JP7369428B2 - Sugar compounds and their uses - Google Patents

Description

The present invention relates to sugar compounds, and more particularly to sugar compounds that can be used to detect peptide-N-glycosidase (PNGase) activity.

"Peptide N-glycanase (PNGase)" and "endo-β-N-acetylglucosaminidase (ENGase)" are sugar chain release enzymes that are widely present in the cytoplasm of eukaryotic cells, and are used for quality control of glycoproteins in the endoplasmic reticulum. It is known that it plays an important role in the mechanism. In recent years, the existence of a genetic disease "Ngly1 deficiency" based on mutations in the PNGase gene (Ngly1) has been revealed (see Non-Patent Document 1), which causes growth retardation, muscle weakness in the limbs, involuntary movements, liver function abnormalities, and electroencephalograms. It has also been revealed that patients exhibit serious symptoms such as abnormalities.

Until now, the activity of Ngly1 (PNGase) has been measured by methods such as detecting the cleavage of sugar chains as a change in molecular weight by SDS-PAGE, a change in retention time by HPLC, and a change in size by TLC. .
However, both methods require complicated detection operations, have low throughput, and are not suitable for processing a large number of samples, making it difficult to perform inhibitor screening on compound libraries. Furthermore, the biggest problem is that the conventional activity detection methods cannot be used to measure intracellular activity.

Need, A. C., et al., J. Med. Genet. 2012, 49, 353-361.

An object of the present invention is to provide a novel compound that can be used for detecting PNGase activity, a method for screening a novel PNGase, and a method for screening a PNGase activity inhibitor.

As a result of intensive studies aimed at solving the above-mentioned problems, the present inventors have discovered that a sugar compound in which a fluorescent group and a quenching group that cause fluorescence resonance energy transfer (FRET) are introduced into a specific polysaccharide structure can be used to detect PNGase activity. The present invention has been completed based on the discovery that the method is effective in the following.
The present invention is as follows.

（式（ｄ－１）中、Ｒ’は水素原子、炭素原子数１～６の炭化水素基、又はアミノ基の保護基を表す。）
（ｉｉ）下記式（ｄ－２）で表される蛍光基と下記式（ａ－１）で表される消光基の組合せ

（式（ｄ－２）中、Ｒは水素原子又はヒドロキシル基の保護基を表す。）
（ｉｉｉ）下記式（ｄ－３）で表される蛍光基と下記式（ａ－１）で表される消光基の組合せ

（式（ｄ－３）中、Ｒは水素原子又はヒドロキシル基の保護基を、Ｒ’は水素原子、炭素原子数１～６の炭化水素基、又はアミノ基の保護基を表す。）
（ｉｖ）下記式（ｄ－４）で表される蛍光基と下記式（ａ－２）で表される消光基の組合せ

（式（ｄ－４）及び（ａ－２）中、Ｒ’はそれぞれ独立して水素原子、炭素原子数１～６の炭化水素基、又はアミノ基の保護基を表す。）
（ｖ）TAMRA（登録商標）である蛍光基とBHQ（登録商標）である消光基の組合せ
〔６〕〔１〕～〔５〕のいずれかに記載の糖化合物を含む、ペプチド－Ｎ－グリコシダーゼ（ＰＮＧａｓｅ）活性検出用組成物。
〔７〕ペプチド－Ｎ－グリコシダーゼ（ＰＮＧａｓｅ）の候補タンパク質またはそれを含む画分に下記式（Ｉ）で表される糖化合物を接触させて、前記糖化合物の分解活性を確認
する活性確認工程
を含む、ペプチド－Ｎ－グリコシダーゼ（ＰＮＧａｓｅ）のスクリーニング方法。
Z²-X² _1-5-(GlcNAc₂-Z¹)Asn-X¹ _0-5 ・・・（Ｉ）
（式（Ｉ）中、X¹ _0-5は０～５個の同一の又は異なるアミノ酸を表し、X² _1-5は１～５個の同一の又は異なるアミノ酸を表し、Z¹は蛍光共鳴エネルギー移動（ＦＲＥＴ）が生じる蛍光基を表し、Z²は前記蛍光基に対応する消光基を表す。）
〔８〕ペプチド－Ｎ－グリコシダーゼ（ＰＮＧａｓｅ）の候補タンパク質またはそれを含む画分に下記式（Ｉ）で表される糖化合物を接触させて、前記糖化合物の分解活性を確認する活性確認工程
を含む、ペプチド－Ｎ－グリコシダーゼ（ＰＮＧａｓｅ）活性の評価方法。
Z²-X² _1-5-(GlcNAc₂-Z¹)Asn-X¹ _0-5 ・・・（Ｉ）
（式（Ｉ）中、X¹ _0-5は０～５個の同一の又は異なるアミノ酸を表し、X² _1-5は１～５個の同一の又は異なるアミノ酸を表し、Z¹は蛍光共鳴エネルギー移動（ＦＲＥＴ）が生じる蛍光基を表し、Z²は前記蛍光基に対応する消光基を表す。）
〔９〕被検化合物をペプチド－Ｎ－グリコシダーゼ（ＰＮＧａｓｅ）に接触させる接触工程、及び
前記被検化合物を接触させたペプチド－Ｎ－グリコシダーゼ（ＰＮＧａｓｅ）に下記式（Ｉ）で表される糖化合物を接触させて、前記糖化合物の分解活性を確認する活性確認工程
を含む、ペプチド－Ｎ－グリコシダーゼ（ＰＮＧａｓｅ）活性阻害剤のスクリーニング方法。
Z²-X² _1-5-(GlcNAc₂-Z¹)Asn-X¹ _0-5 ・・・（Ｉ）
（式（Ｉ）中、X¹ _0-5は０～５個の同一の又は異なるアミノ酸を表し、X² _1-5は１～５個の同一の又は異なるアミノ酸を表し、Z¹は蛍光共鳴エネルギー移動（ＦＲＥＴ）が生じる蛍光基を表し、Z²は前記蛍光基に対応する消光基を表す。） [1] A sugar compound represented by the following formula (I).
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In formula (I), X ¹ _0-5 represents 0 to 5 same or different amino acids, X ² _1-5 represents 1 to 5 same or different amino acids, and Z ¹ represents fluorescence resonance It represents a fluorescent group where energy transfer (FRET) occurs, and ^Z2 represents a quenching group corresponding to the fluorescent group.)
[2] The sugar compound according to [1], wherein the X ¹ _0-5 is Gly-B ¹ -Gly, and the B ¹ is Ser or Thr.
[3] The sugar compound according to [1] or [2], wherein the X ² _1-5 is (β-Ala)-Leu _1-4 .
[4] The sugar compound according to [3], wherein the Leu _1-4 is Leu ₃ .
[5] The combination of the fluorescent group and the quenching group is any of the following (i) to (iv), [1
] - [4] The sugar compound according to any one of [4].
(i) A combination of a fluorescent group represented by the following formula (d-1) and a quenching group represented by the following formula (a-1)

(In formula (d-1), R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)
(ii) A combination of a fluorescent group represented by the following formula (d-2) and a quenching group represented by the following formula (a-1)

(In formula (d-2), R represents a hydrogen atom or a hydroxyl group protecting group.)
(iii) A combination of a fluorescent group represented by the following formula (d-3) and a quenching group represented by the following formula (a-1)

