Synthesis of a glycosylated peptide thioester by the Boc strategy and its application to segment condensation

Biosci. Biotechnol. Biochem., 70 (6), 1338–1349, 2006

Synthesis of a Glycosylated Peptide Thioester by the Boc Strategy

and Its Application to Segment Condensation

y

Eiichiro HAGINOYA,¹Hironobu HOJO,^1;Yuko NAKAHARA,¹Yoshiaki NAKAHARA,^1;

4

Kazuki NABESHIMA,²Bryan P. TOOLE,³and Yasushi WATANABE

¹Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University,

Kanagawa 259-1292, Japan

²Fukuoka University School of Medicine, Fukuoka 814-0180, Japan

³The Medical University of South Carolina, Charleston, SC, USA

⁴National Food Research Institute, Ibaraki 305-8642, Japan

Received November 18, 2005; Accepted January 19, 2006; Online Publication, June 23, 2006

[doi:10.1271/bbb.50621]

The synthesis of a chitobiosylated peptide thioester by

the t-butoxycarbonyl (Boc) strategy is demonstrated.

Boc-Asn carrying benzyl-protected chitobiose was in-

troduced during application of the Boc mode solid-

phase method. HF treatment of the resulting protected

peptide resin gave the desired chitobiosylated peptide

thioester. This thioester was used to prepare the peptide

sequence derived from extracellular matrix metallopro-

teinase inducers (emmprin) (34-94), (34-118) and (22-

118) by the thioester segment condensation method. The

conformation of these glycopeptides is characterized by

circular dichroism (CD) spectral measurement.

by a stronger acid treatment such as with HF. Increasing

interest in such post-translational modiﬁcation as gly-

cosylation has resulted in the development of thioester

preparation by the Fmoc strategy which does not use a

harsh acid treatment for the deprotection step.^5–16)These

methods have been successfully used to prepare pep-

tide thioesters carrying an O-linked single N-acetylga-

lactosamine,⁷⁾seven N-acetylgalactosamines^17,18)and

N-linked core pentasaccharide.^14,15)However, the Fmoc

strategy does not always give satisfactory results. As an

extreme case, Marcaurelle et al. have reported that they

could not obtain a peptide thioester by the Fmoc strategy

when using the safety catch linker because of failure of

thiolysis at the ﬁnal step, whereas the Boc strategy

successfully gave the desired product.¹⁹⁾Thus, depend-

ing on the peptide sequence, the Boc strategy is more

reliable than Fmoc strategy. However, due to the

sensitivity of glycosidic linkages to strong acids, the

Boc strategy has seldom been applied to the preparation

of glycosylated peptide thioesters.^20–23)Its applicability

and limitations still remain unclear. We have brieﬂy

reported the preparation of a chitobiosylated thioester by

the Boc strategy and its application to the synthesis of

the extracellular matrix metalloproteinase inducer,

emmprin^24,25)(34-94), by the thioester method.²³⁾We

report here the full experimental details. In addition, the

glycosylated peptide thioester was further applied to the

synthesis of the larger glycopeptides, emmprin (34-118)

4 as well as (22-118) 5 by using repeated segment

coupling by the thioester method (Fig. 1). The eﬀect of

chain length on the conformation was analyzed by CD

spectral measurement of these glycopeptides.

Key words: glycosylated peptide thioester; solid-phase

synthesis; segment condensation

Peptide thioesters have been widely used as key

intermediates for the preparation of polypeptides by

segment condensation strategies such as the thioester

method^1,2)and native chemical ligation method.^3,4)The

former method involves the thioester group of the N-

terminal partially-protected peptide being activated by

silver ions and the amide bond formed with the C-

terminal peptide. The latter uses the thioesteriﬁcation

reaction between the thioester group of the N-terminal

peptide and the thiol group of the terminal cysteine

residue of the other peptide, this being followed by the

intramolecular S to N acyl migration reaction to form a

native peptide bond. The preparation of peptide thio-

esters has been mainly carried out by the Boc strategy, in

which the peptide chain is elongated by repetitive acid

treatment to remove the Boc group and then deprotected

y

To whom correspondence should be addressed. Tel: +81-463-58-1211 ex. 4173; Fax: +81-463-50-2075; E-mail: hojo@keyaki.cc.u-tokai.ac.jp

or yonak@keyaki.cc.u-tokai.ac.jp

Abbreviations: Acm, acetamidomethyl; Boc, t-butoxycarbonyl; Boc-OSu, N-(t-butoxycarbonyloxy) succinimide; CD, circular dichroism; DCC,

1,3-dicyclohexylcarbodiimide; DIEA, N,N-diisopropylethylamine; DTT, dithiothreitol; Fmoc, 9-ﬂuorenylmethoxycarbonyl; HBTU, O-benzotriazol-

1-yl-N,N,N⁰,N⁰-tetramethyluronium hexaﬂuorophosphate; HOBt, 1-hydroxybenzotriazole; HOOBt, 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine;

Ig, immunoglobulin; MMP, matrix metalloproteinase; NMP, 1-methyl-2-pyrrolidinone; TFA, triﬂuoroacetic acid; THF, tetrahydrofuran

