immunofluorescence staining chamber.

Using microfluidic chips to capture cholangiocarcinoma cells

The microfluidic chip constructed above was used to capture cholangiocarcinoma cells from the blood of healthy or CCA positive patients. Briefly, blood samples collected from 65 CCA patients were individually mixed with red blood cell (RBC) lysis buffer and dispensed into the 5 inlets of the top PDMS layer of the wafer (L1 in Figure 1A), and then Magnetic beads coated with compound of formula (I) or anti-EpCAM antibody were manually pipetted into the blood processing unit and mixed gently for 15 minutes. After the reaction is complete, the cells are recognized and bound by the magnetic beads pre-coated with the compound of formula (I) of the present invention. These magnetic bead-cell complexes were then collected magnetically, washed with IX PBS buffer, and transferred to the immunofluorescence staining unit of the wafer for further analysis.

Statistical analysis: The sample size (n) has been provided in the associated figure description. Details on data correction (patients before and after chemotherapy) are provided in the experimental section. Statistical analysis was performed using OriginPro (OriginLab, Massachusetts, USA).

Example 1 Methods of chemical synthesis of compounds of the present disclosure

The compounds of the present invention were synthesized according to the procedures described in Schemes 1 to 5, respectively. Overall, the tetrasaccharide backbone is assembled from the appropriate disaccharide building blocks in a [2 + 2] fashion, while polymeric [4 + 4] glycosylation provides a fully protected octasaccharide backbone , which undergo further functional group transformations to generate final compounds with varying degrees of O -sulfonation. The glycan molecule was then biotinylated so that the resulting compound 20 (ie, SCH45) could be coupled to the surface of Dynabeads magnetic beads coated with streptavidin.

The structure of the synthetic glycosaminoglycan (GAC) of the present invention is composed of four mutated sulfated disaccharide units, and each saccharide in the disaccharide unit is N-acetyl-α-D-glucosamine ( N-acetyl-α-D-glucosamine, α-D-GlcMAc, abbreviated as "N"), β-D-glucuronic acid (β-D-glucuronic acid, β-D-GlcA, abbreviated as "G") or α-L-iduronic acid (α-L-iduronic acid, α-D-IdoA, abbreviated as "I"), and is bound by α-1→4 to another carbohydrate connect. Accordingly, compound 20 of the present disclosure may be denoted as NINGNING.

Scheme 1 : Synthesis of NI disaccharide donors

Scheme 2 : Synthesis of NG disaccharide receptors

Scheme 3 : Synthesis of NING Tetrasaccharide Backbone

Scheme 4 : Synthesis of NING Tetrasaccharide Donor and Acceptor

Scheme 5 : Synthesis of NINGNING octasaccharide backbone

Scheme 6 : Synthesis of Compounds of the Disclosure

6 -O- Acetyl -4 -O- [2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -4 -O- (2- naphthylmethyl )-α-D- glucopyranosyl ]-2 -O- benzyl -3 -O- benzyl -β-D- glucopyranosyl acetate (6 -O- Acetyl-4 -O- [2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-4 -O- (2-naphthylmethyl)-α- D-glucopyranosyl]-2 -O- benzoyl-3 -O- benzyl-β-D-glucopyranosyl acetate)(7)

Combine thioglycoside 1 (193 mg, 0.22 mmol) with 4-ethanol 6 (86 mg, 0.19 mmol) in anhydrous CH ₂ Cl ₂ (5.6 mL) at room temperature under argon The solution in was stirred with freshly dried 3Å molecular sieves (557 mg) for 1 h. The reaction flask was cooled to –60°C and mixed with N-isobutadiimide (N-idosuccinimide, NIS, 61 mg, 0.26 mmol) and trifluoromethanesulfonic acid (triflic acid, TfOH, 3 μl). , 0.04 mmol) was added to this solution, and the mixture was gradually warmed to –50°C. After stirring for 2 hours, the reaction was quenched by the addition of _Et3N (8 [mu]L, 0.06 mmol), and the mixture was filtered through celite, and the solids _were washed with _CH2Cl2 . The filtrate was washed with 10% Na ₂ S ₂ O _{5 (aq)} (5.0 mL), dried over MgSO ₄ , filtered and concentrated under reduced pressure to give a residue which was subjected to flash column chromatography Purification (flash column chromatography) (ethyl acetate/hexane = 1/4) afforded the expected disaccharide 34 (192 mg, 86%). [α] ²⁵ _D +0.6 ( c 5.34, CHCl ₃ ); IR (thin film): ν 2930, 2107, 1738, 1265, 1111, 1067 cm ^–1 ; ¹ H NMR (600 MHz, CDCl ₃ ): δ 8.02 (d, J = 7.6 Hz, 2H, Bz-H), 7.85–7.81 (m, 1H, Ar-H), 7.79–7.75 (m, 2H, Ar-H), 7.66 (d, J = 7.1 Hz, 2H, Ar-H), 7.64–7.61 (m, 3H, Ar-H), 7.58 (t, J = 7.4 Hz, 1H, Ar-H), 7.51–7.47 (m, 2H, Ar-H), 7.45 (t, J = 7.8 Hz, 2H, Ar-H), 7.42 (d, J = 8.2 Hz, 2H, Ar-H), 7.40–7.34 (m, 2H, Ar-H), 7.34–7.30 (m, 2H, Ar-H), 7.30–7.24 (m, 5H, Ar-H), 7.24–7.20 (m, 3H, Ar-H), 7.18 (d, J = 8.2 Hz, 2H, Ar-H), 5.83 (d, J = 7.9 Hz, 1H, 1-H), 5.52 (d, J = 3.6 Hz, 1H, 1' - H), 5.43 (dd, J = 7.9, 8.5 Hz, 1H, 2-H), 4.95–4.88 (m, 2H, ArCH ₂ ), 4.85–4.80 (m, 2H, ArCH ₂ ), 4.76 (d, J = 10.7 Hz, 1H, ArCH ₂ ), 4.73 (d, J = 10.7 Hz, 1H, ArCH ₂ ), 4.35 (d, J = 12.3 Hz, 1H, 6-H _a ), 4.09 (dd, J = 12.3, 4.1 Hz, 1H, 6-H _b ), 4.04 (dd, J = 8.5, 8.5 Hz , 1H, 3-H), 4.02–3.80 (m, 1H, 6' - H _a ), 3.95 (dd, J = 9.0, 8.5 Hz, 1H, 4-H), 3.92–3.88 (m, 2H, 3 ' -H, 5 ' -H), 3.81–3.77 (m, 1H, 5-H), 3.75 (d, J = 1 1.7 Hz, 1H, 6' - H _b ), 3.62 (d, J = 6.8 Hz, 1H, 4' - H), 3.32–3.25 (m, 1H, 2' - H), 1.99 (s, 3H, Ac ), 1.74 (s, 3H, Ac), 1.04 (s, 9H, t Bu TBDPS); ^{13C NMR (150 MHz, CDCl} ₃ ): δ 170.4 (C), 169.2 (C), 165.0 (C), 137.3 ( C), 136.7 (C), 135.9 (CH), 135.5 (CH), 135.3 (C), 133.6 (CH), 133.3 (C), 133.2 (C), 133.0 (C), 132.8 (C), 131.5 ( CH), 129.8 (CH), 129.7 (CH), 129.5 (CH), 129.1 (C), 128.6 (CH), 128.4 (CH), 128.2 (CH), 127.9 (CH), 127.8 (CH), 127.75 ( CH), 127.70 (CH), 127.6 (CH), 126.3 (CH), 126.2 (CH), 126.0 (CH), 125.5 (CH), 121.8 (C), 97.9 (CH), 91.7 (CH), 82.9 ( CH), 79.9 (CH), 78.0 (CH), 75.2 (CH ₂ ), 74.6 (CH ₂ ), 74.5 (CH ₂ ), 73.4 (CH), 73.2 (CH), 73.0 (CH), 72.6 (CH) , 63.3 (CH ), 62.6 (CH ₂ ), 62.0 (CH ₂ ), 26.9 (CH ₃ ), 20.8 (CH ₃ ), 20.4 (CH ₃ ), 19.3 (C); HRMS ESI: m/z calcd for C ₆₄ H ₆₆ N ₃ O ₁₃ BrSiNa ([M + Na] ⁺ ): 1214.3446, found: 1214.3455.

[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -4 -O- (2- naphthylmethyl )-α-D- glucopyranosyl ]-(1→4)-(6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -β-D- glucopyranosyl acetate ([2-Azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-4 -O- (2-naphthylmethyl)-α-D-glucopyranosyl]-(1→4) -(6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-α-L-idopyranosyl)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 - O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-β-D-glucopyranosyl acetate)(9 )

A solution of glucosinolate 5 (1.2 equiv.) and 4'-ethanol 8 (1.0 equiv.) in dry _{CH2Cl2 (} 10 mL/g total glucosinolate and 4'-ethanol) was mixed with fresh at room temperature. Activated AW-300 molecular sieves (0.5 g/g total of glucosinolate and 4'-ethanol) were stirred for 1 hour and then cooled to -78°C. NIS (2.0 equiv) was added to the mixture and after 5 minutes TfOH (0.1 equiv) was added slowly via a microsyringe. The temperature was gradually raised to -50°C and maintained at this temperature until complete consumption of glucosinolate 5 (approximately 2 hours by TLC plate analysis). _The mixture was then neutralized by adding _Et3N , diluted with _CH2Cl2 and filtered through celite. The resulting solution was washed with ₁₀ % Na2S2O3 ( _{aq )} and _H2O . The organic layers were combined, dried over _MgSO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography to yield tetrasaccharide 9 (81%).

IR (thin film): ν 2930, 2108, 1742, 1266, 1111 cm ^–1 ; ¹ H NMR (600 MHz, CDCl ₃ ): δ 8.03 (dd, J = 8.2, 1.0 Hz, 2H, Bz-H), 8.00 (dd, J = 7.8, 1.1 Hz, 2H, Bz-H), 7.84 (dd, J = 6.1, 3.1 Hz, 1H 2-NAP-H), 7.78-7.72 (m, 2H, Ar-H), 7.69–7.61 (m, 4H, Ar-H), 7.60–7.54 (m, 6H, Ar-H), 7.51–7.43 (m, 4H, Ar-H), 7.41–7.29 (m, 13H, Ar-H) ), 7.28–7.13 (m, 17H, Ar-H), 7.05 (d, J = 8.3 Hz, 1H, Ar-H), 7.00 (d, J = 8.3 Hz, 1H, Ar-H), 5.83 (d , J = 8.0 Hz, 1H, 1-H), 5.44–5.39 (m, 3H, 1 ' -H, 1 '' -H, 2-H), 5.17 (s, 1H, 2 '' -H), 4.90 (d, J = 11.3 Hz, 1H, ArCH ₂ ), 4.89 (d, J = 10.8 Hz, 1H, ArCH ₂ ), 4.84 (s, 2H, ArCH ₂ ), 4.78 (d, J = 10.3 Hz, 1H , ArCH ₂ ), 4.73–4.70 (m, 2H, ArCH ₂ ), 4.69 (d, J = 3.6 Hz, 1H, 1 ''' -H), 4.48 (d, J = 11.3 Hz, 1H, ArCH ₂ ) , 4.39–4.33 (m, 3H, ArCH ₂ , 6-H _a , 5 '' -H), 4.17 (dd, J = 9.6, 8.8 Hz, 1H, 4 ' -H), 4.15-4.08 (m, 3H , ArCH ₂ , 3 '' -H, 6 '' -H _a ), 4.05–3.99 (m, 3H, 3-H, 6 ' -H _a , 6 '' -H _b ), 3.96 (dd, J = 12.1, 4.3 Hz, 1H, 6-H _b ), 3.89–3.80 (m, 4H, 4-H, 6' - H _b , 4 ''' -H, 6 ''' -H _a ), 3.77 (ddd, J = 9.3, 3.8, 2.1 Hz, 1H, 5-H), 3.74–3.70 (m, 2H, 3 ' -H, 5 ''' -H), 3.68–3.63 (m, 2H, 3 ''' -H, 6 ''' -H _b ), 3.55 (s, 1H, 4 '' -H), 3.51 (d, J = 9.6 Hz, 1H, 5 ' -H), 3.29 (dd, J = 10.2, 3.5 Hz, 1H, 2 ''' -H), 3.18 (dd, J = 10.3, 4.0 Hz, 1H, 2 ' -H), 1.99 (s, 3H, Ac), 1.73 (s, 3H, Ac), 1.68 (s, 3H, Ac), 1.05 (s, 9H, t Bu), 0.96 (s, 9H, t Bu); ^{13C NMR ( 150 MHz, CDCl} ₃ ): δ 170.1 (C), 170.0 (C), 169.1 (C), 165.5 (C), 164.9 (C), 137.3 (C), 137.2 (C), 137.0 (C), 136.7 ( C), 135.8 (CH), 135.57 (CH), 135.55 (CH), 135.4 (C), 133.5 (CH), 133.3 (C), 133.2 (C), 133.1 (C), 133.0 (CH), 132.98 ( C), 132.92 (C), 131.4 (C), 132.8 (C), 131.4 (CH), 131.0 (CH), 129.8 (CH), 129.7 (CH), 129.6 (CH), 129.5 (CH), 129.4 ( CH), 129.0 (C), 128.9 (CH), 128.6 (CH), 128.59 (CH), 128.52 (CH), 128.3 (CH), 128.15 (CH), 128.09 (CH), 127.85 (CH), 127.81 ( CH), 127.71 (CH), 127.67 (CH), 127.57 (CH), 127.50 (CH), 127.3 (CH), 126.3 (CH), 126.1 (CH), 125.9 (CH), 125.7 (CH), 121.6 ( C), 121.1 (C) , 98.4 (CH), 97.4 (CH), 97.0 (CH), 91.7 (CH), 82.9 (CH), 80.3 (CH), 78.5 (CH), 77.7 (CH), 75.1 (CH ₂ ), 74.4 (CH ₂ ), 74.2 (CH ₂ ), 74.1 (CH ₂ ), 73.8 (CH), 73.4 (CH), 73.0 (CH), 72.8 (CH, CH ₂ ), 72.64 (CH), 72.59 (CH) , 72.55 ( CH), 68.4 (CH), 64.8 (CH), 63.9 (CH), 63.6 (CH), 62.3 (CH ₂ ), 62.2 (CH ₂ ), 62.0 (CH ₂ ), 26.8 (CH ₃ ), 26.7 (CH ₃ ), 20.8 (CH ₃ ), 20.6 (CH ₃ ), 20.3 (CH ₃ ), 19.36 (C), 19.30 (C); HRMS (MALDI): m/z calcd for C ₁₁₅ H ₁₂₀ Br ₂ N ₆ O ₂₄ Si ₂ Na ([M + Na] ⁺ ): 2209.2178, found: 2209.2236.