(In formula (d-3), R represents a hydrogen atom or a protecting group for a hydroxyl group, and R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)
(iv) A combination of a fluorescent group represented by the following formula (d-4) and a quenching group represented by the following formula (a-2)

(In formulas (d-4) and (a-2), R' each independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)
(v) A combination of a fluorescent group that is TAMRA (registered trademark) and a quenching group that is BHQ (registered trademark) [6] A peptide-N-glycosidase containing the sugar compound according to any one of [1] to [5] (PNGase) Activity detection composition.
[7] An activity confirmation step of contacting a candidate protein of peptide-N-glycosidase (PNGase) or a fraction containing it with a sugar compound represented by the following formula (I) and confirming the decomposition activity of the sugar compound. A screening method for peptide-N-glycosidase (PNGase), comprising:
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In formula (I), X ¹ _0-5 represents 0 to 5 same or different amino acids, X ² _1-5 represents 1 to 5 same or different amino acids, and Z ¹ represents fluorescence resonance It represents a fluorescent group where energy transfer (FRET) occurs, and ^Z2 represents a quenching group corresponding to the fluorescent group.)
[8] An activity confirmation step of contacting a candidate protein of peptide-N-glycosidase (PNGase) or a fraction containing it with a sugar compound represented by the following formula (I) and confirming the decomposition activity of the sugar compound. A method for evaluating peptide-N-glycosidase (PNGase) activity.
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In formula (I), X ¹ _0-5 represents 0 to 5 same or different amino acids, X ² _1-5 represents 1 to 5 same or different amino acids, and Z ¹ represents fluorescence resonance It represents a fluorescent group where energy transfer (FRET) occurs, and ^Z2 represents a quenching group corresponding to the fluorescent group.)
[9] A contacting step of bringing the test compound into contact with peptide-N-glycosidase (PNGase), and adding a sugar compound represented by the following formula (I) to the peptide-N-glycosidase (PNGase) that has been contacted with the test compound. A method for screening a peptide-N-glycosidase (PNGase) activity inhibitor, comprising an activity confirmation step of contacting with the sugar compound to confirm the decomposition activity of the sugar compound.
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In formula (I), X ¹ _0-5 represents 0 to 5 same or different amino acids, X ² _1-5 represents 1 to 5 same or different amino acids, and Z ¹ represents fluorescence resonance It represents a fluorescent group where energy transfer (FRET) occurs, and ^Z2 represents a quenching group corresponding to the fluorescent group.)

According to the present invention, PNGase activity can be detected easily, and new PNGases, PNGase activity inhibitors, etc. can be efficiently screened.

These are the results of tracking the enzyme reaction performed in Example 2 using a microplate reader.

In explaining the details of the present invention, specific examples will be given. However, the present invention is not limited to the following content as long as it does not depart from the spirit of the present invention, and the present invention can be implemented with appropriate changes.

<Sugar compounds>
A sugar compound that is one embodiment of the present invention (hereinafter sometimes abbreviated as "sugar compound of the present invention") is a compound represented by the following formula (I).
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In formula (I), X ¹ _0-5 represents 0 to 5 identical or different amino acid sequences, X ² _1-5 represents 1 to 5 identical or different amino acid sequences, and Z ¹ It represents a fluorescent group in which fluorescence resonance energy transfer (FRET) occurs, and ^Z2 represents a quenching group corresponding to the fluorescent group.)

X ¹ _0-5 in formula (I) represents 0 to 5 identical or different amino acid sequences. The number is preferably 1 to 4, more preferably 2 to 3, and still more preferably 3. Further, specifically, it is preferably Gly-B ¹ -Gly, and B ¹ is preferably Ser or Thr.
Furthermore, X ² _1-5 in formula (I) represents 1 to 5 identical or different amino acid sequences. The number is preferably 2 to 4. Further, specifically, it is preferably (β-Ala)-Leu _1-4 , and the Leu _1-4 is preferably Leu ₃ .
In addition, GlcNAc is contained in two or more molecules in _the sugar compound of formula (I), and is endo-β-N- _{acetylglucosaminidase} (E
The compound represented by formula (I) is not recognized by NGase) and is not cleaved.
Furthermore, in GlcNAc ₂ -Z ¹ in formula (I), a spacer may be provided between GlcNAc ₂ and Z ¹ . Specifically, a high mannose type (Man ₉ ) spacer among N-linked sugar chains is preferred, and Z ¹ may be included at any tip of the branched mannose.

The present inventors have discovered that PNGase has the specificity of selectively cleaving a specific position in formula (I), and the fluorescence resonance energy transfer (FRET) occurs at the position separated by the cleavage. We synthesized a sugar compound with a group (donor) and a quenching group (acceptor) and confirmed that it can actually be used as a FRET probe. For example, the sugar compound in the reaction represented by the following formula has an N-methylanthranyl group as a fluorescent group at the Z ¹ position of formula (I) and a 2, quenching group at the Z ² position of the formula (I). It has a 4-dinitrophenyl group, and when the sugar chain is cleaved by PNGase, the distance between the fluorescent group and the quenching group increases, causing a change in the intensity of fluorescence from the fluorescent group, so PNGase activity can be detected.
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 → Z ² -X ² _1-5 -Asp-X ¹ _0-5 + GlcNAc ₂ -Z ¹
Note that "fluorescent group that causes fluorescence resonance energy transfer (FRET)" and "quenching group corresponding to the fluorescent group" are any combination of a fluorescent group and a quenching group that causes fluorescence resonance energy transfer (FRET). means.

The sugar compound of the present invention is a compound represented by the above formula (I), but the specific type is not particularly limited as long as it corresponds to formula (I), and can be selected as appropriate depending on the purpose of use etc. be able to.

（式（ｄ－１）中、Ｒ’は水素原子、炭素原子数１～６の炭化水素基、又はアミノ基の保護基を表す。） Z ¹ and Z ² represent ``one of which is a fluorescent group that causes fluorescence resonance energy transfer (FRET)'' and ``the other is a quenching group that corresponds to the fluorescent group,'' but the difference between the fluorescent group and the quenching group is Examples of the combination include structures described in "FRET SUBSTRATES" by Bachem et al. and Angew. Chem. Int. Ed. 2006, 45, 4562-4588. Among these, the following (i) to (iv) are preferred as combinations of fluorescent groups and quenching groups.
(i) A combination of a fluorescent group represented by the following formula (d-1) and a quenching group represented by the following formula (a-1)

(In formula (d-1), R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)

（式（ｄ－２）中、Ｒは水素原子又はヒドロキシル基の保護基を表す。） (ii) A combination of a fluorescent group represented by the following formula (d-2) and a quenching group represented by the following formula (a-1)

(In formula (d-2), R represents a hydrogen atom or a hydroxyl group protecting group.)

（式（ｄ－３）中、Ｒは水素原子又はヒドロキシル基の保護基を、Ｒ’は水素原子、炭素原子数１～６の炭化水素基、又はアミノ基の保護基を表す。） (iii) A combination of a fluorescent group represented by the following formula (d-3) and a quenching group represented by the following formula (a-1)

(In formula (d-3), R represents a hydrogen atom or a protecting group for a hydroxyl group, and R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)

（式（ｄ－４）及び（ａ－２）中、Ｒ’はそれぞれ独立して水素原子、炭素原子数１～６の炭化水素基、又はアミノ基の保護基を表す。） (iv) A combination of a fluorescent group represented by the following formula (d-4) and a quenching group represented by the following formula (a-2)

(In formulas (d-4) and (a-2), R' each independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)

(v) Combination of a fluorescent group that is carboxytetramethyl-rhodamine (TAMRA, registered trademark) and a quenching group that is BlackHole Quenchers (BHQ, registered trademark) In addition, TAMRA functions as a fluorescent group in combination with a quenching group that is BHQ. It may be a derivative thereof as long as it does so.
Examples of BHQ include BHQ1 (registered trademark), BHQ2 (registered trademark), and BHQ3 (registered trademark), and derivatives thereof may be used as long as they function as a quenching group in combination with the fluorescent group that is TAMRA. It's okay.