Synthesis of Glycoprotein Carrying Chitobiose

1339

GlcNAc₂

R= GlcNAc

H

1

2

3

34

94

5859

GSKILLTCSLNDSATEVTGHRWLKGGVVLKEDALPGQKTEFKVDSDDQWGEYSCVFLPEPM-NH₂

R

34

8384

GSKILLTCSLNDSATEVTGHRWLK⁵G⁸G⁵⁹VVLKEDALPGQKTEFKVDSDDQWGEYSCVFLPEPM-

GlcNAc₂

118

4

5

GTANIQLHGPPRVKAVKSSEHINE-NH₂

22

AAGTVFTTVEDL-

5859

8384

GSKILLTCSLNDSATEVTGHRWLKGGVVLKEDALPGQKTEFKVDSDDQWGEYSCVFLPEPM-

GlcNAc₂

118

GTANIQLHGPPRVKAVKSSEHINE-NH₂

Fig. 1. Amino Acid Sequence of Emmprin (34-94), (34-118) and (22-118).

The arrows indicate the site of segment coupling.

Boc-Gly-SCH₂CH₂CO-Leu-OCH₂-Pam-resin

ABI433A Peptide synthesizer

Boc/HOBt/DCC 0.1 mmol

Boc-Gly-Ser(Bn)-Lys(ClZ)-Ile-Leu-Leu-Thr(Bn)-Cys(Acm)-Ser(Bn)-Leu-Asn(GlcNAc₂Bn₅)-

Asp(OcHex)-Ser(Bn)-Ala-Thr(Bn)-Glu(OBn)-Val-Thr(OBn)-Gly-His(Bom)-Arg(Tos)-Trp(For)-Leu-

Lys(ClZ)-Gly-SCH₂CH₂CO-Leu-OCH₂-Pam-resin

1) HF treatment, 2) RPHPLC

Gly-Ser-Lys-Ile-Leu-Leu-Thr-Cys(Acm)-Ser-Leu-Asn(R)-Asp-Ser-Ala-Thr-Glu-Val-Thr-Gly-His-Arg-

Trp-Leu-Lys-Gly-SCH₂CH₂CO-Leu (R=GlcNAc₂, GlcNAc)

Boc-OSu

Boc-Gly-Ser-Lys(Boc)-Ile-Leu-Leu-Thr-Cys(Acm)-Ser-Leu-Asn(R)-Asp-Ser-Ala-Thr-Glu-Val-Thr-Gly-

His-Arg-Trp-Leu-Lys(Boc)-Gly-SCH₂CH₂CO-Leu (6, R=GlcNAc₂, 7, R=GlcNAc)

Fig. 2. Synthetic Route for Glycosylated Peptide Thioesters 6 and 7.

Results and Discussion

Gly⁵⁸-Gly⁵⁹was selected as a ligation site, since no

precaution is required to prevent epimerization during

segment coupling. Based on the reported stability of the

chitobiose moiety to an HF treatment,²⁶⁾the N-terminal

chitobiosylated peptide thioester was prepared by the

Boc strategy as shown in Fig. 2. To introduce the

chitobiose moiety, Boc-Asn(GlcNAc₂Bn₅) 11 was pre-

pared by coupling known protected chitobiose unit 9²⁷⁾

to the anhydride of the Boc-Asp derivative as shown in

Fig. 3. Following the previous protocol for peptide

thioester preparation,²³⁾the peptide chain was assembled

on Boc-Gly-SCH₂CH₂CO-Leu-OCH₂-Pam-resin by an

ABI 433A peptide synthesizer with the Boc/HOBt/

DCC protocol. The protected peptide resin correspond-

ing to the sequence of emmprin (45-58) was then reacted

with the benzotriazolyl ester of Boc-Asn(GlcNAc₂Bn₅)

11 (2 eq to the peptide on resin). The remaining

sequence was again introduced by the synthesizer. After

assembly of the sequence (34-58) had been completed, a

part of the resin was stored for the synthesis of (22-58).

The remaining resin was treated with HF at 0 ^ꢀC for

1.5 h. Analysis of the crude peptide by RPHPLC showed

that the desired chitobiosylated peptide thioester had

been obtained in a good yield. In addition to the desired

product, however, a peptide thioester carrying GlcNAc

Emmprin is a heavily glycosylated protein located on

the surface of tumor cells. It stimulates nearby ﬁbro-

blasts to produce MMPs, which are key enzymes for

tumor metastasis.^24,25)In this respect, emmprin is viewed

as a clinical target for the suppression of tumor

metastasis. The functional site is presumed to be the

ﬁrst Ig domain (34-94) which has an N-glycosylation

site at Asn⁴⁴. However, direct evidence that the ﬁrst Ig

domain itself possesses collagenase stimulation activity

has not been presented so far. Moreover, the signiﬁcance

and the structure of the carbohydrate at Asn⁴⁴has not

been clearly elucidated. To clarify these points and the

mechanism for tumor metastasis, we synthesized the

extracellular domains of emmprin carrying carbohy-

drates at Asn⁴⁴.