4- tolyl [2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -4 -O- (2- naphthylmethyl )-α-D- glucopyranosyl ]-(1→4)-(6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -1- Thio -β-D- glucopyranoside (4-Methylphenyl[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-4 -O- (2-naphthylmethyl)-α-D-glucopyranosyl]-(1 →4)-(6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-α-L-idopyranosyl)-(1→4)-[2-azido-3 -O- (4-bromobenzyl) -6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-1-thio-β- D-glucopyranoside) (10)

At room temperature under argon atmosphere, trimethyl(4-methylphenylthio)silane (trimethyl(4-methylphenylthio)silane, TMSSTol, 0.08 mL, 0.39 mmol) and ZnI ₂ (0.13 g, 0.41 mmol) was added to a solution of compound 9 (0.43 g, 0.196 mmol) in anhydrous _{CH2Cl2 (} 5 mL). After complete consumption of the tetrasaccharide ( ₂ hours), the mixture was diluted with _CH2Cl2 (8 mL) and filtered. The filtrate was washed with saturated NaHCO _{3 (aq)} , dried over MgSO ₄ and filtered. The resulting solution was concentrated under reduced pressure, and purified by flash column chromatography (ethyl acetate/hexane=1/5) to obtain glucosinolate 10 (0.38 g, 85%). IR (thin film): ν 2927, 2108, 1733, 1266, 1112, 708 cm ^–1 ; ¹ H NMR (600 MHz, CDCl ₃ ): δ 8.09 (dd, J = 7.9, 1.0 Hz, 2H, Bz-H ), 7.99 (d, J = 8.1, 1.5 Hz, 2H, Bz-H), 7.84 (dd, J = 6.0, 3.4 Hz, 1H, 2-NAP-H), 7.77–7.72 (m, 2H, Ar- H), 7.68–7.52 (m, 10H, Ar-H), 7.51–7.44 (m, 4H, Ar-H), 7.41–7.12 (m, 33H, Ar-H), 7.08–6.98 (m, 6H, Ar-H), 5.43 (d, J = 4. Hz, 1H, 1 ' -H), 5.40 (s, 1H, 1 '' -H), 5.24 (dd, J = 9.7, 8.7 Hz, 1H, 2 -H), 5.19 (s, 1H, 2 '' -H), 4.90 (d, J = 11.3 Hz, 1H, ArCH ₂ ), 4.89 (d, J = 11.0 Hz, 1H, ArCH ₂ ), 4.84 (s , 2H, ArCH ₂ ), 4.75-4.65 (m, 5H, ArCH ₂ , 1-H, 1 ''' -H), 4.49–4.44 (m, 2H, ArCH ₂ , 6-H _a ), 4.38–4.33 (m, 2H, ArCH ₂ , 5 '' -H), 4.17–4.09 (m, 4H, ArCH ₂ , 4 ' -H, 3 '' -H, 6 ' -H _a ), 4.05–4.00 (m, 2H, 5 ''' -H, 6 '' -H _a ), 3.95 (dd, J = 8.3, 8.7 Hz, 1H, 3-H), 3.90 (dd, J = 11.9, 4.7 Hz, 1H, 6- H _b ), 3.88–3.80 (m, 3H, 6 '' -H _b , 4 ''' -H, 6 ''' -H _a ), 3.74–3.63 (m, 5H, 4-H, 3 ' - H, 3 ''' -H, 6 ' -H _b , 6 ''' -H _b ), 3.60 (ddd, J = 9.8, 4.8, 2.4 Hz, 1H, 5-H), 3. 55 (s, 1H, 4 '' -H), 3.48 (d, J = 9.7 Hz, 1H, 5 ' -H), 3.29 (dd, J = 10.2, 3.5 Hz, 1H, 2 ''' -H) , 3.15 (dd, J = 10.3, 4.1 Hz, 1H, 2' - H), 2.32 (s, 3H, SPhCH ₃ ), 1.72 (s, 3H, Ac), 1.69 (s, 3H, Ac), 1.05 ( s, 9H, t Bu), 0.95 (s, 9H, t Bu); ^{13C NMR (150 MHz, CDCl} ₃ ): δ 170.1 (C), 170.0 (C), 165.6 (C), 165.5 (C), 165.1 (C), 138.3 (C), 137.4 (C), 137.3 (C), 137.1 (C), 136.7 (C), 135.83 (CH), 135.82 (CH), 135.57 (CH), 135.54 (CH), 135.4 (C), 133.7 (CH), 133.4 (CH), 133.31 (C), 133.27 (C), 133.2 (C), 133.1 (CH), 133.92 (C), 133.88 (C), 131.4 (C), 131.0 (CH), 129.81 (CH), 129.79 (CH), 129.70 (CH), 129.67 (CH), 129.61 (C), 129.59 (CH), 129.57 (C), 128.9 (CH), 128.6 (CH), 128.53 (CH), 128.51 (CH), 128.3 (CH), 128.16 (C), 128.12 (CH), 128.09 (CH), 127.9 (CH), 127.71 (CH), 127.67 (CH), 127.60 (CH), 127.57 (CH), 127.5 (CH), 127.4 (CH), 126.4 (CH), 126.2 (CH), 126.0 (CH), 125.7 (CH), 121.6 (C), 121.1 (C), 98.5 (CH), 97.4 (CH), 97.0 (CH), 85.9 (CH), 84.7 (CH), 80.3 (CH), 78.6 (CH), 77.7 (CH), 76.4 (CH), 75.1 (CH ₂ ), 74.6 (CH ₂ ), 74.2 (CH ₂ ), 73.9 (CH), 73.1 (CH), 72.91 (CH), 72.86 (CH, CH ₂ ), 72.7 (CH), 72.6 (CH), 68.6 (CH), 64.9 (CH), 63.9 (CH), 63.5 (CH), 62.6 (CH ₂ ), 62.3 (CH ₂ ), 62.1 (CH ₂ ), 62.0 (CH ₂ ), 26.9 (CH ₃ ), 26.8 (CH ₃ ), 21.1 (CH ₃ ), 20.6 (CH ₃ ), 20.4 (CH ₃ ), 19.4 (C), 19.3 (C); HRMS (MALDI): m/z calcd for C ₁₂₀ H ₁₂₄ Br ₂ N ₆ O ₂₂ SSi ₂ Na ([M + Na] ⁺ ): 2273.3711, found: 2273.3777.

4- tolyl [2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-(6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -1- Thio -β-D- glucopyranoside (4-Methylphenyl [2-azido-3 -O- (4-bromobenzyl)-6 -O-tert -butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-(6 -O- acetyl -2 -O- benzoyl-3 -O- benzyl-α-L-idopyrano-syl)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl -2-deoxy-α-D-glucopyranosyl]-(1→4)-6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-1-thio-β-D-glucopyranoside)(11)

To a solution of the thioether-linked tetrasaccharide 10 (0.38 g, 0.17 mmol) in mixed solvent (CH ₂ Cl ₂ /H ₂ O = 19/1, 11.4 mL) was added DDQ (0.06 mL) at room temperature grams, 0.26 mmol). After stirring for ₂ hours, the reaction was quenched by the addition of ₁₀ % Na2S2O3 _{(aq) ,} and the organic mixture was extracted with _CH2Cl2 . The organic layer was washed successively with saturated NaHCO _{3 (aq)} and H ₂ O, dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexane = 1/5) to give 4'''-ethanol 11 (0.31 g, 87%). IR (thin film): ν 2929, 2108, 1733, 1265, 1112, 708 cm ^–1 ; ¹ H NMR (600 MHz, CDCl ₃ ): δ 8.08 (d, J = 7.8 Hz, 2H, Bz-H), 8.02 (d, J = 7.9 Hz, 2H, Bz-H), 7.67–7.55 (m, 9H, Ar-H), 7.49–7.39 (m, 7H, Ar-H), 7.39–7.25 (m, 17H, Ar-H), 7.23–6.15 (m, 9H, Ar-H), 7.12–7.04 (m, 6H, Ar-H), 5.44 (d, J = 4.0 Hz, 1H, 1' - H), 5.42 ( s, 1H, 1 '' -H), 5.23 (dd, J = 9.8, 9.1 Hz, 1H, 2-H), 5.19 (s, 1H, 2 '' -H), 4.90 (d, J = 11.6 Hz , 1H, ArCH ₂ ), 4.84 (d, J = 11.1 Hz, 1H, ArCH ₂ ), 4.75–4.65 (m, 5H, 1-H, 1 ''' -H, ArCH ₂ ), 4.49 (d, J = 11.3 Hz, 2H, ArCH ₂ ), 4.45 (d, J = 10.6 Hz, 1H, 6-H _a ), 4.36 (td, J = 6.5, 2.2 Hz, 1H, 5 '' -H), 4.20 (d , J = 11.1 Hz, 1H, ArCH ₂ ), 4.15 (dd, J = 9.8, 9.2 Hz, 1H, 4'-H), 4.11–4.02 (m, 3H, 6' -H _a , 3 ' ' -H , 6 '' -H _a ), 3.94 (dd, J = 8.8, 8.3 Hz, 1H, 3-H), 3.92–3.84 (m, 3H, 6-H _b , 6 '' -H _b , 6 ''' -H _a ), 3.75–3.64 (m, 6H, 4-H, 3 ' -H, 6 ' -H _b , 4 ''' -H, 5 ''' -H, 6 ''' -H _b ), 3.59 (ddd, J = 9.3, 4.2, 2.0 Hz, 1H, 5-H), 3.57–3.53 (m, 2H, 4 '' -H, 3 ''' -H), 3.4 6–3.50 (m, 1H, 5' -H), 3.18–3.14 (m, 2H, 2'-H, 2 ' '' -H), 2.32 (s, 3H, SPhCH ₃ ), 1.79 (s, 3H , Ac), 1.68 (s, 3H, Ac), 1.04 (s, 9H, t Bu), 0.99 (s, 9H, t Bu); ^{13C NMR (150 MHz, CDCl} ₃ ): δ 170.2 (C), 170.1 (C), 165.5 (C), 165.1 (C), 138.3 (C), 137.32 (C), 137.31 (C), 137.1 (C), 137.0 (C), 135.9 (CH), 135.60 (CH), 135.58 (CH), 135.5 (CH), 133.7 (CH), 133.4 (CH), 133.2 (CH), 132.9 (C), 132.8 (CH), 132.7 (C), 131.4 (C), 131.1 (CH), 129.93 (CH), 129.91 (CH), 129.84 (CH), 129.83 (CH), 129.7 (CH), 129.63 (CH), 129.60 (CH), 129.5 (CH), 129.0 (CH), 128.6 (CH), 128.5 (CH), 128.43 (CH), 128.14 (C), 128.09 (CH), 127.84 (CH), 127.78 (CH), 127.74 (CH), 127.63 (CH), 127.56 (CH), 127.4 (CH), 121.7 (C), 121.1 (C), 98.3 (CH), 97.4 (CH), 97.0 (CH), 85.9 (CH), 84.8 (CH), 79.9 (CH), 78.5 (CH), 76.4 (CH), 74.6 (CH ₂ ), 74.2 (CH ₂ ), 74.1 (CH ₂ ), 73.7 (CH), 73.1 (CH), 72.93 (CH), 72.91 (CH), 72.88 (CH ₂ ), 72.6 (CH), 72.3 ( CH), 71.9 (CH), 68.8 (CH), 65.3 (CH), 63.7 (CH ₂ ), 63.5 (CH), 63.0 (CH), 62.5 (CH ₂ ), 62.2 (CH ₂ ), 62.1 (CH ₂ ), 26.9 (CH ₃ ), 26.8 (CH ₃ ), 21.1 (CH ₃ ), 20.7 (CH ₃ ), 20.4 (CH ₃ ), 19.4 (C), 19.2 (C); HRMS (MALDI): m/z calcd for C ₁₀₉ H ₁₁₆ Br ₂ N ₆ O ₂₂ SSi ₂ Na ([M + Na] ⁺ ): 2133.1855, found: 2133.1912.