Note that R and R' in formulas (d-1) to (d-4) and formulas (a-1) and (a-2) include the following.

Each R is independently a "hydrogen atom" or a "hydroxyl group protecting group." As protecting groups for hydroxyl groups, ether protecting groups such as methyl group, benzyl group, p-methoxybenzyl group, tert-butyl group; acyl protecting groups such as acetyl group, pivaloyl group, benzoyl group; trimethylsilyl group, triethyl group, etc. Examples include silyl ether protecting groups such as a silyl group, a tert-butyldimethylsilyl group, a triisopropylsilyl group, and a tert-butyldiphenylsilyl group.

R' is each independently a "hydrogen atom," a "hydrocarbon group having 1 to 6 carbon atoms," or a "protecting group for an amino group."
"Hydrocarbon group" is not limited to a linear saturated hydrocarbon group, but also a group consisting only of carbon atoms and hydrogen atoms, which may have a carbon-carbon unsaturated bond, a branched structure, or a cyclic structure. shall mean.
Hydrocarbon groups include methyl group (-CH ₃ , -Me), ethyl group (-C ₂ H ₅ , -Et), n-propyl group (- ⁿ C ₃ H ₇ , - ⁿ Pr), i-propyl Group ( ^-i C ₃ H ₇ , - ⁱ Pr), n-butyl group (- ⁿ C ₄ H ₉ , - ⁿ Bu), t-butyl group (- ^t C ₄ H ₉ , - ^t Bu), n- Pentyl group (- ⁿ C ₅ H ₁₁ ), n-hexyl group (- ⁿ C ₆ H ₁₃ , - ⁿ Hex), cyclohexyl group (- ^c C ₆ H ₁₁ , -Cy), phenyl group (- C ₆ H ₅ , -Ph), etc.
Protecting groups for amino groups include t-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), 9-fluorenylmethyloxycarbonyl group (Fmoc), 2,2,2-trichloroethoxycarbonyl group (Troc). ), alkoxycarbonyl protecting groups such as allyloxycarbonyl group (Alloc); acyl protecting groups such as acetyl group and trifluoroacetyl group (Tfa); p-toluenesulfonyl group (Ts), 2-nitrobenzenesulfonyl group (Ns) ) and other alkyl(aryl)sulfonyl groups.

The method for producing the sugar compound of the present invention represented by the above formula (I) is not particularly limited, and can be produced, for example, as shown in Examples.

<Screening method for peptide-N-glycosidase (PNGase) activity inhibitor>
PNGase activity inhibitors are useful because they can be used to investigate the cause of NGLY1 disease. Since the sugar compound of the present invention allows PNGase activity to be easily detected, PNGase that has been brought into contact with a test compound is brought into contact with the sugar compound of the present invention to confirm the decomposition activity of the sugar compound of the present invention. By doing so, PNGase activity inhibitors can be efficiently screened. Note that there is a contact step in which the test compound is brought into contact with PNGase (hereinafter sometimes abbreviated as "contact step"), and a sugar compound represented by formula (I) is brought into contact with the PNGase that has been brought into contact with the test compound. Accordingly, a screening method for a PNGase activity inhibitor that includes an activity confirmation step (hereinafter sometimes abbreviated as "activity confirmation step") of confirming the decomposition activity of a sugar compound is also one aspect of the present invention.

The contact step is a step of bringing the test compound into contact with PNGase. The mass of the test compound to be contacted is usually 7.5 μg or more, preferably 15 μg or more, and usually 150 μg or less, preferably 75 μg or less, per 1 ng of PNGase.

The activity confirmation step is a step in which the sugar compound represented by formula (I) is brought into contact with PNGase that has been brought into contact with the test compound, and the decomposition activity of the sugar compound is confirmed. The mass of the sugar compound represented by formula (I) to be contacted is usually 3 μg or more, preferably 30 μg or more, and usually 500 μg or less, preferably 300 μg or less, per 1 ng of PNGase.

The method for confirming the decomposition activity of the sugar compound represented by formula (I) in the activity confirmation step is not particularly limited, but includes observing changes in the intensity of fluorescence emission based on the fluorescent groups of the sugar compound represented by formula (I). A method of observing the absorption wavelength of ultraviolet rays (UV) based on the quenching group of the sugar compound represented by formula (I) can be mentioned. For example, comparing the sugar compound decomposition activity of PNGase that is not in contact with the test compound and the sugar compound decomposition activity of PNGase that is in contact with the test compound, PNGase that is in contact with the test compound has a higher sugar compound decomposition activity. If the degrading activity of the test compound is inferior, it can be determined that the test compound has an inhibitory effect on PNGase activity (it is a PNGase activity inhibitor).

Specific examples of PNGase targeted by the screening method of the present invention include human cytoplasmic PNGase and PNGase that acts on glycoprotein sugar chains, such as PNGase-F derived from Flavobacterium.

<Screening method for peptide-N-glycosidase (PNGase)>
One aspect of the present invention provides an activity of contacting a candidate protein of peptide-N-glycosidase (PNGase) or a fraction containing the same with a sugar compound represented by formula (I) to confirm the degrading activity of the sugar compound. This is a screening method for peptide-N-glycosidase (PNGase), including a confirmation step.

Since the sugar compound of the present invention allows PNGase activity to be detected easily, the decomposition activity of the sugar compound of the present invention can be detected by contacting a PNGase candidate protein or a fraction containing it with the sugar compound of the present invention. By confirming this, new PNGases can be efficiently screened and PNGase activity can be evaluated.
In addition, for the aspects of the contact step and activity confirmation step in this embodiment, the aspects of the contact step and activity confirmation step in the above-mentioned <Screening method for peptide-N-glycosidase (PNGase) activity inhibitor> are used. However, as a method for confirming the decomposition activity of the sugar compound represented by formula (I) in the activity confirmation step, for example, the decomposition activity of the sugar compound of the PNGase candidate in contact with the test compound is measured, and compared to the control. If the PNGase exhibits significant activity, it can be determined that the PNGase is useful as a PNGase or is a new PNGase.