Synthesis of chitobiosylated Ig domain (34-94)-NH₂

by the thioester method

The ﬁrst Ig domain (34-94) is composed of 61 amino

acid residues, in which Asn⁴⁴is an N-glycosylation site

(Fig. 1). To eﬀectively obtain this long glycopeptide,

the sequence was divided in two, and segment con-

densation by the thioester method was carried out.

1340

E. H^AGINOYAet al.

O

BnO

OBn

BnO

OBn

O

(Boc-Asp-OFm)₂

O

BnO

O

H

N₃

BnO

N

AcNH

O

H₂/ Lindlar catalyst

AcNH

Boc

H

N

9

10

11

COOFm

H

BnO

OBn

O

BnO

N

O

20% piperidine / DMF

AcNH

Boc

H

N

COOH

Fig. 3. Synthetic Procedure for Compound 11.

Fmoc-Rink amide MBHA-resin (Fmoc-NH-resin)

ABI433A peptide synthesizer system software 2.03

FastMoc 0.25 Ω MonPrevPk

Fmoc-Gly-Val-Val-Leu-Lys(Boc)-Glu(OBu^t)-Asp(OBu^t)-Ala-Leu-Pro-Gly-Gln(Trt)-Lys(Boc)-

Thr(Bu^t)-Glu(OBu^t)-Phe-Lys(Boc)-Val-Asp(OBu^t)-Ser(Bu^t)-Asp(OBu^t)-Asp(OBu^t)-Gln(Trt)-

t

Trp(Boc)-Gly-Glu(OBu^t)-Tyr(Bu)-Ser(Bu^t)-Cys(Acm)-Val-Phe-Leu-Pro-Glu(OBu^t)-Pro-Met-

NH-resin

1) Reagent K, 2) ion-exchange chromatography, 3) HPLC

Boc-OSu, DIEA

5% piperidine / DMSO

Gly-Val-Val-Leu-Lys(Boc)-Glu-Asp-Ala-Leu-Pro-Gly-Gln-Lys(Boc)-Thr-Glu-Phe-Lys(Boc)-

Val-Asp-Ser-Asp-Asp-Gln-Trp-Gly-Glu-Tyr-Ser-Cys-Val-Phe-Leu-Pro-Glu-Pro-Met-NH₂

12

Fig. 4. Synthesis of Peptide 12.

was also obtained in about a two-thirds ratio, which

might have been derived from cleavage of the GlcNAc-

GlcNAc bond by the HF treatment. Thus, under the

standard HF treatment conditions (the concentration of

HF was more than 80%), the chitobiose did not retain

complete stability. Treatment with a decreased concen-

tration of an acid, such as a low-HF treatment,²⁸⁾might

overcome this problem. However, it would be worth

noting that the yield of the desired peptide was 6.7%,

which is about 4 times higher than that of the same

peptide thioester carrying an N-linked core pentasac-

charide by the Fmoc strategy (1.8% yield).¹⁴⁾This result

demonstrates that, in the case of a peptide thioester

carrying chitobiose, the Boc strategy would be the

method of choice in respect of the yield of the product.

The resin stored at (34-58) was used for further

elongation by the Boc strategy to obtain the protected

peptide resin of (22-58). HF treatent of this resin gave

the desired peptide and the GlcNAc-truncated peptide in

almost the same ratio as that of the (34-58) peptide

thioester. This result indicates that the stability of

chitobiose to HF seems to have been independent of its

local environment within the peptide. The thioester

method uses activation of the thioester group by silver

ions to acylate the terminal amino group of the amino

component. Thus, other amino and thiol groups were

protected to suppress side reactions. Introduction of the

Boc groups was easily achieved by Boc-OSu in the

presence of DIEA. By following the same procedure,

non-glycosylated peptide thioester 8 was obtained.

C-terminal peptide (59-94) 12 was prepared with the

Fmoc strategy by the FastMoc protocol, which used

HBTU as a coupling reagent, as shown in Fig. 4. The

protected peptide resin was treated with TFA (Reagent

K²⁹⁾), and the crude peptide was puriﬁed by RPHPLC.

Synthesis of this peptide was accompanied by a

signiﬁcant amount of an aspartimide side product (about

30%) which could not be removed by RPHPLC. The

peptide was further puriﬁed by anion-exchange to

remove the by-product and was again desalted by

RPHPLC. This two-step puriﬁcation enabled a highly

pure product to be obtained in a comparable yield to that

of the N-terminal peptide thioester. Protection of the

side-chain amino groups and subsequent removal of the

terminal Fmoc group were then carried out for segment

coupling with the N-terminal peptide thioester.

The segment coupling reaction by the thioester

method was carried out as shown in Fig. 5. Peptides 6

Synthesis of Glycoprotein Carrying Chitobiose

1341

(Boc)₂

Emmprin (34-58)

Acm

O

6

7

8

GlcNAc₂

GlcNAc

H

R¹=

Boc

C-SR²

R¹

(Boc)₃

Emmprin (59-94)

Acm

AgCl + HOOBt

O

C-NH₂

12

H

TFA containing 5% ethanedithiol

Acm Acm

O

C-NH₂

Emmprin (34-94)

R¹

H

1) AgNO₃, DIEA / DMSO, 2) DTT, HCl, 3) RPHPLC, 4) air-oxidation

O

GlcNAc₂

GlcNAc

H

1

2

3

R¹=

Emmprin (34-94)

R¹

C-NH₂

Fig. 5. Synthetic Procedure for Emmprin (34-94)-NH₂1–3.