N - benzyl -N- benzyloxycarbonyl -5- Aminopentyl [2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-(6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -β-D- glucopyranoside ( N -Benzyl -N- benzyloxycarbonyl-5-aminopentyl [2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4 )-(6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-α-L-idopyranosyl)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-β-D-glucopyranoside)(12 )

At room temperature under N atmosphere, glucosinolate 11 ₍ 0.78 g, 0.37 mmol) and N -benzyl- N -benzyloxycarbonyl-5-aminepentanol ( N -benzyl-N - benzyloxycarbonyl-5 -aminopentanol) 4 (0.12 g, 0.37 mmol) in _{CH2Cl2 (} 10 mL) was stirred with freshly dried AW-300 molecular sieves (1 g) for 1 hour. The reaction flask was cooled to -78°C, NIS (0.08 g) and TfOH (27 mL) were added to the solution, and the mixture was gradually warmed to room temperature. After complete consumption of glucosinolates according to TLC analysis ( ₂ hours), _Et3N was added to quench the reaction, the mixture was filtered through celite, and the solid was washed with _CH2Cl2 . The filtrate was washed with 10% Na ₂ S ₂ O _{3 (aq)} , dried over MgSO ₄ and concentrated under reduced pressure to give a residue which was subjected to flash column chromatography (ethyl acetate/hexane= 1/3) was purified to yield the expected tetrasaccharide 12 (0.76 g, 89%). IR (thin film): ν 2926, 2108, 1737, 1266, 1112, 701 cm ^–1 ; ¹ H NMR (600 MHz, CDCl ₃ ): δ 8.03–7.97 (m, 4H, Bz-H), 7.67–7.62 (m, 4H, Ar-H), 7.61–7.55 (m, 4H, Ar-H), 7.50–7.39 (m, 6H, Ar-H), 7.38–7.26 (m, 23H, Ar-H), 7.26 –7.20 (m, 7H, Ar-H), 7.20–7.15 (m, 6H, Ar-H), 7.11–7.05 (m, 5H, Ar-H), 5.48 (d, J = 3.7 Hz, 1H, 1 ' -H), 5.42 (s, 1H, 1 '' -H), 5.25 (t, J = 8.3 Hz, 1H, 2-H), 5.19 (s, 1H, 2 '' -H), 5.12 (d , J = 7.4 Hz, 2H, ArCH ₂ ), 4.91 (d, J = 11.6 Hz, 1H, ArCH ₂ ), 4.84 (d, J = 11.2 Hz, 1H, ArCH ₂ ), 4.75–4.65 (m, 4H, ArCH ₂ , 1 '' -H), 4.53–4.43 (m, 3H, ArCH ₂ , 1-H), 4.41–4.32 (m, 4H, ArCH ₂ , 6-H _a , 5 '' -H), 4.21 –4.14 (m, 2H, ArCH ₂ , 4’-H), 4.11–4.02 (m, 3H, 6’-H _a , 3 ’ ’ -H, 6 ’ ’ -H _a ), 3.99–3.88 (m, 3H, 3-H, 6-H _b , 6 ''' -H _a ), 3.85 (d, J = 12.0 Hz, 6 '' -H _b ), 3.77 (t, J = 9.0 Hz, 1H, 4- H), 3.75–3.66 (m, 5H, 3' -H, 6'- _Hb , 4 ''' -H, 5 ''' -H, 6 ' '' - _Hb ), 3.66–3.61 (m , 1H), 3.58–3.48 (m, 4H, 5-H, 5' -H, 3 ''' -H), 3.39–3.26 (m, 1H), 3.18–3.14 (m, 2H, 2' - H , 2 ''' -H), 3.08–2.89 (m, 2H), 2.46 (d, J = 2.6 Hz, 1H, 4 ''' -OH), 1.79 (s, 3H, Ac), 1.66–1.59 (m, 2H), 1.63 (s, 3H, Ac), 1.50–1.25 (m, 4H), 1.03 (s, 9H, t Bu), 0.98 (s, 9H, t Bu); ^{13C NMR (150 MHz, CDCl} ₃ ) : δ 170.2 (C), 170.1 (C), 165.5 (C), 165.0 (C), 156.6/156.0 (C), 137.9 (C), 137.8 (C), 137.4 (C), 137.3 (C), 137.1 (C), 137.0 (C), 136.8/136.7 (C), 135.9 (CH), 135.6 (CH), 135.56 (CH), 135.52 (CH), 133.3 (C), 133.2 (CH), 132.9 (C) , 132.8 (C), 132.7 (C), 131.4 (CH), 131.1 (CH), 129.93 (CH), 129.91 (CH), 129.8 (CH), 129.7 (CH), 129.62 (CH), 129.60 (CH) , 128.9 (CH), 128.56 (CH), 128.51 (CH), 128.45 (CH), 128.3 (CH), 128.1 (CH), 127.85 (CH), 127.78 (CH), 127.72 (CH), 127.61 (CH) , 127.56 (CH), 127.4 (CH), 127.23 (CH), 127.17 (CH), 127.09 (CH), 121.6 (C), 121.1 (C), 100.8 (CH), 98.2 (CH), 97.2 (CH) , 96.9 (CH), 83.3 (CH), 79.9 (CH), 78.5 (CH), 74.3 (CH ₂ ), 74.2 (CH ₂ ), 74.10 (CH ₂ ), 73.96 (CH), 73.7 (CH), 73.1 (CH), 72.93 (CH), 72.88 (CH ₂ ), 72.86 (CH), 72.6 (CH), 72.5 (CH ₂ ), 72.3 (CH ), 71.9 (CH), 69.7 (CH ₂ ), 69.6 (CH ₂ ), 68.8 (CH), 67.0 (CH ₂ ), 65.3 (CH), 63.6 (CH ₂ ), 63.5 (CH), 63.0 (CH) , 62.5 (CH ₂ ), 62.2 (CH ₂ ), 62.1 (CH ₂ ), 53.7 (CH ₂ ), 50.4 (CH ₂ ), 50.1 (CH ₂ ), 47.0 (CH ₂ ), 46.0 (CH ₂ ), 21.7 (CH ₃ ), 29.2 (CH ₃ ), 28.9 (CH ₂ ), 27.6/27.2 (CH ₂ ), 26.9 (CH ₃ ), 26.8 (CH ₃ ), 23.0 (CH ₂ ), 20.7 (CH ₃ ), 20.3 (CH ₃ ), 19.4 (C), 19.2 (C); HRMS (ESI): m/z calcd for C ₁₂₂ H ₁₃₃ Br ₂ N ₇ O ₂₅ Si ₂ Na ([M + Na] ⁺ ): 2332.7154, found : 2332.7119.

N - benzyl -N- benzyloxycarbonyl -5- Aminopentyl [2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -4 -O- (2- naphthylmethyl )-α-D- glucopyranosyl ]-(1→4)-(6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-(6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -β-D- glucopyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-(6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-6 -O- Acetyl -2 -O- benzyl -3 -O- benzyl -β-D- glucopyranoside ( N -Benzyl -N- benzyloxycarbonyl-5-aminopentyl[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-4 -O- (2-naphthylmethyl)-α -D-glucopyranosyl]-(1→4)-(6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-α-L-idopyranosyl)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert -butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-(6 -O- acetyl-2 -O- benzoyl-3 -O - benzyl-β-D-glucopyranosyl)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]- (1→4)-(6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-α-L-idopyranosyl)-(1→4)-[2-azido-3 -O- (4- bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-6 -O- acetyl-2 -O- benzoyl-3 -O- benzyl-β-D- glucopyranoside) (13)

Dissolve glucosinolate 10 (1.4 equiv.) and 4'''-ethanol 12 (1.0 equiv.) in anhydrous _{CH2Cl2 (} 10 mL/g total glucosinolate and 4'''-ethanol) at room temperature The solution was stirred with freshly activated AW-300 molecular sieves (0.5 g/g total of glucosinolate and 4'''-ethanol) for 1 hour, then cooled to -40°C, followed by the addition of NIS (1.6 equiv.). After stirring for 5 minutes, TfOH (0.4 equiv) was slowly added to the solution via a microsyringe. The reaction was gradually warmed to room temperature and kept stirring until the glucosinolate was completely consumed (about 2 hours by thin layer chromatography analysis). _The mixture was neutralized by adding _Et3N , diluted with _CH2Cl2 and filtered through celite. The resulting solution was washed with ₁₀ % Na2S2O3 ( _{aq )} and _H2O . The organic layers were combined, dried over _MgSO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography to give octasaccharide 13 (72%).

[α] ²⁶ _D –2.3 ( c 5.0, CHCl ₃ ); IR (thin film): ν 2928, 2108, 1741, 1698, 1264, 1112, 1038, 701 cm ^–1 ; ¹ H NMR (600 MHz, CDCl _{3 )} ): δ 8.02–7.97 (m, 4H, Bz-H), 7.96–7.92 (m, 2H, Bz-H), 7.85–7.82 (m, 1H, Ar-H), 7.81–7.78 (m, 2H, Ar-H), 7.76–7.71 (m, 3H, Ar-H), 7.69–7.64 (m, 4H, Ar-H), 7.63–7.60 (m, 2H, Ar-H), 7.58–7.47 (m, 15H, Ar-H), 7.44–7.40 (m, 2H, Ar-H), 7.40–7.33 (m, 14H, Ar-H), 7.33–7.18 (m, 44H, Ar-H), 7.18–7.09 ( m, 12H, Ar-H), 7.07–6.97 (m, 8H, Ar-H), 5.46 (d, J = 4.0 Hz, 1H, 1' - H), 5.44 (d, J = 4.0 Hz, 1H, 1 ''''' -H), 5.41 (s, 1H, 1 '' -H), 5.29-5.21 (m, 3H, 2-H, 2 '''' -H, 1 '''''' -H), 5.18 (bs, 1H, 2 '' -H), 5.15 (dd, J = 3.4, 2.8 Hz, 1H, 2 '''''' -H), 5.12 (d, J = 7.9 Hz, 2H, PhCH ₂ in Cbz), 4.91–4.84 (m, 4H, 1 '''' -H, ArCH ₂ ), 4.83 (s, 2H, NaphCH ₂ ), 4.79 (d, J = 11.1 Hz, 1H, ArCH ₂ ), 4.74–4.66 (m, 6H, 1 ''' -H, ArCH ₂ ), 4.60 (d, J = 3.5 Hz, 1H, 1 ''''''' -H), 4.53 (d, J = 10.1 Hz, 1H, ArCH ₂ ), 4.49–4.42 (m, 3H, 1-H, ArCH ₂ ), 4.40–4.32 (m, 5H), 4.18–4.04 (m, 8H) , 4.04–3.99 (m, 3H), 3.97 (t, J = 3.8 Hz, 1H, 3 '''''' -H), 3.92 (t, J = 8.8 Hz, 1H, 3-H), 3.90– 3.73 (m, 11H), 3.73–3.63 (m, 7H), 3.63–3.55 (m, 2H), 3.55–3.48 (m, 3H), 3.46–3.42 (m, 2H), 3.40–3.27 (m, 3H) , 5 '''' -H-5, 5 ''''''' -H, CH ₂ in linker), 3.27 (dd, J = 10.2, 3.5 Hz, 1H, 2 ''' -H), 3.15 –3.10 (m, 2H, 2 ' -H, 2 ''''' -H), 3.08–2.89 (m, 2H, CH ₂ in linker), 1.70 (s, 3H, Ac), 1.59 (bs, 5H , Ac, CH ₂ in linker), 1.46 (s, 3H, Ac), 1.38 (s, 3H, Ac), 1.49–1.25 (m, 4H, CH ₂ in linker), 1.10 (s, 9H, t Bu) , 1.04 (s, 9H, t Bu), 0.93 (s, 18H, t Bu × 2); ^{13C NMR (150 MHz, CDCl} ₃ ): δ 170.11 (C), 170.09 (C), 170.0 (C), 169.4 (C), 165.54(C), 165.46 (C), 165.0 (C), 164.8 (C), 156.6/156.0 (C), 137.94 (C), 137.90 (C), 137.8 (C), 137.6 (C) , 137.40 (C), 137.36 (C), 137.19 (C), 137.15 (C), 137.1 (C), 136.9 (C), 136.8 (C), 136.0 (CH), 135.89 (CH), 135.86 (CH) , 135.8 (CH), 135.62 (CH), 135.56 (CH), 135.52 (CH), 135.45 (C), 133.7 (C), 133.4 (CH), 133.34 (CH), 133.27 (C), 133.2 (CH) , 133.20 (C), 133.18 (C), 133.1 (CH), 133.0 (C), 132.9 (C), 132.8 (C), 132.2 (C), 131.4 (CH), 131.1 (CH), 131.04 (CH), 131.03 (CH), 130.2 (CH), 130.0 (CH), 129.83 (CH), 129.77 (CH), 129.7 (CH), 129.6 (CH), 129.5 (CH), 129.4 (CH), 129.15 (CH), 129.09 (CH), 129.01 (CH), 128.96 (C), 128.9 (CH), 128.8 (CH), 128.6 (CH), 128.54 (CH), 128.46 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 128.13 (CH), 128.10 (CH), 128.08 (CH), 127.9 (CH), 127.8 (CH), 127.72 (CH), 127.69 (CH), 127.65 (CH), 127.61 (CH), 127.59 (CH), 127.56 (CH), 127.5 (CH), 127.43 (CH), 127.38 (CH), 127.24 (CH), 127.18 (CH), 127.1 (CH), 126.4 (CH), 126.2 (CH), 126.0 (CH), 125.7 (CH), 125.3 (CH), 121.6 (C), 121.1 (C), 121.0 (C), 100.8 (CH), 99.8 (CH), 98.5 (CH), 98.3 (CH), 97.4 (CH), 97.3 (CH), 96.99 (CH), 96.96 (CH), 83.6 (CH), 83.3 (CH), 80.4 (CH), 78.4 (CH), 78.2 (CH), 78.0 (CH), 77.7 (CH), 76.0 (CH), 75.1 (CH ₂ ), 74.7 (CH ₂ ), 74.4 (CH), 74.3 (CH ₂ ), 74.2 (CH ₂ ), 74.0 (CH/CH ₃ ), 73.9 (CH), 73.5 (CH) , 73.4 (CH), 73.2 (CH), 73.1 (CH/CH ₂ ), 72.94 (C H), 72.85 (CH), 72.83 (CH ₂ ), 72.77 (CH), 72.6 (CH), 72.4 (CH), 72.2 (CH), 69.7/69.6 (CH ₂ ), 69.1 (CH), 68.5 (CH ), 67.1 (CH ₂ ), 65.5 (CH), 64.8 (CH), 64.0 (CH), 63.5 (CH), 63.4 (CH), 63.1 (CH), 62.7 (CH ₂ ), 62.4 (CH ₂ ), 62.3 (CH ₂ ), 62.0 (CH ₂ ), 61.9 (CH ₂ ), 61.4 (CH ₂ ), 60.9 (CH ₂ ), 50.4/50.1 (CH ₂ ), 47.0/46.0 (CH ₂ ), 29.0 (CH ₂ ), 27.6/27.2 (CH ₂ ), 26.93 (CH ₃ ), 26.87 (CH ₃ ), 26.81 (CH ₃ ), 26.75 (CH ₃ ), 26.7 (CH ₃ ), 23.0 (CH ₂ ), 21.4 (CH ₃ ), 20.7 (CH ₃ ), 20.34 (CH ₃ ), 20.32 (CH ₃ ), 19.9 (CH ₃ ), 19.42 (C), 19.37 (C), 19.32 (C), 19.28 (C); HRMS (MALDI) : m/z calcd for C ₂₃₅ H ₂₄₉ Br ₄ N ₁₃ O ₄₇ Si ₄ Na ([M + Na] ⁺ ): 4462.5806, found: 4462.5928.