The present invention will be described in more detail with reference to Examples below, but changes can be made as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

化合物9 （10.17 g, 17.5 mmol）をジクロロメタン（30 mL）に溶解し、トリエチルシ
ラン（8.4 mL, 52.6 mmol）、ボロントリフルオリドエチルエーテルコンプレックス（2.22 mL, 17.5 mmol）を加えて-78℃で1時間撹拌し、0℃に昇温した。42時間撹拌したのち-40℃にてトリエチルアミン（3.6 mL, 26 mmol）を加えて反応を終了させた。酢酸エチルで希釈し、1M塩酸、飽和食塩水、飽和炭酸水素ナトリウム水溶液、飽和食塩水の順に洗浄した。有機層を硫酸マグネシウムで乾燥させ、溶媒を減圧留去した。得られた残渣をシリカゲルクロマトグラフィー（ヘキサン : 酢酸エチル= 100 : 1 ～ 4 : 1, v : v）を用いて精製し、化合物10 （8.98 g, 88%）を得た。
R_f =0.43（n-hexane : AcOEt = 1 : 1, v : v）
¹H-NMR （400 MHz, CHLOROFORM-D） δ 7.83-6.86 （m, 20H）, 5.58-5.50 （m, 1H）, 5.00-4.68 （m, 1H）, 4.63-4.42 （m, 3H）, 4.28-4.20 （m, 2H）, 3.87-3.77 （m, 3H
）, 3.71-3.66 （m, 1H）, 2.89 （d, J = 2.5 Hz, 1H）, 1.69-1.60 （m, 1H） <Example 1: Production of sugar compound represented by formula (I)>
(Synthesis of Phenyl-3,6-di-O-benzyl-2-deoxy-2-phtalimide-1-thio-β-D-glucopyranoside (Compound 10))

Compound 9 (10.17 g, 17.5 mmol) was dissolved in dichloromethane (30 mL), triethylsilane (8.4 mL, 52.6 mmol) and boron trifluoride ethyl ether complex (2.22 mL, 17.5 mmol) were added, and the mixture was incubated at -78°C. The mixture was stirred for an hour and the temperature was raised to 0°C. After stirring for 42 hours, triethylamine (3.6 mL, 26 mmol) was added at -40°C to terminate the reaction. The mixture was diluted with ethyl acetate and washed successively with 1M hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate solution, and saturated brine. The organic layer was dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified using silica gel chromatography (hexane:ethyl acetate = 100:1 to 4:1, v:v) to obtain compound 10 (8.98 g, 88%).
R _f =0.43 (n-hexane: AcOEt = 1: 1, v: v)
¹ H-NMR (400 MHz, CHLOROFORM-D) δ 7.83-6.86 (m, 20H), 5.58-5.50 (m, 1H), 5.00-4.68 (m, 1H), 4.63-4.42 (m, 3H), 4.28 -4.20 (m, 2H), 3.87-3.77 (m, 3H)
), 3.71-3.66 (m, 1H), 2.89 (d, J = 2.5 Hz, 1H), 1.69-1.60 (m, 1H)

化合物10 （1.05 g, 1.81 mmol）をジクロロメタン（20 mL）に溶解し、ピリジン（620
μL, 7.7 mmol）、トリフルオロメタンスルホン酸無水物（600 μL, 3.55 mmol）を加え、室温で60分撹拌した。飽和重曹水、クロロホルムで希釈し、飽和重曹水、飽和食塩水で順次洗浄した。硫酸マグネシウムで乾燥後、溶媒を減圧留去した。ジメチルホルムアミド（17 mL）に溶かし、硝酸ナトリウム(1.19 g, 17.2 mmol)を加え、室温で60分撹拌した。クロロホルムで希釈し、1 M塩酸、飽和重曹水、飽和食塩水の順で洗浄した。溶媒を減圧
留去した。得られた残渣をシリカゲルクロマトグラフィー（トルエン : 酢酸エチル=400 : 1 ～ 5 / 1 v : v）により精製し、化合物12 （701 mg, 2段階収率67%）を得た。
R_f =0.39（toluene : AcOEt = 5 : 1, v : v）
¹H-NMR （400 MHz, CHLOROFORM-D） δ 7.86-6.92 （m, 23H）, 5.51 （d, J = 10.5 Hz,
1H）, 4.63-4.52 （m, 4H）, 4.32-4.25 （m, 2H）, 4.20 （t, J = 2.4 Hz, 1H）, 3.86-3.80 （m, 3H）, 2.58 （d, J = 1.1 Hz, 1H）, 1.55 （s, 2H） (Synthesis of Phenyl-3,6-di-O-benzyl-2-deoxy-2-phtalimide-1-thio-β-D-galactopyranoside (Compound 12))

Compound 10 (1.05 g, 1.81 mmol) was dissolved in dichloromethane (20 mL) and pyridine (620
μL, 7.7 mmol) and trifluoromethanesulfonic anhydride (600 μL, 3.55 mmol) were added, and the mixture was stirred at room temperature for 60 minutes. The mixture was diluted with saturated aqueous sodium bicarbonate and chloroform, and washed successively with saturated aqueous sodium bicarbonate and saturated brine. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. It was dissolved in dimethylformamide (17 mL), added with sodium nitrate (1.19 g, 17.2 mmol), and stirred at room temperature for 60 minutes. The mixture was diluted with chloroform and washed with 1 M hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated saline in this order. The solvent was removed under reduced pressure. The obtained residue was purified by silica gel chromatography (toluene:ethyl acetate=400:1 to 5/1 v:v) to obtain compound 12 (701 mg, 2-step yield: 67%).
R _f =0.39 (toluene : AcOEt = 5 : 1, v : v)
¹ H-NMR (400 MHz, CHLOROFORM-D) δ 7.86-6.92 (m, 23H), 5.51 (d, J = 10.5 Hz,
1H), 4.63-4.52 (m, 4H), 4.32-4.25 (m, 2H), 4.20 (t, J = 2.4 Hz, 1H), 3.86-3.80 (m, 3H), 2.58 (d, J = 1.1 Hz) , 1H), 1.55 (s, 2H)

化合物12 （4.90 g, 8.42 mmol）をジクロロメタン（50 mL）に溶かし、ピリジン（3.1
mL, 37.9 mmol）、トリフルオロメタンスルホン酸無水物（2.20 mL, 13 mmol）を加えた。室温で60分撹拌したのち、飽和重曹水で希釈し反応を止めた。飽和重曹水、飽和食塩水の順で洗浄し、溶媒を減圧留去した。得られた残渣をトルエン（50 mL）に溶かし、テト
ラブチルアンモニウムアジド（3.59 g, 12.6 mmol）を加え、室温で2日間撹拌した。酢酸エチルで希釈し、飽和重曹水、飽和食塩水の順で洗浄した。得られた残渣をシリカゲルクロマトグラフィー（ヘキサン : 酢酸エチル＝5 : 1, v : v）にて精製し、化合物5 （4.32 g, 84%）を得た。
R_f =0.55（toluene : AcOEt = 10 : 1, v : v）
¹H-NMR （400 MHz, CHLOROFORM-D） δ7.80-7.15 （m, 15H）, 7.01-6.83 （m, 5H）, 5.49-5.43 （m, 1H）, 4.81 （dd, J = 11.8, 4.7 Hz, 1H）, 4.65 （dd, J = 12.0, 4.7 Hz, 1H）, 4.57 （dd, J = 11.8, 4.7 Hz, 1H）, 4.39 （dd, J = 11.9, 4.6 Hz, 1H）, 4
.27-4.23 （m, 2H）, 3.84-3.75 （m, 3H）, 3.53 （ddd, J = 10.2, 4.1, 1.7 Hz, 1H）, 1.59-1.54 （m, 2H） (Synthesis of Phenyl-4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phtalimide-1-thio-β-D-glucopyranoside (Compound 5))