R²denotes-CH₂CH₂CO-Leu for 6 and 7, and -CH₂CH₂CO-Nle-NH₂for 8.

and 12 were dissolved in DMSO containing HOOBt and

DIEA. The thioester group was then activated by silver

ions. The coupling reaction was almost completed

within 6 h without signiﬁcant side reactions. The Boc

and Acm groups of the peptide were respectively

removed by TFA and AgNO₃. The peptide was puriﬁed

by RPHPLC, and the disulﬁde bond was formed by air-

oxidation in 1% AcONH₄(pH 7.8) overnight. Final

product 1 was successfully obtained after RPHPLC

puriﬁcation. The chitobiose unit was stable throughout

the segment coupling reaction. The glucosaminylated

and non-glycosylated Ig domains (34-94)-NH₂2, 3 were

prepared by following the same procedure (Fig. 6).

7131.9

a

(7132.0)

6929.0

(6928.8)

b

c

6725.1

(6725.6)

40

30

20

Synthesis of chitobiosylated emmprin (34-118)-NH₂4

and (22-118)-NH₂5 by the thioester method

The thioester method was further applied to the larger

glycopeptides, emmprin (34-118) 4 and (22-118) 5, by

repetition of the segment coupling as shown in Fig. 7.

To achieve repetition of the coupling, the terminal

amino group of middle segment 14 was orthogonally

protected by the Fmoc group to the side chain amino

group. This peptide thioester was prepared by the Boc

strategy. The N-terminal glycine was introduced by

using Fmoc-Gly. C-terminal peptide 15 was synthesized

by the Fmoc strategy according to the same procedure as

that used for peptide 12.

To synthesize of glycopeptide 4, peptides 14 and 15

were condensed by the same procedure as that used for

(34-94)-NH₂, using activation of the thioester group by

silver ions. The reaction was completed without signiﬁ-

cant side reactions within 12 h. The terminal Fmoc

group was then removed by piperidine. The crude

peptide obtained was used for the second coupling

reaction with glycopeptide thioester 6 without further

puriﬁcation. This reaction also proceeded smoothly

(Fig. 8a). After the Boc and Acm groups had been

removed (Fig. 8b), the crude peptide was puriﬁed by

0

10

20

Retention time (min)

Fig. 6. RPHPLC Proﬁles of Emmprin (34-94)-NH₂.

a) glycopeptide 1, b) glycopeptide 2, c) peptide 3. Elution

conditions: column, Mightysil RP-18GP (4:6 ꢁ 150 mm) at a ﬂow

rate of 1 ml min^ꢂ1; eluent A, distilled water containing 0.1% TFA;

eluent B, acetonitrile containing 0.1% TFA.

RPHPLC to obtain glycopeptide 17. This reduced form

of the product was air-oxidized to form a disulﬁde bond

(Fig. 8c). After HPLC puriﬁcation, ﬁnal product 4 was

successfully obtained with high purity as shown in

Fig. 8d.

The thioester method was further applied to the

synthesis of (22-118) which was composed of 97 amino

acid residues. Ala²²of this peptide is presumed to be the

N-terminal of the native emmprin. Peptides 14 and 15

were condensed as already described. Obtained crude

peptide 16 was further coupled with glycopeptide

thioester 13 by the thioester method. The reaction was

1342

E. H^AGINOYAet al.

(Boc)₃

59-83

(Boc)₂

84-118

Acm

O

C NH₂

Boc

SR

Fmoc

SR

14

H

15

(GlcNAc)₂

(34-58): 6, (22-58): 13

1) AgCl, HOOBt, DIEA,

2) 20% piperidine / DMSO, 3) RPHPLC

(Boc)₅

Acm

O

59-118

C NH₂16

H

1) AgCl, HOOBt, DIEA, 2) 10% ethanedithiol-TFA, 3) AgNO

O

₃, DIEA, 4) purification

(34-118): 17

(22-118): 18

H

C NH₂

(GlcNAc)₂

1) Air-oxidation in 0.1M AcONH₄(pH 8.0), 2) purification

O

C NH₂

(34-118): 4

(22-118): 5

H

(GlcNAc)₂

Fig. 7. Synthetic Procedure for Chitobiosylated Emmprin (34-118)-NH₂4 and (22-118)-NH₂5.

50

40

30

a

b

a

20

50

40

30

20

b

c

50

d

40

30

20

d

0

10

20

30

Retention time (min)

0

10

20

30

Retention time (min)

Fig. 8. RPHPLC Proﬁles of the Glycopeptides.

a) TFA-treated reaction mixture of the coupling between peptides

6 and 16, b) crude peptide 17, c) crude peptide 4, d) puriﬁed peptide

4. The elution conditions were the same as those described in Fig. 6.

Fig. 9. HPLC Proﬁles of the Glycopeptides.

a) TFA-treated reaction mixture of the coupling between peptides

13 and 16, b) crude peptide 18, c) crude peptide 5, d) puriﬁed

peptide 5. The elution conditions were the same as those described

in Fig. 6.

completed within 12 h without serious side reactions as

shown in Fig. 9a. Removal of the Boc and Acm groups

was then carried out as described for glycopeptide 4.