N - benzyl -N- benzyloxycarbonyl -5- Aminopentyl [2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -4 -O- (2- naphthylmethyl )-α-D- glucopyranosyl ]-(1→4)-(3 -O- benzyl -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-(3 -O- benzyl -β-D- glucopyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-(3 -O- benzyl -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-3 -O- benzyl -β-D- glucopyranoside ( N -Benzyl -N- benzyloxycarbonyl-5-aminopentyl[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-4 -O- (2-naphthylmethyl)-α -D-glucopyranosyl]-(1→4)-(3 -O- benzyl-α-L-idopyranosyl)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 -O -tert -butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-(3 -O- benzyl-β-D-glucopyranosyl)-(1→4)-[2-azido-3 -O - (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-(3 -O- benzyl-α-L-idopyranosyl)-(1→4 )-[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-3 -O- benzyl-β- D-glucopyranoside) (14)

NaOMe (5 equiv per acyl group) was added to a solution of octasaccharide 13 (1.68 g, 0.38 mmol) in mixed solvent (MeOH/CH ₂ Cl ₂ = 1/1) at room temperature under _N2 in solution. After stirring for 16 hours, DOWEX 50WX4-200 resin was added to neutralize the reaction. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain deacylated compound 14 (1.23 g, 85%). [α] ²⁷ _D +8.6 ( c 5.0, CHCl ₃ ); IR (thin film): ν 3476, 2929, 2109, 1684, 1254, 1070, 1030, 700 cm ^–1 ; ¹ H NMR (600 MHz, CDCl _{3 )} ): δ 7.83–7.80 (m, 1H, Ar-H), 7.78–7.711 (m, 2H, Ar-H), 7.71–7.68 (m, 4H, Ar-H), 7.68–7.65 (m, 2H, Ar-H), 7.61–7.59 (s, 1H, Ar-H), 7.49–7.47 (m, 2H, Ar-H), 7.45–7.33 (m, 39H, Ar-H), 7.33–7.27 (m, 13H, Ar-H), 7.27–7.25 (m, 3H, Ar-H), 7.23–7.18 (m, 5H, Ar-H), 7.12–7.16 (m, 7H, Ar-H), 7.04 (d, J = 8.2 Hz, 2H, Ar-H), 7.00 (d, J = 8.2 Hz, 2H, Ar-H), 5.64 (bs, 1H), 5.61 (d, J = 3.8 Hz, 1H), 5.23 (s , 1H), 5.21–5.13 (m, 3H), 5.09 (d, J = 10.7 Hz, 1H), 5.05 (d, J = 3.7 Hz, 1H), 5.01 (d, J = 4.1 Hz, 1H), 4.95 –4.87 (m, 3H), 4.84–4.81 (m, 3H), 4.79–4.74 (m, 3H), 4.68–4.55 (m, 5H), 4.54–4.46 (m, 5H), 4.34–4.24 (m, 1H), 4.19–4.14 (m, 2H), 4.13–4.10 (m, 1H), 4.10–4.05 (m, 1H), 4.05–3.99 (m, 2H), 3.99–3.92 (m, 2H), 3.92– 3.89 (m, 2H), 3.87–3.80 (m, 10H), 3.77–3.72 (m, 3H), 3.71–3.64 (m, 5H), 3.62–3.51 (m, 11H), 3.48–3.39 (m, 4H) ), 3.38–3.36 (m, 1H), 3.34–3.16 (m, 7H), 3.11–3. 06 (m, 1H, CH ₂ in linker), 3.05–2.99 (m, 1H, CH ₂ in linker), 1.66–1.49 (m, 6H, CH ₂ in linker), 1.10 (s, 9H, t Bu), 1.08 (s, 9H, t Bu), 1.05 (s, 9H, t Bu), 1.04 (s, 9H, t Bu); ^{13C NMR (150 MHz, CDCl} ₃ ): δ 156.7/156.3 (C), 138.4 ( C), 137.7 (C), 137.5 (C), 137.4 (C), 136.8 (C), 136.7 (C), 136.6 (C), 136.00 (CH), 135.97 (CH), 135.91 (CH), 135.87 ( CH), 135.8 (CH), 135.7 (CH), 135.61 (CH), 135.57 (CH), 135.3 (C), 133.33 (C), 133.28 (C), 133.2 (C), 133.1 (C), 133.0 ( C), 132.9 (C), 131.51 (CH), 131.47 (CH), 131.2 (CH), 129.9 (CH), 129.8 (CH), 129.71 (CH), 129.67 (CH), 129.6 (CH), 129.4 ( CH), 129.3 (CH), 129.2 (CH), 128.52 (CH), 128.47 (CH), 128.4 (CH), 128.35 (CH), 128.31 (CH), 128.21 (CH), 128.18 (CH), 127.90 ( CH), 127.85 (CH), 127.81 (CH), 127.76 (CH), 127.70 (CH), 127.68 (CH), 127.6 (CH), 127.5 (CH), 127.3 (CH), 127.2 (CH), 126.2 ( CH), 126.1 (CH), 125.9 (CH), 125.5 (CH), 121.8 (C), 121.7 (C), 121.5 (C), 102.9/102.7 (CH), 102.1 (CH), 99.9 (CH), 99.8 (CH), 97.3 (CH), 97.1 (CH), 94.8 (CH), 94.2 (CH), 84.9 (CH), 84.6 (CH), 81.1 (CH), 79.4 (CH), 78.9 (CH), 78.6 (CH), 77.9 (CH), 77.2 (CH), 76.1 (CH), 75.8 (CH), 75.2 (CH), 75.1 (CH ₂ ), 74.9 (CH ₂ ), 74.74 (CH/CH ₂ ), 74.66 (CH ₂ ), 74.6 (CH), 74.3 (CH ₂ ), 73.9 (CH ₂ ), 73.0 (CH), 72.90 (CH), 72.85 (CH), 72.6 (CH), 72.4 (CH), 72.3 (CH/CH ₂ ), 72.2 (CH ₂ ), 72.0 (CH), 70.4 (CH ), 70.0 (CH ₂ ), 69.9 (CH), 67.2 (CH ₂ ), 66.8 (CH), 66.7 (CH), 66.4 (CH), 66.2 (CH), 63.8 (CH), 63.6 (CH), 63.34 (CH), 63.31 (CH), 62.4 (CH ₂ ), 62.3 (CH ₂ ), 62.1 (CH ₂ ), 62.0 (CH ₂ ), 61.5 (CH ₂ ), 61.31 (CH ₂ ), 61.26 (CH ₂ ) , 53.7 (CH ₂ ), 50.4/50.2 (CH ₂ ), 46.9/45.9 (CH ₂ ), 29.6 (CH ₂ ), 29.2 (CH ₂ ), 28.8 (CH ₂ ), 27.7 (CH ₂ ), 26.88 (CH 2 ) ₃ ), 26.86 (CH ₃ ), 26.84 (CH ₃ ), 23.1 (CH ₂ ), 19.42 (C), 19.38 (C), 19.31 (C); HRMS (MALDI): m/z calcd for C ₁₉₉ H ₂₂₅ Br ₄ N ₁₃ O ₃₉ Si ₄ Na ([M + Na] ⁺ ): 3877.9968, found: 3877.9971.

N - benzyl -N- benzyloxycarbonyl -5- Aminopentyl [2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -4 -O- (2- naphthylmethyl )-α-D- glucopyranosyl ]-(1→4)-( methyl 3 -O- benzyl -α-L- Iduranuronate )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-( methyl 3 -O- benzyl -β-D- glucopyranosyl ester )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-( methyl 3 -O- benzyl -α-L- Iduranuronate )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-6 -O- tert-Butyldiphenylsilyl -2- deoxygenation -α-D- glucopyranosyl ]-(1→4)- methyl 3 -O- benzyl -β-D- Methyl glucopyranosate ( N -Benzyl -N- benzyloxycarbonyl-5-aminopentyl[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-4 -O- (2-naphthylmethyl)-α -D-glucopyranosyl]-(1→4)-(methyl 3 -O- benzyl-α-L-idopyranosyluronate)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 - O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-(methyl 3 -O- benzyl-β-D-glucopyranosyluronate)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-(methyl 3 -O- benzyl-α-L-idopyranosyluronate)-( 1→4)-[2-azido-3 -O- (4-bromobenzyl)-6 -O-tert- butyldiphenylsilyl-2-deoxy-α-D-glucopyranosyl]-(1→4)-methyl 3 -O- benzyl-β-D-glucopyranosiduronate) (15)