Compound 12 (4.90 g, 8.42 mmol) was dissolved in dichloromethane (50 mL) and pyridine (3.1
mL, 37.9 mmol) and trifluoromethanesulfonic anhydride (2.20 mL, 13 mmol) were added. After stirring at room temperature for 60 minutes, the reaction was stopped by diluting with saturated aqueous sodium bicarbonate solution. The mixture was washed with saturated sodium bicarbonate solution and saturated brine in that order, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in toluene (50 mL), tetrabutylammonium azide (3.59 g, 12.6 mmol) was added, and the mixture was stirred at room temperature for 2 days. The mixture was diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate and saturated brine in that order. The obtained residue was purified by silica gel chromatography (hexane:ethyl acetate=5:1, v:v) to obtain Compound 5 (4.32 g, 84%).
R _f =0.55 (toluene : AcOEt = 10 : 1, v : v)
¹ H-NMR (400 MHz, CHLOROFORM-D) δ7.80-7.15 (m, 15H), 7.01-6.83 (m, 5H), 5.49-5.43 (m, 1H), 4.81 (dd, J = 11.8, 4.7 Hz, 1H), 4.65 (dd, J = 12.0, 4.7 Hz, 1H), 4.57 (dd, J = 11.8, 4.7 Hz, 1H), 4.39 (dd, J = 11.9, 4.6 Hz, 1H), 4
.27-4.23 (m, 2H), 3.84-3.75 (m, 3H), 3.53 (ddd, J = 10.2, 4.1, 1.7 Hz, 1H), 1.59-1.54 (m, 2H)

化合物5 （720 mg, 1.24 mmol）、化合物6 （836 mg, 1.38 mmol）、N-ヨードスクシンイミド（461 mg, 2.05 mmol）を乾燥モレキュラーシーブズ4Å雰囲気下、ジクロロメタン（12.4 mL）に溶かした。-78℃にて、トリフルオロメタンスルホン酸（60.7 μL, 683 μL）を加え、2時間撹拌した。TLCにて反応の終了を確認し、トリエチルアミンを加えて反
応を停止した。反応液を酢酸エチルで希釈し、チオ硫酸ナトリウム、飽和炭酸水素ナトリウム水溶液、飽和食塩水にて順次洗浄した。有機層を硫酸マグネシウムによって乾燥後、溶媒を減圧留去した。得られた残渣をシリカゲルクロマトグラフィー（ヘキサン : 酢酸
エチル=20 : 1 ～ 1 : 1）および再結晶（熱イソプロパノール）にて精製し、化合物4 （993 mg, 74%）を得た。
R_f =0.43（toluene : EcOEt = 8 : 1, v : v）
¹H NMR （600 MHz, CDCl₃）:δ8.25-6.73 （m, 48H; aromatic H）, 5.22 （d, J = 7.9 Hz, 1H, H-1b）, 4.91 （d, J = 8.6 Hz, 1H, H-1b）, 4.81（dd, J = 23.0 Hz, 2H, CH₂-Ph）, 4.67 （d, J = 12.4 Hz, 1H, CH₂-Ph）, 4.54 （s, 2H, CH₂-Ph）, 4.48 （s, 2H, CH₂-Ph）, 4.45 （d, J = 12.7 Hz, 1H, CH₂-Ph）, 4.40 （d, J = 12.0 Hz, 1H, CH₂-Ph）, 4.34 （d, J = 12.4 Hz, 1H, CH₂-Ph）, 4.23-4.08 （m, 5H, H-2a, H-3a, H-4a, H-2b, H-3b）, 3.83-3.80 （t, 1H, H-4b） 3.68 （d, J = 11.0 Hz, 1H, H-6b）, 3.59 （dd, J = 11.3, 3.4 Hz, 1H,H-6’b）, 3.54 （d, J = 10.3 Hz, 1H, H-6a）, 3.41 （dd, J = 11.0, 3.8 Hz, 1H,H-6’a）,3.28 （dd, J = 9.8, 2.6 Hz, 1H, H-5a）,3.13 （dd, J = 10.3, 1.7 Hz, 1H, H-5b）ppm,
¹³C NMR （150 MHz, CDCl₃） δ 128.27-127.26（aromatic C）, 97.15 （C-1b）, 96.92
（C-1a）, 78.15 （C-3b）, 77.32 （CH₂-Ph）, 77.11 （CH₂-Ph）, 76.89 （CH₂-Ph）,
76.65 （C-3a）, 75.72 （C-4a）, 74.83 （CH₂-Ph）, 74.52（C-5a）, 74.39（C-5b）,
74.24 （CH₂-Ph）, 73.35 （CH₂-Ph）, 72.79 （CH₂-Ph）, 70.57 （CH₂-Ph）, 68.37
（C-6b）, 68.22 （C-6a）, 63.52 （C-4b）, 56.54 （C-2b）, 55.77（C-2a）ppm. (Benzyl-4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phtalimide-β-D-glucopyranoside-(1-4)-3,6-di-O-benzyl -2-deoxy-2-phtalimide-β-D-glucopyranoside (compound 4) synthesis)

Compound 5 (720 mg, 1.24 mmol), compound 6 (836 mg, 1.38 mmol), and N-iodosuccinimide (461 mg, 2.05 mmol) were dissolved in dichloromethane (12.4 mL) under an atmosphere of 4 Å dry molecular sieves. At -78°C, trifluoromethanesulfonic acid (60.7 μL, 683 μL) was added and stirred for 2 hours. Completion of the reaction was confirmed by TLC, and triethylamine was added to stop the reaction. The reaction solution was diluted with ethyl acetate and washed successively with sodium thiosulfate, saturated aqueous sodium bicarbonate solution, and saturated brine. After drying the organic layer with magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel chromatography (hexane:ethyl acetate=20:1 to 1:1) and recrystallization (hot isopropanol) to obtain Compound 4 (993 mg, 74%).
R _f =0.43 (toluene : EcOEt = 8 : 1, v : v)
¹ H NMR (600 MHz, CDCl ₃ ): δ8.25-6.73 (m, 48H; aromatic H), 5.22 (d, J = 7.9 Hz, 1H, H-1b), 4.91 (d, J = 8.6 Hz, 1H, H-1b), 4.81 (dd, J = 23.0 Hz, 2H, CH ₂ -Ph), 4.67 (d, J = 12.4 Hz, 1H, CH ₂ -Ph), 4.54 (s, 2H, CH ₂ - Ph), 4.48 (s, 2H, CH ₂ -Ph), 4.45 (d, J = 12.7 Hz, 1H, CH ₂ -Ph), 4.40 (d, J = 12.0 Hz, 1H, CH ₂ -Ph), 4.34 (d, J = 12.4 Hz, 1H, CH ₂ -Ph), 4.23-4.08 (m, 5H, H-2a, H-3a, H-4a, H-2b, H-3b), 3.83-3.80 (t , 1H, H-4b) 3.68 (d, J = 11.0 Hz, 1H, H-6b), 3.59 (dd, J = 11.3, 3.4 Hz, 1H,H-6'b), 3.54 (d, J = 10.3 Hz, 1H, H-6a), 3.41 (dd, J = 11.0, 3.8 Hz, 1H,H-6'a),3.28 (dd, J = 9.8, 2.6 Hz, 1H, H-5a),3.13 (dd , J = 10.3, 1.7 Hz, 1H, H-5b) ppm,
¹³ C NMR (150 MHz, CDCl ₃ ) δ 128.27-127.26 (aromatic C), 97.15 (C-1b), 96.92
(C-1a), 78.15 (C-3b), 77.32 (CH ₂ -Ph), 77.11 (CH ₂ -Ph), 76.89 (CH ₂ -Ph),
76.65 (C-3a), 75.72 (C-4a), 74.83 (CH ₂ -Ph), 74.52 (C-5a), 74.39 (C-5b),
74.24 (CH ₂ -Ph), 73.35 (CH ₂ -Ph), 72.79 (CH ₂ -Ph), 70.57 (CH ₂ -Ph), 68.37
(C-6b), 68.22 (C-6a), 63.52 (C-4b), 56.54 (C-2b), 55.77 (C-2a) ppm.