This reaction proceeded smoothly (Fig. 9b). In contrast

to glycopeptide 17, the isolation of 18 was diﬃcult, as it

was highly adsorbed to the HPLC column. The reason

for this low recovery might be attributable to the N-

terminal sequence being abundant in hydrophobic amino

acids. The puriﬁcation was therefore carried out by gel

ﬁltration chromatography using 50% aqueous acetoni-

trile containing 0.1% TFA. The disulﬁde bond formation

was ﬁrst attempted in an ammonium acetate buﬀer

Synthesis of Glycoprotein Carrying Chitobiose

1343

Fig. 10. CD Spectra of the Synthetic Glycopeptides.

(pH 8.0). However, most of the peptide was precipi-

tated. This precipitate was dissolved in 6 ^Mguanidine

hydrochloride containing DTT. Glycopeptide 18 was

recovered by gel ﬁltration chromatography and oxidized

for 2 days in ammonium acetate (pH 8.0) containing

6 ^Mguanidine hydrochloride (Fig. 9c). The solution was

loaded into a gel chromatography column and the

desired product 5 was obtained (Fig. 9d). The MALDI-

TOF mass analysis, and an amino acid composition

analysis conﬁrmed that the desired product had been

successfully synthesized.

is not yet known. However, the result might indicate that

other factors beside the peptide chain length also

contributed to the folding of this peptide. Emmprin

retains large carbohydrates with a molecular weight of

about 30 kDa, which is comparable to the molecular

weight of its polypeptide chain, 27 kDa. This fact might

show that, at the small chitobiose level, glycosylation at

Asn⁴⁴only altered the conformation around Asn⁴⁴to

exert emmprin activity, but did not stabilize the entire Ig

domains to take a particular conformation. It is therefore

possible that a larger oligosaccharide chain would be

required to assume the correct three-dimensional struc-

ture of emmprin. It might also be possible that further

elongation of the peptide chain could induce a more

stable comformation. Synthesis of the Ig domain carry-

ing a more complex carbohydrate as well as synthesis of

a longer extracellular chain are required to clarify this

point.

CD spectrum of the synthetic emmprin glycopeptides

The synthetic glycopeptides were dissolved in a

sodium phosphate buﬀer (pH 7.0), and the secondary

structure was characterized by CD spectral measure-

ment. As shown in Fig. 10, there was a distinct

diﬀerence between non-glycosylated peptide 3 and

glycosylated peptides 1, 2, 4 and 5. The biological

activity of peptides 1, 2 and 3 was preliminarily

measured by Nabeshima et al. (unpublished data). The

results show that the MMP stimulation activity increased

as the sugar chain became longer from GlcNAc 2 to

chitobiose 1, whereas the Ig domain itself, 3, had

no activity. This result corresponds well with the CD

spectrum. The [ꢀ] values for peptide 4 and 5 were

considerably larger in the negative direction than those

of glycopeptides 1 and 2, indicating that a more stable

conformation was formed by elongation of the peptide

chain. However, the spectra of these glycopeptides

indicate that the peptide did not assume a structure rich

in ꢁ-strand, which has been observed for immunoglo-

bulins and Ig-like domains.³⁰⁾The reason for this result

In conclusion, a chitobiosylated peptide thioester was

prepared by the Boc strategy and used for the prepara-

tion of chitobiosylated emmprin (34-94)-NH₂. The

larger glycopeptides, (34-118)-NH₂and (22-118)-NH₂,

were successfully prepared by repetitive segment cou-

pling, which demonstrates the usefulness of the thioester

method for the synthesis of glycoproteins. Elongation of

peptide chain contributed to a stabilized conformation.

The biological activity of these glycosylated peptides is

under study.

Experimental

Optical rotation values were determined with a DIP-

1344

E. HAGINOYA et al.

370 polarimeter (Jasco, Tokyo, Japan) for solutions in

1

J ¼ 9:0 Hz, Asn ꢁ-NH), 9.09 (d, 1H, J ¼ 9:0 Hz, Ac-

NH-b), 9.07 (d, 1H, J ¼ 10:7 Hz, Ac-NH-a), 5.82 (brt,

1H, J ¼ 9:3 Hz, H-1a), 4.94 (d, 1H, J ¼ 11:2 Hz, Ph-

CH₂-), 4.46 (m, 1H, H-3a), 4.23 (m, 1H, H-2a), 3.39 (dd,

1H, J ¼ 6:4, 15.6 Hz, Asn ꢁH), 3.27 (dd, 1H, J ¼ 4:4,

15.6 Hz, Asn ꢁH), 2.21 (s, 3H, CH₃CO), 2.13 (s, 3H,

CH₃CO), 1.46 (s, 9H, t-Bu). HRFABMS m=z ðM þ HÞ^þ:

calcd. for C₆₀H₇₃N₄O₁₅, 1089.5072; found, 1089.5114.