At room temperature, bis(acetoxy)iodobenzene (BAIB, 331 mg, 1.03 mmol) was added to compound 14 (330 mg, 0.09 mmol) in a mixed solvent (CH 2Cl2/ _H2O = _2/1 , ₃ mL). After 5 minutes, the 2,2,6,6-tetramethyl-1-piperidinyloxyl free radical (2,2,6,6-tetramethyl-1-piperidinyloxyl free radical, TEMPO, 7 mg, 0.04 mmol ear) was added to the mixture, which was then kept stirring for 8 hours. To quench the reaction, 10% Na2S2O3( _{aq )} ( ₃ mL ₎ was added and the desired material was extracted with _CH2Cl2 . The combined organic layers were washed with brine, dried over anhydrous _MgSO4 , filtered and concentrated under reduced pressure. To a solution of this residue in THF (2.5 mL) was then added LiOH (0.5 vol molar in water, 1.7 mL, 0.9 mmol) at room temperature. After 4 hours, the reaction was neutralized by addition of DOWEX 50WX-200 resin, filtered and concentrated under reduced pressure. To a solution of crude uronate in _CH2Cl2 ( ₃ mL) was added an ethereal solution of _CH2N2 ( ₃ mL, 1.28 mmol) and the reaction was quenched at room temperature. The mixture was stirred for 12 hours. The reaction solution was quenched with acetic acid, and the resulting mixture was concentrated under reduced pressure to obtain a crude residue. Purification by flash column chromatography (ethyl acetate/hexane = 1/5) gave compound 42 (220 mg, 65% overall). [α] ²² _D +8.6 ( c 4.0, CHCl ₃ ); IR (thin film): ν 3514, 2931, 2110, 1754, 1215, 1112, 1013, 701 cm ^–1 ; ¹ H NMR (600 MHz, CDCl _{3 )} ): δ 7.88–7.80 (m, 1H, Ar-H), 7.75–7.66 (m, 16H, Ar-H), 7.65–7.62 (m, 2H, Ar-H), 7.59–7.57 (m, 1H, Ar-H), 7.50–7.44 (m, 5H, Ar-H), 7.40–7.31 (m, 38H, Ar-H), 7.31–7.23 (m, 23H, Ar-H), 7.18–7.14 (m, 1H, Ar-H), 7.14–7.11 (m, 2H, Ar-H), 7.10–7.06 (m, 2H, Ar-H), 7.05–7.02 (m, 2H, Ar-H), 5.54–5.49 ( m, 2H), 5.38 (s, 2H), 5.20–5.14 (m, 2H), 5.05–4.99 (m, 2H), 4.99–4.91 (m, 3H), 4.90–4.85 (m, 1H), 4.82– 4.73 (m, 6H), 4.69–4.62 (m, 3H), 4.61–4.53 (m, 4H), 4.51–4.45 (m, 5H), 4.30–4.21 (m, 1H), 4.16–4.09 (m, 2H ), 4.09–4.03 (m, 2H), 4.01–3.92 (m, 5H), 3.91–3.84 (m, 7H), 3.83–3.77 (m, 6H), 3.75–3.71 (m, 3H), 3.70–3.65 (m, 3H), 3.63–3.57 (m, 1H), 3.56–3.50 (m, 2H), 3.50–3.45 (m, 3H), 3.42–3.30 (m, 9H), 3.27–3.22 (m, 6H) , 3.19–3.13 (m, 5H), 1.74–1.45 (m, 6H), 1.10 (s, 9H, t Bu), 1.073 (s, 9H, t Bu), 1.070 (s, 9H, t Bu), 1.06 (s, 9H, t Bu); ^{13C NMR (150 MHz, CDCl} ₃ ): δ 169.0 (C), 168.6(C), 168.1 (C), 156.7/156.3 (C), 138.3 (C), 138.23 (C), 138.16 (C), 137.8 (C), 137.14 (C), 137.12 (C) , 137.0 (C), 136.92 (C), 136.86 (C), 136.8 (C), 136.7 (C), 136.60 (C), 136.57 (C), 136.0 (CH), 135.9 (CH), 135.82 (CH) , 135.79 (CH), 135.6 (CH), 135.54 (CH), 135.49 (CH), 133.5 (C), 133.4 (C), 133.3 (C), 133.21 (C), 133.18 (C), 133.10 (C) , 133.08 (C), 133.0 (C), 132.93 (C), 132.90 (C), 132.87 (C), 132.75 (C), 131.5 (CH), 131.4 (CH), 131.11 (CH), 131.08 (CH) , 130.4 (CH), 130.0 (CH), 129.9 (CH), 129.74 (CH), 129.72 (CH), 129.68 (CH), 129.65 (CH), 129.63 (CH), 129.60 (CH), 129.1 (CH) , 129.0 (CH), 128.61 (CH), 128.58 (C), 128.5 (CH), 128.4 (CH), 128.34 (CH), 128.29 (CH), 128.21 (CH), 128.17 (CH), 128. (CH) ), 128.0 (CH), 127.9 (CH), 127.84 (CH), 127.81 (CH), 127.71 (CH), 127.66 (CH), 127.55 (CH), 127.53 (CH), 127.32 (CH), 127.28 (CH) ), 127.17 (CH), 126.2 (CH), 125.9 (CH), 125.8 (CH), 125.2 (CH), 121.8 (C), 121.7 (C), 121.08 (C), 121.06 (C), 103.1 (CH) ), 102.8 (CH), 102.2 (CH), 101.0 (CH), 100.8 (CH), 97.20 (CH), 97.15 (CH), 95.4 (CH), 95.2 (CH), 84.2 (CH), 84.0 (CH), 80.9 (CH), 79.2 (CH), 78.3 (CH), 78.1 (CH), 77.6 (CH), 76.1 (CH), 75.1 (CH ₂ ), 74.92 (CH ₂ ), 74.89 (CH ₃ ), 74.87 (CH ₂ ), 74.8 (CH ), 74.7 ( CH ₂ ), 74.6 (CH), 74.5 (CH ₂ ), 74.4 (CH ₂ ), 74.2 (CH), 74.0 (CH), 73.7 (CH), 73.6 (CH), 73.1 (CH ₂ ), 72.9 (CH ₂ ), 72.7 (CH), 72.55 (CH), 72.49 (CH ₂ ), 72.4 (CH/CH ₂ ), 72.3 (CH), 72.2 (CH ₂ ), 72.1 (CH), 72.0 (CH), 71.4 ( CH), 70.4/70.0 (CH ₂ ), 67.4 (CH), 67.2 (CH ₂ ), 67.1 (CH), 66.9 (CH), 66.6 (CH), 63.7 (CH), 63.2 (CH), 63.0 (CH) ), 62.1 (CH ₂ ), 61.9 (CH ₂ ), 61.1 (CH ₂ ), 53.8 (CH ₂ ), 52.3 (CH ₃ ), 52.1 (CH ₃ ), 51.7 (CH ₃ ), 51.6 (CH ₃ ), 50.4/50.2 (CH ₂ ), 46.9/45.9 (CH ₂ ), 31.9 (CH ₂ ), 29.7 (CH ₂ ), 29.6 (CH ₂ ), 29.3 (CH ₂ ), 26.92 (CH ₃ ), 29.0/28.8 ( CH ₂ ), 27.0 (CH ₃ ), 26.94 (CH ₃ ), 26.89 (CH ₃ ), 23.3 (CH ₂ ), 23.0 (CH ₂ ), 22.70 (CH ₂ ), 22.67 (CH ₂ ), 22.6 (CH ₂ ), 19.42 (C), 19.41 (C), 19.37 (C); HRMS (MALDI): m/z calcd for C ₂₀₃ H ₂₂₅ B r ₄ N ₁₃ O ₄₃ Si ₄ Na ([M + Na] ⁺ ): 3990.0383, found: 3990.0508.

N - benzyl -N- benzyloxycarbonyl -5- Aminopentyl [2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -4 -O- (2- naphthylmethyl )-α-D- glucopyranosyl ]-(1→4)-( methyl 3 -O- benzyl -α-L- Iduranuronate )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-( methyl 3 -O- benzyl -β-D- glucopyranosyl ester )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -α-D- glucopyranosyl ]-(1→4)-( methyl 3 -O- benzyl -α-L- Iduranuronate )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -α-D- glucopyranosyl ]-(1→4)- methyl 3 -O- benzyl -β-D- Methyl glucopyranosate ( N -Benzyl -N- benzyloxycarbonyl-5-aminopentyl [2-azido-3 -O- (4-bromobenzyl)-2-deoxy-4 -O- (2-naphthylmethyl)-α-D-glucopyranosyl]-(1 →4)-(methyl 3 -O- benzyl-α-L-idopyranosyluronate)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-2-deoxy-α-D-glucopyranosyl] -(1→4)-(methyl 3 -O- benzyl-β-D-glucopyranosyluronate)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-2-deoxy-α-D -glucopyranosyl]-(1→4)-(methyl 3 -O- benzyl-α-L-idopyranosyluronate)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-2-deoxy- α-D-glucopyranosyl]-(1→4)-methyl 3 -O- benzyl-β-D-glucopyranosiduronate)(16)

A 70% solution of HF in pyridine (500 equiv) was added to compound 15 (210 mg, 0.05 mmol) in a mixed solvent (pyridine/THF = 1/1, v/v, 40 mL/g octosaccharide) )middle. The mixture was warmed to room temperature and stirred for 3 days. The solution was concentrated under reduced pressure, and the residue was purified by flash column chromatography to give compound 16 (135 mg, 83%). [α] ²⁷ _D –29.7 ( c 2.0, CHCl ₃ ); IR (thin film): ν 2924, 2109, 1748, 1027, 699 cm ^–1 ; ¹ H NMR (600 MHz, CDCl ₃ ): δ 7.81–7.79 (m, 1H, Ar-H), 7.78–7.74 (m, 2H, Ar-H), 7.63 (bs, 1H, Ar-H), 7.48–7.45 (m, 2H, Ar-H), 7.43–7.41 (m, 2H, Ar-H), 7.38–7.34 (m, 18H, Ar-H), 7.33–7.30 (m, 8H, Ar-H), 7.30–7.26 (m, 7H, Ar-H), 7.25 -7.22 (m, 4H, Ar-H), 7.20-7.18 (m, 2H, Ar-H), 7.16-7.13 (m, 1H, Ar-H), 7.10–7.07 (m, 2H, Ar-H) , 7.04–7.00 (m, 4H, Ar-H), 5.56 (bs, 1H), 5.47 (d, J = 3.5 Hz, 1H), 5.23 (d, J = 8.5 Hz, 2H), 5.19–5.12 (m , 2H), 5.11–5.03 (m, 1H), 4.99–4.95 (m, 2H), 4.92 (d, J = 10.9 Hz, 1H), 4.89 (d, J = 3.5 Hz, 1H), 4.86 (d, J = 11.8 Hz, 1H), 4.81–4.77 (m, 3H), 4.74–4.72 (m, 3H), 4.71 (d, J = 5.0 Hz, 1H), 4.69 (d, J = 5.0 Hz, 1H), 4.61–4.56 (m, 3H), 4.56–4.50 (m, 3H), 4.49–4.42 (m, 4H), 4.31–4.24 (m, 1H), 4.07 (t, J = 9.0 Hz, 1H), 4.03 ( t, J = 9.0 Hz, 1H), 4.00 (bs, 1H), 3.98–3.93 (m, 3H), 3.92–3.85 (m, 4H), 3.85–3.81 (m, 3H), 3.81–3.78 (m, 2H), 3.78–3.75 (m, 3H), 3.73–3.69 (m, 8H), 3.68–3.65 (m, 3H), 3.65–3.62 (m, 3H), 3.62–3.59 (m, 3H), 3.58–3.55 (m, 2H), 3.51 (s, 3H), 3.50–3.49 (m, 1H), 3.49 –3.46 (m, 1H), 3.45–3.43 (m, 5H), 3.42–3.41 (m, 1H), 3.40–3.37 (m, 4H), 3.24–3.18 (m, 3H), 1.34–1.25 (m, 6H, CH ₂ in linker); ^{13C NMR (150 MHz, CDCl} ₃ ): δ 169.5 (C), 169.4 (C), 168.8 (C), 168.5 (C), 156.7/156.3 (C), 138.12 (C) , 138.06 (C), 138.0 (C), 137.7 (C), 137.0 (C), 136.8 (C), 136.72 (C), 136.65 (C), 136.52 (C), 136.47 (C), 135.0 (C) , 133.1 (C), 132.9 (C), 131.45 (CH), 131.37 (CH), 131.14 (CH), 131.10 (CH), 129.52 (CH), 129.46 (CH), 129.0 (CH), 128.9 (CH) , 128.6 (CH), 128.5 (CH), 128.40 (CH), 128.36 (CH), 128.3 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.2 (CH) , 127.1 (CH), 126.28 (CH), 126.27 (CH), 126.1 (CH), 125.4 (CH), 121.7 (C), 121.5 (C), 121.14 (C), 121.09 (C), 103.2 (CH) , 103.1 (CH), 102.9 (CH), 100.9 (CH), 100.7 (CH), 97.4 (CH), 97.2 (CH), 96.0 (CH), 95.4 (CH), 83.9 (CH), 80.7 (CH) , 79.3 (CH), 78.4 (CH), 78.3 (CH), 76.2 (CH), 75.0 (CH ₂ ), 74.9 (CH ₂ ), 7 4.8 (CH ₂ ), 74.73 (CH ₂ ), 74.66 (CH ), 74.6 (CH ₃ ), 74.5 (CH ), 74.1 (CH), 73.9 (CH ), 73.50 (CH ₂ ), 73.47 (CH ₂ ), 73.1 (CH), 72.9 (CH), 72.6 (CH ₂ ), 72.5 (CH ₂ ), 72.2 (CH), 72.1 (CH), 71.9 (CH), 71.6 (CH ₂ ), 71.0 (CH ₂ ), 70.4 (CH ₂ ), 70.3 (CH ₂ ), 70.0 (CH ₂ ), 67.7 (CH), 67.5 (CH), 67.2 (CH ₃ /CH ₂ ), 67.1 (CH), 66.8 (CH), 63.5 (CH) , 63.4 (CH), 63.3 (CH), 61.8 (CH ₂ ), 61.0 (CH ₂ ), 60.9 (CH ₂ ), 60.3 (CH ₂ ), 52.7 (CH ₃ ), 52.6 (CH ₃ ), 52.14 (CH ) ₃ ), 52.13 (CH ₃ ), 50.4/50.2 (CH ₂ ), 46.9/45.9 (CH ₂ ), 31.9 (CH ₂ ), 31.6 (CH ₂ ), 30.0 (CH ₃ ), 29.63 (CH ₂ ), 29.59 (CH ₂ ), 29.3 (CH ₂ ), 29.0 (CH ₂ ), 28.7 (CH ₂ ), 27.6 (CH ₂ ), 27.1 (CH ₂ ), 27.0 (CH ₂ ), 23.2 (CH ₂ ), 23.0 (CH 2 ) ₂ ), 22.6 (CH ₂ ), 19.2 (C), 14.1 (CH ₂ ), 13.9 (C); HRMS (MALDI): m/z calcd for C ₁₃₉ H ₁₅₃ Br ₄ N ₁₃ O ₄₃ Na [M + Na ] ⁺ : 3036.4155, found: 3036.4241.