化合物4（246 mg, 229 μmol）をn-ブタノール（5 mL）に溶解し、エチレンジアミン（1.5 mL）を加え、90℃にて45時間撹拌したのち、溶媒を減圧留去した。得られた残渣の水分をトルエンと共沸させた後、ピリジン（10 mL）に溶解し、無水酢酸（5 mL）を加えた
。40℃にて18時間撹拌したのち、メタノール（10 mL）を加えて反応を終了させた。クロ
ロホルムで希釈し、1M塩酸、飽和食塩水、飽和炭酸水素ナトリウム水溶液、飽和食塩水の順に洗浄した。有機層を硫酸マグネシウムで乾燥させ、溶媒を減圧留去した。得られた残渣をシリカゲルクロマトグラフィー（クロロホルム : メタノール＝30 : 1, v : v）を用いて精製し、化合物13 （157 mg, 2段階収率76%）を得た。
R_f=0.51（chloroform : methanol = 15 : 1, v : v）
¹H-NMR （400 MHz, CHLOROFORM-D） δ 7.39-7.20 （m, 30H）, 6.19 （d, J = 8.7 Hz, 1H）, 4.94 （d, J = 8.5 Hz, 1H）, 4.82 （d, J = 11.9 Hz, 2H）, 4.70-4.36 （m, 10H）, 4.08-4.04 （m, 1H）, 3.95 （t, J = 5.3 Hz, 1H）, 3.81-3.58 （m, 8H）, 3.47 （t, J = 9.8 Hz, 1H）, 3.13-3.09 （m, 1H）, 1.92 （d, J = 12.6 Hz, 3H）, 1.76-1.69 （m, 3H）, 1.64 （d, J = 2.7 Hz, 2H） (Benzyl-2-acetamide-4-azide-3,6-di-O-benzyl-2,4-di-deoxy-β-D-glucopyranosyl-(1-4)-2-acetamide-3.6-di-O -Synthesis of benzyl-2-deoxy-β-D-glucopyranoside (compound 13))

Compound 4 (246 mg, 229 μmol) was dissolved in n-butanol (5 mL), ethylenediamine (1.5 mL) was added, and the mixture was stirred at 90°C for 45 hours, and then the solvent was distilled off under reduced pressure. After the water in the obtained residue was azeotroped with toluene, it was dissolved in pyridine (10 mL), and acetic anhydride (5 mL) was added. After stirring at 40°C for 18 hours, methanol (10 mL) was added to terminate the reaction. It was diluted with chloroform and washed successively with 1M hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate solution, and saturated brine. The organic layer was dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified using silica gel chromatography (chloroform:methanol=30:1, v:v) to obtain compound 13 (157 mg, 2-step yield: 76%).
R _f =0.51 (chloroform : methanol = 15 : 1, v : v)
¹ H-NMR (400 MHz, CHLOROFORM-D) δ 7.39-7.20 (m, 30H), 6.19 (d, J = 8.7 Hz, 1H), 4.94 (d, J = 8.5 Hz, 1H), 4.82 (d, J = 11.9 Hz, 2H), 4.70-4.36 (m, 10H), 4.08-4.04 (m, 1H), 3.95 (t, J = 5.3 Hz, 1H), 3.81-3.58 (m, 8H), 3.47 (t , J = 9.8 Hz, 1H), 3.13-3.09 (m, 1H), 1.92 (d, J = 12.6 Hz, 3H), 1.76-1.69 (m, 3H), 1.64 (d, J = 2.7 Hz, 2H)

化合物13 （98.1 mg, 109 μmol）を水（18 mL）、tert-ブタノール（18 mL）に溶解し、水酸化パラジウム炭素を加え、水素雰囲気下で40℃にて2日間撹拌した。セライトろ過
後、溶媒を減圧留去した。得られた残渣を逆相カラムクロマトグラフィー（milliQ水）によって精製し、化合物14 （31.04 mg, 64%）を得た。
¹H-NMR （400 MHz, D₂O） δ 5.00 （d, J = 2.2 Hz, 1H）, 4.39-4.36 （m, 1H）, 3.73-3.16 （m, 15H）, 2.56 （t, J = 9.5 Hz, 1H）, 1.90-1.85 （m, 6H） (Synthesis of 4-acetamide-2-amino-2,4-di-deoxy-β-D-glucopyranosyl-(1-4)-2-acetamide-2-deoxy-D-glucopyranose (compound 14))

Compound 13 (98.1 mg, 109 μmol) was dissolved in water (18 mL) and tert-butanol (18 mL), palladium hydroxide on carbon was added, and the mixture was stirred at 40° C. for 2 days under a hydrogen atmosphere. After filtering through Celite, the solvent was distilled off under reduced pressure. The obtained residue was purified by reverse phase column chromatography (milliQ water) to obtain Compound 14 (31.04 mg, 64%).
¹ H-NMR (400 MHz, D ₂ O) δ 5.00 (d, J = 2.2 Hz, 1H), 4.39-4.36 (m, 1H), 3.73-3.16 (m, 15H), 2.56 (t, J = 9.5 Hz, 1H), 1.90-1.85 (m, 6H)

化合物16（20.0 μL, 181 μmol）を1,4-ジオキサン（500 μL）に溶解させ、ペンタフルオロフェノール（56.6 mg, 308 μmol）を加えたのち、0℃でジシクロヘキシルカルボ
ジイミド（56.4 mg, 273 μmol）を加え、室温で2時間反応させた。析出した副生成物を
ろ別したのち、精製せずに次の反応に用いた。 (Synthesis of perfluorophenyl hex-5-ynoate (compound 17))

Compound 16 (20.0 μL, 181 μmol) was dissolved in 1,4-dioxane (500 μL), pentafluorophenol (56.6 mg, 308 μmol) was added, and dicyclohexylcarbodiimide (56.4 mg, 273 μmol) was added at 0°C. was added and allowed to react at room temperature for 2 hours. After filtering out the precipitated by-product, it was used in the next reaction without purification.