CHCl₃. H-NMR spectra were recorded with an AL400

spectrometer (Jeol, Tokyo, Japan). Chemical shifts are

expressed in ppm downﬁeld from the signal for the

internal Me₄Si standard in pyridine-d₅. TLC was

performed on 60 F₂₅₄silica gel (Merck). MALDI-TOF

mass spectra were recorded with a Voyager-DE PRO

spectrometer (Applied Biosystems, CA, USA). The

amino acid composition was determined with a LaCh-

rom amino acid analyzer (Hitachi, Tokyo, Japan) after

hydrolysis with 6 ^MHCl at 150 ^ꢀC for 2 h in an

evacuated sealed tube. Calculation of the peptide content

was based on the amino acid analysis data. Compound 9

was prepared as reported previously.²⁷⁾

Boc-[Asn(GlcNAc_n)⁴⁴, Cys(Acm)⁴¹, Lys(Boc)^36;57]-

emmprin (34-58)-SCH₂CH₂CO-Leu (6, n ¼ 2; 7, n ¼

1). Starting from Boc-Gly-SCH₂CH₂CO-Leu-OCH₂-

Pam-resin (0.74 mmol/g, 0.14 g), the peptide chain

was elongated by a 433A peptide synthesizer (Applied

Biosystems, CA, USA), using 0.1 mmol Boc/HOBt/

DCC. The brief procedure for this protocol is as follows:

1) TFA deprotection (25% TFA/CH₂Cl₂for 3 min, 50%

TFA/CH₂Cl₂for 11 min), 2) CH₂Cl₂wash (ꢁ4), 3)

DIEA neutralization (5% DIEA/NMP for 0.5 min ꢁ2),

4) NMP wash (ꢁ5), 5) Boc-amino acid HOBt ester

(1 mmol) in a 15% DMSO-NMP solution for 8 min

and then for 4 min in the presence of 3.8 eq of DIEA,

6) capping (10% Ac₂O, 5% DIEA in NMP for 5 min),

7) CH₂Cl₂wash (ꢁ4). This protocol enabled Boc-

Gly-Ser(Bn)-Lys(ClZ)-Ile-Leu-Leu-Thr(Bn)-Cys(Acm)-

Ser(Bn)-Leu-Asn(GlcNAc₂Bn₅)-Asp(OcHex)-Ser(Bn)-

Ala-Thr(Bn)-Glu(OBn)-Val-Thr(Bn)-Gly-His(Bom)-

Arg(Tos)-Trp(For)-Leu-Lys(ClZ)-Gly-SCH₂CH₂CO-

Leu-OCH₂-Pam-resin (0.58 g) to be obtained. To intro-

duce Asn⁴⁴, Boc-Asn(GlcNAc₂Bn₅) 11 (220 mg, 0.2

mmol), 1 ^MHOBt/NMP (0.2 ml) and 1 M DCC/NMP

(0.2 ml) were mixed for 30 min, and the benzotriazolyl-

active ester generated was added to the resin. A part of

the resin (200 mg) was treated with HF (5.4 ml) con-

taining anisole (0.4 ml) and 1,4-butanedithiol (0.8 ml)

at 0 ^ꢀC for 1.5 h. After HF had been evaporated in vacuo,

the crude peptide was washed with ether (ꢁ3), extracted

with 50% aqueous acetonitrile containing 0.1% TFA

and lyophilized. The crude peptide obtained (120 mg)

was puriﬁed by RPHPLC, and [Asn(GlcNAc₂)⁴⁴,

Cys(Acm)⁴¹]-emmprin (34-58)-SCH₂CH₂CO-Leu (2.3

mmol, 6.7% based on the Gly content of the initial resin)

and [Asn(GlcNAc)⁴⁴, Cys(Acm)⁴¹]-emmprin (34-58)-

SCH₂CH₂CO-Leu (1.4 mmol, 4.1% based on the Gly

content of the initial resin) were separately collected.

[Asn(GlcNAc₂)⁴⁴, Cys(Acm)⁴¹]-emmprin (34-58)-

SCH₂CH₂CO-Leu (6.5 mmol) was dissolved in DMSO

(0.2 ml), and Boc-OSu (7.6 mg, 35 mmol) and DIEA (6.2

ml, 36 mmol) were added. After the reaction mixture had

stood for 2 h at room temperature, the product was

precipitated by ether, washed twice with ethyl acetate

and lyophilized from the dioxane suspension to give

peptide 6 (4.8 mmol, 74%). MALDI-TOF MS: found,

m=z 3665.1 ðM þ HÞ^þ; calcd. for ðM þ HÞ^þ, 3664.8.

Amino acid analysis: Asp_1:99Thr_2:78Ser_2:59Glu_1:04Gly₃-

Ala_1:02Val_1:40Ile_0:90Leu_5:01Lys_1:97His_1:01Arg_1:01. Peptide

7 was obtained by the same procedure in a 92% yield.

MALDI-TOF MS: found, m=z 3461.8 ðM þ HÞ^þ; calcd.