N - benzyl -N- benzyloxycarbonyl -5- Aminopentyl [2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -4 -O- (2- naphthylmethyl )-6 -O- sulfonate group -α-D- glucopyranosyl )-(1→4)-( methyl 3 -O- benzyl -2 -O- sulfonate group -α-L- Iduranuronate )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -6 -O- sulfonate group -α-D- glucopyranosyl ]-(1→4)-( methyl 3 -O- benzyl -2 -O- sulfonate group -β-D- glucopyranosyl ester )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -6 -O- sulfonate group -α-D- glucopyranosyl )-(1→4)-( methyl 3 -O- benzyl -2 -O- sulfonate group -α-L- Iduranuronate )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -6 -O- sulfonate group -α-D- glucopyranosyl )-(1→4)- methyl 3 -O- benzyl -2 -O- sulfonate group -β-D- Methyl glucopyranonate octasodium salt ( N -Benzyl- N -benzyloxycarbonyl-5-aminopentyl[2-azido-3 -O- (4-bromobenzyl)-2-deoxy-4 -O- (2-naphthylmethyl)-6 -O- sulfonato-α-D -glucopyranosyl)-(1→4)-(methyl 3 -O- benzyl-2 -O- sulfonato-α-L-idopyranosyluronate)-(1→4)-[2-azido-3 -O- (4-bromobenzyl )-2-deoxy-6 -O- sulfonato-α-D-glucopyranosyl]-(1→4)-(methyl 3 -O- benzyl-2 -O- sulfonato-β-D-glucopyranosyluronate)-(1→4 )-[2-azido-3 -O- (4-bromobenzyl)-2-deoxy-6 -O- sulfonato-α-D-glucopyranosyl)-(1→4)-(methyl 3 -O- benzyl-2 - O- sulfonato-α-L-idopyranosyluronate)-(1→4)-[2-azido-3 -O- (4-bromobenzyl)-2-deoxy-6 -O- sulfonato-α-D-glucopyranosyl)-( 1→4)-methyl 3 -O- benzyl-2 -O- sulfonato-β-D-glucopyranosiduronate octasodium salt)(17)

Compound 16 (190 mg, 0.06 mmol) and SO ₃ Et ₃ N ( ₄ equiv of octasaccharide per OH group) were prepared in DMF (10 mL/g octasaccharide) at 60°C under N atmosphere ) was continuously stirred for 1 day. The reaction flask was cooled to room temperature, phosphate buffer (pH=7, 2.0 mL) was added, and the mixture was stirred for an additional 30 minutes. The resulting solution was concentrated in vacuo and the residue was purified with Sephadex LH-20 using MeOH as eluent to give O-sulfonated compound 17 (226 mg, 94%). IR (thin film): ν 3464, 2111, 1747, 1236, 1068, 1005, 701 cm ^–1 ; ¹ H NMR (600 MHz, CD ₃ OD): δ 7.81–7.72 (m, 3H, Ar-H), 7.70 (bs, 1H, Ar-H), 7.55–7.49 (m, 6H, Ar-H), 7.46–7.37 (m, 18H, Ar-H), 7.36–7.33 (m, 5H, Ar-H), 7.32–7.26 (m, 9H, Ar-H), 7.25–7.21 (m, 4H, Ar-H), 7.21–7.17 (m, 3H, Ar-H), 7.04 (d, J = 8.5 Hz, 2H, Ar-H), 7.01 (d, J = 8.4 Hz, 2H, Ar-H), 5.56 (s, 1H), 5.48–5.42 (m, 3H), 5.32 (d, J = 10.0 Hz, 1H), 5.19 –5.09 (m, 5H), 5.04 (d, J = 3.0 Hz, 1H), 4.99–4.94 (m, 2H), 4.93–4.91 (m, 1H), 4.85–4.77 (m, 8H, ArCH ₂ ), 4.73–4.70 (m, 2H), 4.69–4.68 (m, 1H), 4.67–4.63 (m, 2H), 4.59 (s, 2H), 4.55–4.48 (m, 4H), 4.45–4.38 (m, 3H ), 4.37–4.31 (m, 4H), 4.30–4.25 (m, 3H), 4.25–4.20 (m, 5H), 4.15–4.10 (m, 2H), 4.06 (t, J = 9.5 Hz, 1H), 4.04–3.99 (m, 2H), 3.97 (dd, J = 8.5, 4.9 Hz, 1H), 3.91–3.86 (m, 4H), 3.86–3.83 (m, 1H), 3.80 (s, 3H), 3.76– 3.71 (m, 2H), 3.71–3.64 (m, 6H), 3.64–3.59 (m, 3H), 3.55 (s, 3H), 3.48–3.46 (m, 1H), 3.45 (s, 3H), 3.43– 3.41 (m, 1H), 3.41–3.38 (m, 1H), 3.30–3.27 (m, 2H) , 3.26–3.18 (m, 2H), 1.64–1.50 (m, 4H, CH ₂ in linker), 1.41–1.29 (m, 2H, CH ₂ in linker); ^{13C NMR (150 MHz,} CD ₃ OD): δ 172.6 (C), 172.2 (C), 171.8 (C), 170.9 (C), 158.6/158.1 (C), 139.8 (C), 139.6 (C), 139.5 (C), 139.4 (C), 139.1 (C) ), 138.83 (C), 138.81 (C), 138.3 (C), 138.2 (C), 137.2 (C), 134.8 (C), 134.5 (C), 132.40 (CH), 132.37 (CH), 132.36 (CH) ), 132.2 (CH), 131.6 (CH), 130.7 (CH), 130.52 (CH), 130.46 (CH), 130.4 (CH), 129.3 (CH), 129.8 (CH), 129.73 (CH), 129.66 (CH) ), 129.6 (CH), 129.5 (CH), 129.29 (CH), 129.25 (CH), 129.11 (CH), 129.05 (CH), 129.0 (CH), 128.8 (CH), 128.7 (CH), 128.5 (CH) ), 128.4 (CH), 127.7 (CH), 127.2 (CH), 127.1 (CH), 126.9 (CH), 122.33 (C), 122.26 (C), 122.2 (C), 122.1 (C), 102.7 (CH ), 101.8 (CH), 99.6 (C), 98.8 (CH), 98.6 (CH), 98.5 (CH), 98.4 (CH), 98.2 (CH), 84.1 (CH), 83.8 (CH), 81.7 (CH) ), 80.4 (CH), 80.3 (CH), 80.2 (CH), 80.0 (CH), 79.6 (CH), 78.9 (CH), 78.8 (CH), 77.0 (CH), 76.1 (CH ₂ ), 75.65 ( CH ₂ ), 75.6 (CH ₂ ), 75.4 (CH ₂ /CH), 75.2 (CH), 75.0 (CH), 74.9 ( _CH2 ), 74.8 ( _CH2 ), 74.2 ( _CH2 ), 73.6 ( _CH2 ), 73.5 ( _CH2 ), 73.4 (CH), 73.2 (CH), 72.5 (CH), 72.2 (C), 72.0 (C), 71.9 (C), 71.8 (CH), 71.5 (CH), 70.5 (CH), 70.32 (CH), 70.26 (CH ₂ ), 70.1 (CH ₂ ), 68.6 (CH ₂ ), 68.4 ( CH ₂ ), 68.1 (CH ), 68.0 (CH ), 67.3 (CH ₂ ), 67.03 (CH ₂ ), 66.98 (CH ₂ ), 66.6 (CH ₂ ), 65.4 (CH ), 65.0 (CH ), 54.1 ( CH), 53.6 (CH), 51.6 (CH ₂ ), 51.5 (CH ₂ ), 47.7 (CH ₂ ), 30.1 (CH ₂ ), 30.0 (CH ₂ ), 29.0 (CH ₂ ), 28.6 (CH ₂ ), 24.32 (CH ₂ ), 24.25 (CH ₂ ); LRMS (ESI): m/z calcd for C ₁₃₉ H ₁₄₅ Br ₄ N ₁₃ Na ₁₀ O ₆₇ S ₈ ([M –8H + 10Na] ²⁺ ): 1937.9, found: 1937.6.

O - sulfonate group -α-D- glucopyranosyl ]-(1→4)-(3 -O- benzyl -2 -O- sulfonate group -β-D- glucopyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -6 -O- sulfonate group -α-D- glucopyranosyl ]-(1→4)-(3 -O- benzyl -2 -O- sulfonate group -α-L- iduropyranosyl )-(1→4)-[2- Azido -3 -O- (4- Bromobenzyl )-2- deoxygenation -6 -O- sulfonate group -α-D- glucopyranosyl ]-(1→4)-3 -O- benzyl -2 -O- sulfonate group -β-D- Glucuropyranoside dodeca sodium salt ( O -sulfonato-α-D-glucopyranosyl]-(1→4)-(3- O -benzyl-2- O -sulfonato-β-D-glucuronopyranosyl)-(1→4)-[2-azido-3 - O- (4-bromobenzyl)-2-deoxy-6- O -sulfonato-α-D-glucopyranosyl]-(1→4)-(3- O -benzyl-2- O -sulfonato-α-L-iduronopyranosyl )-(1→4)-[2-azido-3- O -(4-bromo-benzyl)-2-deoxy-6- O -sulfonato-α-D-glucopyranosyl]-(1→4)-3- O -benzyl-2- O -sulfonato-β-D-glucuronopyranoside dodecasodium salt)(18)

_CHCl3 (2.4 mL), MeOH (8.8 mL), NaOH (5 vol molar solution in _H2O , 1.2 mL) and _H2O (1.2 mL) were added to compound 17 (1.2 mL) at room temperature 155 mg, 0.05 mmol). The mixture was kept stirring for 2 days, and the reaction solution was neutralized with 1 N HCl _(aq) . The mixture was concentrated in vacuo and the residue was purified by gel permeation chromatography using Sephadex LH-20 resin and MeOH as eluent. The appropriate fractions were combined and passed through a column of AG 50W-X8 Na ⁺ resin containing MeOH to give compound 18 (150 mg, 96%).