化合物14（10.0 mg, 23.6 μmol）をジメチルスルホキシドに溶解し、活性エステル（19.7 mg, 70.9 μmol）、飽和重曹水（50 μL）を加え、4℃に2日間静置し、凍結反応させたのち、酢酸エチルで3回洗浄した。逆相カラムクロマトグラフィー（milliQ水/メタノール）によって精製し、化合物15（8.80 mg, 72%）を得た。
¹H-NMR （400 MHz, D₂O） δ 5.01 （d, J = 3.1 Hz, 1H）, 4.53-4.51 （m, 0H）, 4.42
（dd, J = 8.4, 3.6 Hz, 1H）, 3.75-3.32 （m, 13H）, 2.56 （s, 4H）, 2.24 （t, J = 7.4 Hz, 2H）, 2.06 （td, J = 7.0, 5.0 Hz, 2H）, 1.87 （d, J = 12.6 Hz, 7H）, 1.67-1.56 （m, 2H） (Synthesis of 2-acetamide-2,4-di-deoxy-4-hexiolylamino-β-D-glucopyranosyl-(1-4)-2-acetamide-2-deoxy-D-glucopyranose (compound 15))

Compound 14 (10.0 mg, 23.6 μmol) was dissolved in dimethyl sulfoxide, active ester (19.7 mg, 70.9 μmol) and saturated sodium bicarbonate solution (50 μL) were added, and the mixture was allowed to stand at 4°C for 2 days to undergo a freezing reaction. , and washed three times with ethyl acetate. Purification was performed by reverse phase column chromatography (milliQ water/methanol) to obtain Compound 15 (8.80 mg, 72%).
¹ H-NMR (400 MHz, D ₂ O) δ 5.01 (d, J = 3.1 Hz, 1H), 4.53-4.51 (m, 0H), 4.42
(dd, J = 8.4, 3.6 Hz, 1H), 3.75-3.32 (m, 13H), 2.56 (s, 4H), 2.24 (t, J = 7.4 Hz, 2H), 2.06 (td, J = 7.0, 5.0 Hz, 2H), 1.87 (d, J = 12.6 Hz, 7H), 1.67-1.56 (m, 2H)

化合物18（5.01 g, 12.2 mmol）を1,4-ジオキサン（50 mL）、ジメチルホルムアミド（2 mL）に溶解させ、ペンタフルオロフェノール（2.70 g, 14.7 mmol）を加え、0℃でジシクロヘキシルカルボジイミド（3.02 g, 14.6 mmol）を加え、室温で2時間反応させた。析出した副生成物をろ別したのち、再結晶（ヘキサン / 酢酸エチル = 3 / 1）で精製し、
化合物19（4.42 g, 63 %）を得た。 (Synthesis of 1-(tert-butyl) 4-(perfluorophenyl) (((9H-fluoren-9-yl)methoxy)carbonyl)aspartate (compound 19))

Compound 18 (5.01 g, 12.2 mmol) was dissolved in 1,4-dioxane (50 mL) and dimethylformamide (2 mL), pentafluorophenol (2.70 g, 14.7 mmol) was added, and dicyclohexylcarbodiimide (3.02 mmol) was dissolved at 0 °C. g, 14.6 mmol) and allowed to react at room temperature for 2 hours. After filtering out the precipitated by-products, it was purified by recrystallization (hexane/ethyl acetate = 3/1).
Compound 19 (4.42 g, 63%) was obtained.

化合物15 （7.11 mg, 13.7 μmol）を飽和炭酸水素アンモニウム水溶液 （2 mL）に溶
解させ、45℃で24時間撹拌した。溶液を水で希釈し、溶媒が半分になるまで減圧蒸留することを5回繰り返し、炭酸水素アンモニウムを除き、凍結乾燥した。残渣をジメチルスル
ホキシド（250 μL）に溶解させ、ジイソプロピルエチルアミン（3.2 μL, 18.3 μmol）、Fmoc-Asp（PFP）-OtBu（17.6 mg, 30.4 μmol）を加えて-70℃にて２時間静置して凍結反応させた。酢酸エチルで洗浄し、凍結乾燥したのち、得られた残渣を逆相カラムクロマ
トグラフィー（milliQ水 / アセトニトリル＝100 / 0～50 / 50）にて精製し、化合物2
（6.35 mg, 6.98 μmol, 46%）を得た。
¹H-NMR （400 MHz, DMSO-D6） δ7.89-7.80 （m, 5H）, 7.66 （d, J = 7.3 Hz, 2H）, 7.45-7.26 （m, 6H）, 4.81-4.74 （m, 1H）, 4.37 （d, J = 8.2 Hz, 1H）, 4.29-4.14
（m, 5H）, 3.25-3.20 （m, 1H）, 2.74 （t, J = 2.5 Hz, 1H）, 2.63-2.37 （m, 16H）, 2.16-2.09 （m, 3H）, 1.83-1.71 （m, 7H）, 1.65-1.58 （m, 2H）, 1.34-1.31 （m, 11H）, 1.18-1.10 （m, 2H） (Synthesis of 2-acetamide-2,4-di-deoxy-4-hexiolylamino-β-D-glucopyranosyl-(1-4)-2-acetamide-2-deoxy-D-glucopyranosyl-L-asparagine (compound 2) )

Compound 15 (7.11 mg, 13.7 μmol) was dissolved in a saturated aqueous ammonium bicarbonate solution (2 mL) and stirred at 45°C for 24 hours. The solution was diluted with water and distilled under reduced pressure until the solvent was reduced to half, which was repeated 5 times to remove ammonium bicarbonate and freeze-dry. The residue was dissolved in dimethyl sulfoxide (250 μL), diisopropylethylamine (3.2 μL, 18.3 μmol) and Fmoc-Asp(PFP)-OtBu (17.6 mg, 30.4 μmol) were added, and the mixture was left standing at -70°C for 2 hours. The cells were frozen and reacted. After washing with ethyl acetate and freeze-drying, the resulting residue was purified by reverse phase column chromatography (milliQ water/acetonitrile = 100/0 to 50/50) to obtain compound 2.
(6.35 mg, 6.98 μmol, 46%) was obtained.
¹ H-NMR (400 MHz, DMSO-D6) δ7.89-7.80 (m, 5H), 7.66 (d, J = 7.3 Hz, 2H), 7.45-7.26 (m, 6H), 4.81-4.74 (m, 1H), 4.37 (d, J = 8.2 Hz, 1H), 4.29-4.14
(m, 5H), 3.25-3.20 (m, 1H), 2.74 (t, J = 2.5 Hz, 1H), 2.63-2.37 (m, 16H), 2.16-2.09 (m, 3H), 1.83-1.71 (m , 7H), 1.65-1.58 (m, 2H), 1.34-1.31 (m, 11H), 1.18-1.10 (m, 2H)

(Synthesis of sugar compound 1)
Synthesis of Peptide The peptide ((β-Ala)-Leu-Leu-Leu-(GlcNAc ₂ -Z ¹ )Asn-Gly-Thr-Gly) was synthesized according to the general Fmoc solid phase synthesis method. Fmoc amino acid, O-(1H-benzoriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole monohydrate (HOBt・H ₂ O), N,N-diisopropylethylamine (DIEA) ), 1-methyl-2-pyrrolidone (NMP), and piperidine were purchased from Watanabe Chemical. (7-Azabenzotriazol-1-yloxy)tripyrrolidinoophosphonium hexafluorophosphonate (PyAOP) was purchased from Carboshynth. 1-hydroxy-7-azabenzotriazole (HOAt) was purchased from Arc Pharm.
40 mg (20 μmol) of H-Rink amide ChemMatrix Resin (Sigma Aldrich; 0.5 mmol/g) was weighed, and an appropriate amount of NMP was added to the synthesis column to swell it. NMP was removed by filtration. Fmoc-Gly-OH (3 eq.), HBTU (3 eq.), HOBt・H ₂ O (3 eq.), and DIEA (6 eq.) were dissolved in NMP (0.4 mL) and applied to the synthesis column. Added. The mixture was stirred by vortexing at appropriate times and allowed to react for about 40 minutes. The progress of the reaction was determined by the Kaiser test, and if positive, the coupling reaction was performed again. If the Kaiser test was negative, the Fmoc group was deprotected. An appropriate amount of 20% piperidine/NMP solution was added, stirred by vortexing at appropriate times, and filtered off after 1 minute. An appropriate amount of 20% piperidine/NMP solution was added, and the mixture was stirred by vortexing at appropriate times. After reacting for 15 minutes, it was washed 5 times with NMP. Fmoc-Thr(tBu)-OH was coupled in the same manner as above. Similarly, after introducing Fmoc-Gly-OH, the resin was divided into halves. For this half amount of resin (10 μmol),
Compound 2 (1.4 eq.), PyAOP (4 eq.), HOAt (4 eq.), 2,4,6-trimethylpyridine (5.4 eq.)
was dissolved in NMP (0.1 mL) and added to the synthesis column. The mixture was stirred at appropriate times in an incubator at 40°C and allowed to react for 1 hour and 20 minutes. Although the Kaiser test was slightly positive, we proceeded to the next residue. Fmoc-Leu-OH was performed similarly to the Fmoc-Gly-OH coupling described above. This was repeated three times to extend three leucine residues. Following similar coupling of Fmoc-(β-Ala)-OH, the Fmoc group was deprotected.