N²-(tert-Butoxycarbonyl)-N⁴-[2-acetamido-3,4,6-tri-

O-benzyl-2-deoxy-ꢁ-^D-glucopyranosyl-(1!4)-2-acet-

amido-3,6-di-O-benzyl-2-deoxy-ꢁ-D-glucopyranosyl]-

L-asparagine ﬂuorenylmethyl ester (10). A mixture of

Boc-Asp-OFm (180 mg, 0.44 mmol) and DCC (50 mg,

0.24 mmol) was dissolved in CH₂Cl₂(0.44 ml) at 0 ^ꢀC,

and the solution was stirred for 30 min. The precipitate

was ﬁltered, and the ﬁltrate was concentrated in vacuo.

The residue was dissolved in THF (5.2 ml) and added

to a mixture of compound 9 (100 mg, 0.11 mmol) and

a Lindlar catalyst (50 mg) under Ar. After the mixture

had been stirred for 43 h under an H₂atmosphere, the

catalyst was ﬁltered through Celite. The ﬁltrate was

puriﬁed on silica gel with CHCl₃–methanol (96:4) and

then by gel chromatography on LH-60 with CHCl₃–

methanol (1:1) to give compound 10 (140 mg, 0.11

mmol, 96%). R_f0.41 (95:5 CHCl₃–methanol). ½ꢂꢃ_D

1

ꢂ3:2^ꢀ(c 1.0, CHCl₃). H-NMR (400 MHz, pyridine-d₅,

TMS), ꢃ: 10.25 (d, 1H, J ¼ 9:3 Hz, Asn ꢁ-NH), 9.08 (d,

1H, J ¼ 8:3 Hz, Ac-NH-b), 9.03 (d, 1H, J ¼ 9:5 Hz, Ac-

NH-a), 8.24 (d, 1H, J ¼ 8:8 Hz, Asn ꢂ-NH), 5.81 (brt,

1H, J ¼ 9:5 Hz, H-1a), 5.45 (m, 1H, Asn ꢂH), 4.20 (m,

1H, H-2b), 4.07 (brt, 1H, J ¼ 8:6 Hz, H-3a), 4.01 (dd,

1H, J ¼ 4:0, 11.7 Hz, H-6a), 3.92 (brt, 1H, J ¼ 9:2 Hz,

H-4b), 3.65 (m, 1H, H-5b), 3.34 (dd, 1H, J ¼ 6:4, 15.6

Hz, Asn ꢁH), 3.25 (dd, 1H, J ¼ 5:6, 15.6 Hz, Asn ꢁH),

2.20 (s, 3H, CH₃CO), 2.12 (s, 3H, CH₃CO), 1.50 (s, 9H,

t-Bu). HRFABMS m=z ðM þ HÞ^þ: calcd. for

C₇₄H₈₃N₄O₁₅, 1267.5855; found, 1267.5905.

N²-(tert-Butoxycarbonyl)-N⁴-[2-acetamido-3,4,6-tri-

O-benzyl-2-deoxy-ꢁ-^D-glucopyranosyl-(1!4)-2-acet-

amido-3,6-di-O-benzyl-2-deoxy-ꢁ-^D-glucopyranosyl]-

L-asparagine (11). To a solution of compound 10 (50

mg, 39 mmol) in DMF (1.6 ml) was added piperidine (0.4

ml), and the solution was stirred for 30 min. The solvent

was removed in vacuo, and the residue was dissolved in

CHCl₃–methanol containing acetic acid (1:1:0.01). The

solution was loaded onto LH-60 with CHCl₃–methanol

(1:1). The major fraction was collected to give com-

pound 11 (49 mg, quantitative). R_f0.38 (9:1:0.1 CHCl₃–

methanol–AcOH). ½ꢂꢃ_Dþ11:0^ꢀ(c 0.5, CHCl₃). ¹H-

NMR (400 MHz, pyridine-d₅, TMS), ꢃ: 10.14 (d, 1H,

Synthesis of Glycoprotein Carrying Chitobiose

1345

for ðM þ HÞ^þ, 3461.8. Amino acid analysis: Asp_1:97

-

dissolved in DMSO (0.15 ml), and Boc-OSu (8.3 mg,

39 mmol) and DIEA (6.7 ml, 39 mmol) were added. The

solution was kept at room temperature for 3 h, and the

product was precipitated by ether, washed with ethyl

acetate and lyophilized from the dioxane suspension to

give peptide 8 (3.6 mmol, 97%). MALDI-TOF MS:

found, m=z 3258.0 ðM þ HÞ^þ; calcd. for ðM þ HÞ^þ,

Thr_2:73Ser_2:59Glu_1:01Gly₃Ala_1:00Val_1:30Ile_0:88Leu_5:08

Lys_1:97His_1:01Arg_0:97

.