¹ H NMR (600 MHz, CD ₃ OD): δ 7.81–7.72 (m, 4H, Ar-H), 7.50–7.47 (m, 4H, Ar-H), 7.46–7.42 (m, 7H, Ar-H) ), 7.40–7.37 (m, 5H, Ar-H), 7.36–7.32 (m, 5H, Ar-H), 7.30–7.27 (m, 10H, Ar-H), 7.27–7.24 (m, 7H, Ar -H), 7.24–7.21 (m, 5H, Ar-H), 7.21–7.19 (m, 3H, Ar-H), 7.19–7.16 (m, 3H, Ar-H), 5.64 (s, 1H), 5.59 (s, 1H), 5.52 (d, J = 3.1 Hz, 1H), 5.49 (d, J = 2.8 Hz, 1H), 5.30-5.24 (m, 3H), 5.19-5.09 (m, 4H), 5.05 –4.96 (m, 5H), 4.89–4.86 (m, 2H), 4.81–4.78 (m, 2H), 4.68–4.62 (m, 6H, ArCH ₂ ), 4.60–4.54 (m, 7H), 4.53–4.49 (m, 4H), 4.41–4.36 (m, 4H), 4.33–4.27 (m, 3H), 4.25–4.18 (m, 6H), 4.16–4.12 (m, 3H), 4.10–4.04 (m, 3H) , 4.03–3.98 (m, 3H), 3.97–3.93 (m, 3H), 3.92–3.89 (m, 2H), 3.88–3.83 (m, 3H), 3.69 (t, J = 9.4 Hz, 1H), 3.60 –3.49 (m, 2H), 3.40–3.36 (m, 1H), 3.29–3.18 (m, 3H), 3.13 (d, J = 9.1 Hz, 1H), 1.60–1.47 (m, 4H, CH ₂ in linker ), 1.40–1.28 (m, 2H, CH ₂ in linker); ^{13C NMR (150 MHz,} CD ₃ OD): δ 176.4 (C), 176.1 (C), 175.9 (C), 158.6/158.0 (C), 140.0 (C), 139.9 (C), 139.5 (C), 139.4 (C), 139. 3 (C), 139.2 (C), 139.14 (C), 139.06 (C), 139.0 (C), 138.2 (C), 138.1 (C), 137.2 (C), 134.8 (C), 134.5 (C), 132.4 (CH), 132.3 (CH), 132.2 (CH), 131.7 (CH), 131.0 (CH), 130.4 (CH), 129.8 (CH), 129.72 (CH), 129.67 (CH), 129.6 (CH), 129.5 (CH), 129.44 (CH), 129.39 (CH), 129.3 (CH), 129.23 (CH), 129.17 (CH), 129.1 (CH), 129.04 (CH), 128.99 (CH), 128.92 (CH), 128.86 (CH), 128.8 (CH), 128.7 (CH), 128.5 (CH), 128.4 (CH), 128.3 (CH), 128.1 (CH), 127.6 (CH), 127.1 (CH), 126.9 (CH), 122.2 (C), 122.09 (C), 122.07 (C), 102.8 (CH), 102.0 (CH), 98.3 (C), 98.0 (CH), 97.1 (CH), 96.9 (CH), 96.5 (CH), 85.2 (CH), 85.0 (CH), 82.0 (CH), 81.8 (CH), 81.4 (CH), 80.3 (CH), 79.3 (CH), 78.9 (CH), 78.6 (CH), 78.5 (CH), 78.3 (CH), 78.1 (CH), 76.9 (CH), 76.1 (CH ₂ ), 75.9 (CH ), 75.5 (CH ₂ ), 75.3 (CH ₂ ), 73.2 (CH ₂ /CH), 73.0 (CH ₂ /CH), 72.6 (CH), 72.4 (CH), 71.65 (CH), 71.56 (CH), 71.2 (CH ₂ ), 71.0 (CH), 70.1 (CH), 69.31 (CH), 69.27 (CH), 68.9 (CH ₂ ), 68.5 (CH ₂ ), 68.4 (CH ₂ ), 67.5 (CH ₂ ), 65.7 (CH 2 ), 65.5 ( CH), 65.2 (CH), 65.1 (CH), 51.7 (CH ₂ ), 51.4 (CH ₂ ), 50.0 (CH ₂ ), 47.8 (CH ₂ ), 30.2 (CH ₂ ), 29.2 (CH ₂ ), 28.6 (CH ₂ ), 24.2 (CH ₂ ); HRMS (MALDI): m/z calcd for C ₁₃₅ H ₁₃₃ Br ₄ N ₁₃ O ₆₇ S ₈ Na ₁₄ ([M + 14Na – 12H] ²⁺ ): 1953.7903, found : 1953.7806.

( 2R,3S,4S,5R,6R )-3-((( 2R,3R,4R,5S,6R )-3- Amine -4,5- dihydroxy -6-(( Sulfonyloxy ) methyl ) tetrahydro -2H - pyran -2- base ) Oxygen )-6-((( 2R,3S,4R,5R,6R )-5- Amine -6-((( 2S,3S,4S,5R,6R )-6-((( 2R,3S,4R,5R,6R )-5- Amine -6-((( 2S,3S,4S,5R,6R )-6-((( 2S,3S,4S,5R,6R )-5- Amine -6-((( 2S,3S,4S,5R,6R )-6-(((5- Aminopentyl ) Oxygen )-2- carboxyl group -4- hydroxyl -5-( Sulfonyloxy ) tetrahydro -2H - pyran -3- base ) Oxygen )-4- hydroxyl -2-(( Sulfonyloxy ) methyl ) tetrahydro -2H - pyran -3- base ) Oxygen )-2- carboxyl group -4- hydroxyl -5-( Sulfonyloxy ) tetrahydro -2H - pyran -3- base ) Oxygen )-4- hydroxyl -2-(( Sulfonyloxy ) methyl ) tetrahydro -2H - pyran -3- base ) Oxygen )-2- carboxyl group -4- hydroxyl -5-( Sulfonyloxy ) tetrahydro -2H - pyran -3- base ) Oxygen )-4- hydroxyl -2-(( Sulfonyloxy ) methyl ) tetrahydro -2H - pyran -3- base ) Oxygen )-4- hydroxyl -5-( Sulfonyloxy ) tetrahydro -2H - pyran -2- Carboxylate ((2 R ,3 S ,4 S ,5 R ,6 R )-3-(((2 R ,3 R ,4 R ,5 S ,6 R )-3-amino-4,5-dihydroxy-6 -((sulfonatooxy)methyl)tetrahydro- 2H -pyran-2-yl)oxy)-6-((((2 R ,3 S ,4 R ,5 R ,6 R )-5-amino-6-(( (2 S ,3 S ,4 S ,5 R ,6 R )-6-(((2 R ,3 S ,4 R ,5 R ,6 R )-5-amino-6-(((2 R , 3 S ,4 S ,5 R ,6 R )-6-((((2 R ,3 S ,4 R ,5 R ,6 R )-5-amino-6-(((2 S ,3 S ,4 S ,5 R ,6 R )-6-((5-aminopentyl)oxy)-2-carboxylato-4-hydroxy-5-(sulfonatooxy)tetrahydro-2 H -pyran-3-yl)oxy)-4-hydroxy -2-((sulfonatooxy)methyl)tetrahydro-2 H -pyran-3-yl)oxy)-2-carboxylato-4-hydroxy-5-(sulfonatooxy)tetrahydro-2 H -pyran-3-yl)oxy)- 4-hydroxy-2-((sulfonatooxy)methyl)tetrahydro-2 H -pyran-3-yl)oxy)-2-carboxylato-4-hydroxy-5-(sulfonatooxy)tetrahydro-2 H -pyran-3-yl) oxy)-4-hydroxy-2-((sulfonatooxy)methyl)tetrahydro-2 H -pyran-3-yl)oxy)-4-hydroxy-5-(sulfonatooxy)tetrahydro-2 H -pyran-2-carboxylate)( 19)

A solution of compound 18 and Pd(OH) ₂ on carbon (Pd(OH) ₂ on carbon, 3 g/g octosaccharide) in phosphate buffer (pH = 7.0, 15 mL/g octosaccharide) (necessary MeOH may be added to increase the solubility of the octasaccharide) in a hydrogen balloon, and the mixture was stirred at room temperature for 2 days. The entire mixture was filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified on a Sephadex G-25 column with water as eluent and then Na ⁺ exchanged with AG 50WX8 (Na ⁺ form) cation exchange resin. The appropriate fractions were lyophilized to yield the desired product 19 (80%).

¹ H NMR (600 MHz, D ₂ O) δ 5.52 (d, J = 3.8 Hz, 1H), 5.46 (d, J = 3.7 Hz, 1H), 5.31 (t, J = 4.4 Hz, 1H), 5.16 ( d, J = 4.1 Hz, 1H), 4.84 (d, J = 1.8 Hz, 1H), 4.80 (brs, 1H), 4.52 (d, J = 7.8 Hz, 1H), 4.47 (d, J = 11.0 Hz, 1H), 4.29–4.25 (m, 4H), 4.19 – 4.07 (m, 7H), 4.01 (dd, J = 8.8, 7.9 Hz, 1H), 3.97 – 3.91 (m, 2H), 3.90 – 3.70 (m, 14H), 3.64 (t, J = 9.8 Hz, 1H), 3.62 – 3.59 (m, 1H), 3.47 (t, J = 9.7 Hz, 1H), 3.28 (dd, J = 10.7, 3.8 Hz, 1H), 3.23 – 3.16 (m, 2H), 2.91 (t, J = 7.3 Hz, 2H), 1.63 – 1.53 (m, 4H), 1.44 – 1.36 (m, 2H).; ¹³ C NMR (150 MHz, D ₂ O) ) δ 175.3, 175.0, 174.6, 174.6, 100.5, 100.0, 98.8, 98.5, 96.4, 96.0, 91.7, 91.4, 80.0, 79.6, 77.1, 76.9, 76.1, 76.0, 75.8, 75.1, 75.0, 74.9, 72.9, 72.7, 70.8, 70.4, 70.2, 70.1, 69.7, 69.5, 69.3, 68.9, 68.6, 67.7, 67.2, 66.1, 66.0, 65.4, 62.8, 54.1, 53.9, 27.9, 26.1, 21.8. ; HRMS (ESI): m/z calcd for C ₅₃ H ₈₇ N ₅ Na ₄ O ₆₅ S ₈ ([M+10H-8Na] ²⁺ ): 1090.5501, found: 1090.5498.

compound 20 :

At room temperature, freshly prepared activated biotin ester (activated biotin ester) was added to the octasaccharide amine compound 19 in DMF (220 μl/1 mg), Et ₃ N (25 μl/1 mg) containing 10% water. biotin ester, 75 μl/1 mg in 0.1 vol molar DMF, 15 equiv). Stirring was continued for a further 16 hours at room temperature. The solvent was evaporated using a stream of _N2 and purified using exclusive column chromatography with G-10 sephadex format, followed by Na ⁺ exchange with a cation exchange resin column of AG 50WX8 (Na ⁺ form). . The appropriate fractions were lyophilized to yield the desired compound 20 (white foamy solid, 94%).

¹ H NMR (600 MHz, D ₂ O) δ 5.37 – 5.26 (m, 1H), 5.10 (s, 1H), 5.08 – 5.04 (m, 1H), 4.62 – 4.60 (m, 1H), 4.54 – 4.50 ( m, 3H), 4.50 – 4.41 (m, 2H), 4.35 – 4.32 (m, 3H), 4.30 – 4.20 (m, 4H), 4.13 (brs, 3H), 4.06 – 4.00 (m, 1H), 3.97 ( brs, 2H), 3.93–3.88 (m, 4H), 3.83 – 3.76 (m, 2H), 3.76 – 3.63 (m, 21H), 3.70 – 3.53 (m, 120H), 3.59 – 3.51 (m, 20H), 3.52 – 3.43 (m, 1H), 3.31–3.29 (m, 10H), 3.26–3.23 (m, 6H), 3.09 (t, J = 6.8 Hz, 1H), 3.02 (d, J = 13.3 Hz, 1H) , 2.95 – 2.85 (m, 4H), 2.83 (dd, J = 13.3, 4.7 Hz, 1H), 2.70 (s, 2H), 2.68 (s, 1H), 2.62 – 2.54 (m, 2H), 2.52–2.50 (m, 2H), 2.42 (t, J = 6.2 Hz, 1H), 2.38–2.36 (m, 4H), 2.20–2.17 (m, 10H), 1.68–1.40 (m, 21H), 1.34–1.31 (m , 10H).; ¹³ C NMR (150 MHz, D ₂ O) δ 180.2, 180.1, 176.9, 176.9, 175.2, 174.6, 174.4, 174.2, 174.0, 173.7, 165.3, 165.3, 163.1, 9.0.4, 9. , 98.9, 97.1, 96.9, 94.4, 94.0, 80.3, 79.9, 76.5, 76.2, 75.5, 74.2, 71.6, 71.0, 70.4, 69.6, 69.6, 69.5, 69.5, 69.4, 69.4, 69.3, 69.3, 68.8, 67.9, 66.8 , 66.7, 66.7, 66.5 , 66.4, 66.2, 65.0, 64.4, 62.1, 60.2, 59.5, 59.5, 58.4, 55.3, 55.3, 55.1, 53.6, 53.2, 52.9, 50.2, 40.9, 39.7, 39.7, 39.4, 38.9, 38.9, 37.6, 37.1, 36.1 , 35.9, 35.8, 35.7, 35.6, 35.4, 35.4, 28.5, 28.3, 27.9, 27.8, 27.7, 27.7, 27.6, _25.1 , 25.1, _{22.4 .;} O ₁₁₀ S ₁₃ ([M+7H-12Na] ^5- ): 979.0973, found: 979.0985.

Example 2 Using Pre-Coated Examples 1 Magnetic beads of the compound to detect CTC

Following the procedure described in the "Materials and Methods" section, this example constructed a microfluidic wafer for capturing CTCs from 65 advanced or metastatic CCA samples and pre-coated with the compound of Example 1 (i.e., MB-SCH45) or anti-EpCAM antibody (ie, MB-anti-EpCAM, as control) magnetic beads to capture CTCs in all 65 blood samples.