Introduction of BHQ Add a solution of BHQ-2 Carboxylic Acid (1 eq.), PyAOP (3 eq.), HOAt (3 eq.), and DIEA (4 eq.) in NMP (0.1 mL). , and reacted at 40°C for 1 hour. After washing with NMP, it was washed with dichloromethane and dried in a desiccator. Approximately 0.5 mL of a mixed solution of 95% (v/v) trifluoroacetic acid (TFA), 2.5% (v/v) triisopropyl silane, and 2.5% (v/v) water was added to the dried resin and stirred at room temperature. . After 2 hours, the solution was collected and added to about 20 mL of diethyl ether to cause precipitation. The precipitate was collected by centrifugation, washed three times with diethyl ether, and centrifuged repeatedly. The thus obtained crude peptide into which BHQ-2 had been introduced was dried in a desiccator.

Introduction of TAMRA
The TAMRA group was introduced as follows. Tetramethylrhodamine 5-carboxyamido-(6-azidohexanyl)), 5-isomer (approximately 0.9 μmol) was dissolved in trifluoroethanol (TFE; 400 μL), and BHQ-2-introduced crude peptide (approximately 0.4 mg, 0.23 μmol) was added. and dissolved. Here, 50
20 μL of an mM copper (II) sulfate aqueous solution and 100 μL of a 200 mM sodium ascorbate aqueous solution were sequentially added. After about 2 hours, high performance liquid chromatography (HPLC; Shimadzu LC-10Avp system), Cosmosil
Analysis was performed using a C18 AR-II (Nacalai Tesque, 4.6 mm x 150 mm) column to confirm completion of the reaction. Purification was performed using a Cosmosil C18 AR-II (10 mm x 250 mm) column. The TAMRA-introduced sugar compound 1 thus obtained was freeze-dried.

<Example 2: Detection of peptide N-glycanase (PNGase) activity>
30 μL of probe sugar compound 1 solution (1.0 μM, 2.5 μM, 1.0 μM), Flavobacterium-derived PNGase-F (manufactured by BioLabs) 2 μL (1.25 U/μL), 4 μL GlycoBuffer, 4 μL Np- A final 50 μL of reaction mixture was prepared using 40 and water, and changes in fluorescence intensity ((ex)546 nm, (em)579 nm) of the reaction mixture were measured using a microplate reader (Molecular Devices). The tracking results are shown in Figure 1. As the enzymatic reaction progressed, the fluorescence intensity increased, confirming the cleavage of the substrate.
Note that this hydrolysis reaction is the reaction shown below. However, in the formula below, Z ¹ is TAMRA
and Z ² represents BHQ-2. )
Z ² -(β-Ala)-Leu-Leu-Leu-(GlcNAc ₂ -Z ¹ )Asn-Gly-Thr-Gly
→ GlcNAc ₂ -Z ¹ + Z ² -(β-Ala)-Leu-Leu-Leu-Asn-Gly-Thr-Gly

The sugar compound of the present invention can be used for searching for novel PNGases and efficiently screening for PNGase activity inhibitors.

Claims (6)

A sugar compound represented by the following formula (I).
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In the formula (I), X ¹ _0-5 represents Gly-Thr-Gly , X ² _1-5 represents (β-Ala)-Leu ₃ , and Z ¹ represents fluorescence resonance energy transfer (FRET). represents a fluorescent group, and ^Z2 represents a quenching group corresponding to the fluorescent group.) The sugar compound according to claim 1 , wherein the combination of the fluorescent group and the quenching group is any of the following (i) to (iv).
(i) A combination of a fluorescent group represented by the following formula (d-1) and a quenching group represented by the following formula (a-1)

(In formula (d-1), R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)
(ii) A combination of a fluorescent group represented by the following formula (d-2) and a quenching group represented by the following formula (a-1)

(In formula (d-2), R represents a hydrogen atom or a hydroxyl group protecting group.)
(iii) A combination of a fluorescent group represented by the following formula (d-3) and a quenching group represented by the following formula (a-1)

(In formula (d-3), R represents a hydrogen atom or a protecting group for a hydroxyl group, and R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)
(iv) A combination of a fluorescent group represented by the following formula (d-4) and a quenching group represented by the following formula (a-2)

(In formulas (d-4) and (a-2), R' each independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)
(v) Combination of a fluorescent group that is TAMRA (registered trademark) and a quenching group that is BHQ (registered trademark) A composition for detecting peptide-N-glycosidase (PNGase) activity, comprising the sugar compound according to claim 1 or 2 . A peptide comprising an activity confirmation step of contacting a candidate protein of peptide-N-glycosidase (PNGase) or a fraction containing the same with a sugar compound represented by the following formula (I) and confirming the degrading activity of the sugar compound. -N-glycosidase (PNGase) screening method.
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In the formula (I), X ¹ _0-5 represents Gly-Thr-Gly , X ² _1-5 represents (β-Ala)-Leu ₃ , and Z ¹ represents fluorescence resonance energy transfer (FRET). represents a fluorescent group, and ^Z2 represents a quenching group corresponding to the fluorescent group.) A peptide comprising an activity confirmation step of contacting a candidate protein of peptide-N-glycosidase (PNGase) or a fraction containing the same with a sugar compound represented by the following formula (I) and confirming the degrading activity of the sugar compound. -A method for evaluating N-glycosidase (PNGase) activity.
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In the formula (I), X ¹ _0-5 represents Gly-Thr-Gly , X ² _1-5 represents (β-Ala)-Leu ₃ , and Z ¹ represents fluorescence resonance energy transfer (FRET). represents a fluorescent group, and ^Z2 represents a quenching group corresponding to the fluorescent group.) a contacting step of bringing the test compound into contact with peptide-N-glycosidase (PNGase); and contacting the peptide-N-glycosidase (PNGase) with which the test compound has been contacted with a sugar compound represented by the following formula (I). A screening method for a peptide-N-glycosidase (PNGase) activity inhibitor, comprising an activity confirmation step of confirming the decomposition activity of the sugar compound.
Z ² -X ² _1-5 -(GlcNAc ₂ -Z ¹ )Asn-X ¹ _0-5 ...(I)
(In the formula (I), X ¹ _0-5 represents Gly-Thr-Gly , X ² _1-5 represents (β-Ala)-Leu ₃ , and Z ¹ represents fluorescence resonance energy transfer (FRET). represents a fluorescent group, and ^Z2 represents a quenching group corresponding to the fluorescent group.)

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