The experimental results found that MB-SCH45 was able to isolate CTCs from all samples regardless of disease status. The number of CTCs isolated from the above samples using MB-SCH45 and MB-anti-EpCAM, along with details about the stage of tumor metastasis and metastatic organs are listed in Table 1. Notably, at the time points observed, CTCs were detectable by radiography even in the blood of patients without distant metastases (Nos in Table 1: 5, 14, 23 , 30, 32, 43 and 60). CTCs were observed to range in diameter from 5 to 20 microns (data not shown). Most cells exhibit a high nucleus-to-cytoplasmic ratio, but not all cells do. This experiment also documented significant heterogeneity in cell shape between patients, as has been documented in other studies. Experimental data showed that MB- ^SCH45 and MB-anti-EpCAM could separate ≥1 and ≥3 CTCs in blood, respectively. In addition, it was observed that some stage IV patients had the lowest CTC concentrations, but non-metastatic patients had a large number of CTCs.

surface 1 isolated per milliliter of blood CTC quantity. Table and provide information about diseases, metastatic organs, tumor staging and use MB-SCH45 or MB- anti- -EpCAM to separate CTC Quantity and other related information Sample # disease metastatic organ Staging separated CTC quantity MB-SCH45 MB- anti- - EpCAM 1 intrahepatic cholangiocarcinoma Distal lymph nodes IV 12 28 2 intrahepatic cholangiocarcinoma Peritoneal dissemination IV 3 10 3 hilar cholangiocarcinoma cervical lymph nodes IV 4 20 4 intrahepatic cholangiocarcinoma Lymph node metastasis and peritoneal dissemination IV 45 122 5 intrahepatic cholangiocarcinoma No distant metastasis III twenty four 90 6 intrahepatic cholangiocarcinoma lymph nodes III 30 98 7 intrahepatic cholangiocarcinoma Peritoneal dissemination IV 3 10 8 intrahepatic cholangiocarcinoma brain metastases IV 6 15 9 hilar cholangiocarcinoma cervical lymph nodes IV 6 20 10 intrahepatic cholangiocarcinoma liver metastases IV 38 126 11 intrahepatic cholangiocarcinoma Lymph node metastasis and peritoneal dissemination IV 50 121 12 intrahepatic cholangiocarcinoma lymph nodes IV 15 45 13 intrahepatic cholangiocarcinoma lymph nodes III 4 15 14 intrahepatic cholangiocarcinoma No distant metastasis III 12 15 15 intrahepatic cholangiocarcinoma lymph nodes III 60 120 16 hilar cholangiocarcinoma adrenal glands IV 70 104 17 intrahepatic cholangiocarcinoma Peritoneal dissemination IV 47 102 18 intrahepatic cholangiocarcinoma Peritoneal dissemination IV 84 126 19 intrahepatic cholangiocarcinoma lymph nodes III 47 123 20 intrahepatic cholangiocarcinoma lymph nodes III 9 30 twenty one hilar cholangiocarcinoma cervical lymph nodes IV 7 30 twenty two intrahepatic cholangiocarcinoma lymph nodes IV 6 20 twenty three intrahepatic cholangiocarcinoma No distant metastasis III 5 20 twenty four intrahepatic cholangiocarcinoma Distal lymph nodes IV 3 10 25 intrahepatic cholangiocarcinoma lymph node recurrence III 30 50 26 intrahepatic cholangiocarcinoma Peritoneal dissemination IV 38 75 27 intrahepatic cholangiocarcinoma lymph nodes III 38 121 28 intrahepatic cholangiocarcinoma liver metastases IV 35 80 29 hilar cholangiocarcinoma lymph nodes III 16 50 30 intrahepatic cholangiocarcinoma No distant metastasis III 3 20 31 Cholangiocarcinoma lymph nodes IV 3 7 32 intrahepatic cholangiocarcinoma No distant metastasis IV 7 25 33 intrahepatic cholangiocarcinoma liver IV 13 25 34 intrahepatic cholangiocarcinoma Peritoneal dissemination IV 17 30 35 Cholangiocarcinoma lymph nodes III 1 5 36 Cholangiocarcinoma lymph nodes IV 30 50 37 Cholangiocarcinoma lymph nodes III 19 35 38 Cholangiocarcinoma lymph nodes IV 10 25 39 intrahepatic cholangiocarcinoma Peritoneal dissemination IV 4 7 40 intrahepatic cholangiocarcinoma liver IV twenty two 55 41 Cholangiocarcinoma lymph nodes IV 1 3 42 Cholangiocarcinoma lymph nodes IV 6 12 43 intrahepatic cholangiocarcinoma No distant metastasis III 2 5 44 intrahepatic cholangiocarcinoma lymph nodes IV 3 8 45 intrahepatic cholangiocarcinoma No distant metastasis IV 6 16 46 intrahepatic cholangiocarcinoma liver IV 2 6 47 intrahepatic cholangiocarcinoma Peritoneal dissemination IV 27 64 48 Cholangiocarcinoma lymph nodes III 11 twenty four 49 intrahepatic cholangiocarcinoma lymph nodes IV 1 4 50 Cholangiocarcinoma lymph nodes III 1 3 51 intrahepatic cholangiocarcinoma liver IV 55 98 52 Cholangiocarcinoma lymph nodes IV 72 128 53 intrahepatic cholangiocarcinoma liver III 97 200 54 intrahepatic cholangiocarcinoma lymph nodes IV 12 36 55 intrahepatic cholangiocarcinoma lymph nodes IV 1 3 56 intrahepatic cholangiocarcinoma lymph nodes IV 5 12 57 Cholangiocarcinoma lymph nodes III 5 10 58 intrahepatic cholangiocarcinoma liver IV 9 twenty two 59 intrahepatic cholangiocarcinoma liver IV 1 3 60 Multiple intrahepatic cholangiocarcinoma No distant metastasis IV 4 9 61 intrahepatic cholangiocarcinoma lymph nodes IV 1 4 62 intrahepatic cholangiocarcinoma lymph nodes IV 5 12 63 Cholangiocarcinoma lymph nodes III 1 4 64 intrahepatic cholangiocarcinoma liver III twenty three 59 65 Multiple intrahepatic cholangiocarcinoma liver IV 15 37

Immunofluorescence images of some samples are shown in Figure 2. It is worth mentioning that the samples are randomly selected and there is no specific order. CTC clusters were observed with both MB-SCH4 and MB-anti-EpCAM in some stage IV patients. Some clusters were relatively small, while others contained dozens of tumor cells. When MB-anti-EpCAM was used for positive enrichment, CTC clusters were more pronounced, as shown in Figure 2 (bottom 4 panels). Such cluster-forming CTCs are known to have relatively high metastatic potential and should be investigated in detail in future work.

Example 3 CTCs for assessing disease status

In this example, the correlation between CTC counts and response to treatment based on standard radiology was investigated by analyzing the medical records of 5 patients with advanced or metastatic CCA (ie, patients A to E). Details of treatment strategies and therapies for these five patients are summarized in Table 2. The number of CTCs and tumor size before treatment were considered to be 100%, and the above data were corrected for post-treatment data such as individual cell number distribution and tumor size measurements. Corrected CTC isolation results using MB-SCH45, MB-anti-EpCAM and flow cytometry (FACS), and computed tomography (CT) scan tumor measurements are shown in FIG. 3 . It was found that CTCs could be successfully detected in all patients even after several lines of chemotherapy. The total number of CTCs decreased after the first round of treatment compared to before treatment in all cases, a phenomenon consistent with the radiographic response results in all tested cases. After several lines of chemotherapy, an increase in the number of CTCs was observed in patients A, C, and D, confirming the radiographic findings that these patients had increased tumor size. These results imply not only that CTCs are predictive of treatment response in all patients, but can also be used to predict patient response to chemotherapy, often before imaging results are available. The data using MB-SCH45 were comparable (patients A, B and D), or even better (patients C and E) compared to FACS analysis. A similar trend was observed with MB-anti-EpCAM. Summarizing the above, these experimental results demonstrate the prognostic and predictive relevance of the persistence of CTCs during chemotherapy, suggesting that the compounds of formula (I) of the present invention have the potential to be used to monitor the treatment of CCA.

Table 2 Information about patients A to E Patient / Age / Gender disease Staging Types of chemotherapy Patient A/47/Female Intrahepatic cholangiocarcinoma with peritoneal spread IV S- ^1a , chrysanthemum folic acid, oxaliplatin and gemcitabine (SLOG) Patient B/65/Male Cholangiocarcinoma with lymph node metastasis IV Gemcitabine and Cisplatin Patient C/83/Female Intrahepatic cholangiocarcinoma without distant metastasis III Radiation therapy in parallel with S-1 oral chemotherapy Patient D/46/Male Multiple intrahepatic cholangiocarcinoma with vascular invasion IV S-1, chrysanthemum folic acid, oxaliplatin and gemcitabine (SLOG) Patient E/66/Female Cholangiocarcinoma with lymph node metastasis IV S-1, chrysanthemum folic acid, oxaliplatin and gemcitabine (SLOG) ^a : S-1 is a combination of tegafur, 5-chloro-2,4-dihydropyrimidine (CDHP) and potassium oxazine.

It should be understood that the above description of the embodiments is only an exemplary embodiment, and those with ordinary knowledge in the technical field to which the present invention pertains can make various changes and modifications. The above specification, examples and experimental data provide a complete description of the structure and use of the present disclosure as exemplary embodiments. While various embodiments of the present disclosure have been described above with a certain degree of characterization, or with reference to one or more individual embodiments, those of ordinary skill in the art to which this invention pertains can still do so without departing from the spirit of the present disclosure. and scope, numerous modifications are made to the disclosed embodiments.

L1: Liquid channel layer

L2: Air Control Layer

L3: Liquid channel layer

L4: Thin PDMS layer

L5: Double-sided tape

(i): Hemolysis receiving inlet

(ii): Sediment collection area

(iii): Waste liquid collection outlet

(I): Blood Processing Unit

(II): Immunofluorescence staining unit

1-13: Solenoid valve connection

C ₁ : Cell pellet collection unit

C ₂ : Waste liquid collection unit

C ₃ : Magnetic Bead Storage Unit

C ₄ -C ₅ : 1 x PBS storage unit

_C6 : storage unit for secondary antibodies

C ₇ : Waste liquid collection unit

C8: Anti- _CK17 primary antibody storage unit

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various example systems, methods, and other illustrative implementations of various aspects of the present invention. The present disclosure will become more apparent upon reference to the following detailed description and accompanying drawings, wherein:

Figure 1A : Schematic of a microfluidic wafer. The wafer has layers L1 to L5, representing liquid channel layer, air control layer, liquid channel layer, thin PDMS layer, and double-sided tape, respectively . The detailed structures of the L1, L2 and L3 layers are also listed in the figure, where (i) represents the receiving inlet for hemolysis, (ii) represents the sediment collection area, and (iii) represents the outlet for waste collection. (I) represents the blood processing unit, and (II) represents the immunofluorescence staining unit. Thirteen connections to electromagnetic valves (EMV) are used for pneumatic control of the wafer, as shown in 1 to 13. The 8 microchambers/reagent storage units are designated C ₁ to C ₈ . C ₁ : cell pellet collection unit, C ₂ : waste liquid collection unit, C ₃ : magnetic bead storage unit, C ₄ and C ₅ : 1×phosphate-buffered saline (PBS) storage unit, C ₆ : storage unit for secondary antibodies (ie, first with DyLight 488 antibody and CD45 _- PE antibody, followed by staining with DAPI), C7: waste collection unit, and C8: storage unit for anti- _CK17 primary antibody.

the first 1B Figure: No. 1A Figure Microfluidics Physical photo of the chip.

Figure 2 : Photographs of immunofluorescence staining of CTC clusters captured with MB-SCH45 or MB -anti- EpCAM . Two representations of phases III and IV with negative depletion of anti-CD45 antibody and positive enrichment with MB-SCH45 (upper 16 panels) and with MB-anti-EpCAM (lower 20 panels) sample. The bottom 4 panels show these CTC clusters after being enriched with MB-anti-EpCAM.

Figure 3 : Predictive significance of CTC counts . The correlation between CTC counts and clinical response to chemotherapy was tested in 5 patients with advanced or metastatic CCA. (A) to (E) Panels represent patients A to E. Supplementary panels I-III in each panel represent corrected CTC counts isolated by MB-SCH45, MB-anti-EpCAM, and flow assisted cell sorting (FACS), respectively. Supplementary panel IV represents adjusted tumor size using radiography.

Claims (9)

a compound of formula (I),

Wherein, R ₁ and R ₂ are respectively H or -SO ₃ M; and M is a monovalent cation and is selected from the group consisting of sodium ion, potassium ion, lithium ion and ammonium ion. The compound of claim 1, wherein in the formula (I), R ₁ and R ₂ are individually -SO ₃ M, and M is a sodium ion. A method for detecting a circulating cholangiocarcinoma (CCA) cell from an ex vivo biological sample, wherein the ex vivo biological sample is from an individual suspected of suffering from CCA, the method comprising: (a) contacting the ex vivo biological sample with the compound described in claim 1; and (b) in an immunoassay, detecting the circulation of the compound described in claim 1 with the ex vivo biological sample A complex formed by CCA cells; wherein the presence of the complex indicates that the individual suffers from CCA. The method of claim 3, wherein in the formula (I), R ₁ and R ₂ are individually -SO ₃ M, and M is a sodium ion. The method of claim 3, wherein the compound of claim 1 is coupled to streptavidin, and the streptavidin is pre-conjugated to the outer portion of a magnetic bead on the surface. The method of claim 3, wherein the ex vivo biological sample is selected from the group consisting of blood, plasma, serum, urine, sputum, saliva, tissue samples, biological specimens, and tissue lysates. The method of claim 6, wherein the ex vivo biological sample is blood. The method of claim 7, further comprising, before step (a), pre-treating the blood with a lysis buffer, so as to lyse the red blood cells therein. The method of claim 3, wherein the individual suffers from stage III or IV CCA, or metastatic CCA.

Images