Boc-[Asn(GlcNAc₂)⁴⁴, Cys(Acm)⁴¹, Lys(Boc)^36;57]-

emmprin (22-58)-SCH₂CH₂CO-Leu (13). Using the

resin stored at Gly³⁴during the synthesis of 6 and 7

(13 mmol), the peptide chain was further elongated

by the 0.1 mmol Boc/HOBt/DCC protocol to obtain

Boc-Ala-Ala-Gly-Thr(Bn)-Val-Phe-Thr(Bn)-Thr(Bn)-

Val-Glu(OBn)-Asp(OcHex)-Leu-Gly-Ser(Bn)-Lys(ClZ)-

Ile-Leu-Leu-Thr(Bn)-Cys(Acm)-Ser(Bn)-Leu-Asn(Glc-

NAc₂Bn₅)-Asp(OcHex)-Ser(Bn)-Ala-Thr(Bn)-Glu(OBn)-

Val-Thr(Bn)-Gly-His(Bom)-Arg(Tos)-Trp(For)-Leu-

Lys(ClZ)-Gly-SCH₂CH₂CO-Leu-OCH₂-Pam-resin (86

mg). This resin (86 mg) was treated with HF (2.5 ml)

containing anisole (0.1 ml) and 1,4-butanedithiol (0.4

ml) at 0 ^ꢀC for 1.5 h. After HF had been evaporated in

vacuo, the crude peptide was washed with ether (ꢁ3),

extracted with 50% aqueous acetonitrile containing

0.1% TFA and lyophilized. The analysis of the crude

peptide showed that the desired product and GlcNAc-

truncated peptide thioester had been obtained in 3:2

ratio. After puriﬁcation by RPHPLC, [Asn(GlcNAc₂)⁴⁴,

Cys(Acm)⁴¹]-emmprin (22-58)-SCH₂CH₂CO-Leu (50

nmol, 0.4% based on the Gly content of the initial

resin) was obtained. [Asn(GlcNAc₂)⁴⁴, Cys(Acm)⁴¹]-

emmprin (22-58)-SCH₂CH₂CO-Leu (50 nmol) was

dissolved in DMSO (20 ml), and Boc-OSu (0.3 mg,

1.4 mmol) and DIEA (0.2 ml, 1.2 mmol) were added.

After the reaction mixture had stood for 2 h at room

temperature, the product was precipitated by ether,

washed twice with ethyl acetate and lyophilized from

the dioxane suspension to give peptide 13 (quantitative).

MALDI-TOF MS: found, m=z 4868.8 ðM þ HÞ^þ; calcd.

3257.7. Amino acid analysis: Asp_2:02Thr_2:80Ser_2:68

Glu_1:04Gly₃Ala_1:03Val_1:02Ile_0:88Leu_3:93Lys_2:11His_0:97

-

Arg_1:01

.

[Cys(Acm)⁸⁷, Lys(Boc)^63;71;75]-emmprin (59-94)-NH₂

(12). Starting from Fmoc-Rink amide-MBHA-resin

(0.54 mmol/g, 0.46 g), the peptide chain was elongated

by an ABI 433A peptide synthesizer, using the FastMoc

protocol. The brief procedure for this protocol (0.25

mmol scale) is as follows: 1) piperidine deprotection

(18% piperidine/NMP for 3 min and 20% piperidine/

NMP for 3 min. If the Fmoc removal is incomplete,

the deprotection is repeated), 2) NMP wash (ꢁ5), 3)

coupling with Fmoc-amino acid (1 mmol) activated

by HBTU/HOBt (0.9 mmol each) and DIEA (2 mmol)

for 20 min. The protected peptide resin, Fmoc-Gly-Val-

Val-Leu-Lys(Boc)-Glu(OBu^t)-Asp(OBu^t)-Ala-Leu-Pro-

Gly-Gln(Trt)-Lys(Boc)-Thr(Bu^t)-Glu(OBu^t)-Phe-Lys

(Boc)-Val-Asp(OBu^t)-Ser(Bu^t)-Asp(OBu^t)-Asp(OBu^t)-

Gln(Trt)-Trp(Boc)-Gly-Glu(OBu^t)-Tyr(Bu^t)-Ser(Bu^t)-

Cys(Acm)-Val-Phe-Leu-Pro-Glu(OBu^t)-Pro-Met-NH-

resin (1.9 g), was obtained by this protocol. A part of the

resin (400 mg) was treated with Reagent K²⁹⁾(5.0 ml) for

2 h at room temperature. TFA was removed in a nitrogen

stream, and the peptide was precipitated by ether. The

peptide was dissolved in a 0.1 ^Msodium phosphate

buﬀer (pH 6.0) and loaded into an anion-exchange

column (ICE DEAE-825, 8 mm ꢁ 75 mm; Showa Den-

ko, Tokyo, Japan) which had been equilibrated with

0.1 ^Msodium phosphate (pH 6.0), before eluting with an

increasing NaCl concentration in the buﬀer. The peptide

was desalted by RPHPLC to give Fmoc-[Cys(Acm)⁸⁷]-

emmprin (59-94)-NH₂(5.6 mmol, 10% based on the

amino groups in the initial resin). This peptide (5.6

mmol) was dissolved in DMSO (0.2 ml), and Boc-OSu

(12 mg, 56 mmol) and DIEA (9.4 ml, 54 mmol) were

added. After the solution had been kept at room

temperature for 5 h, the product was precipitated by

ether and washed with ethyl acetate. The obtained

peptide was dissolved in DMSO (0.2 ml) containing 5%

piperidine. The solution was kept at room temperature

for 1 h, and the product was precipitated by ether and

dried. The peptide was washed with 0.1% TFA and

lyophilized to give peptide 12 (4.8 mmol, 8.9% based on

the amino groups in the initial resin). MALDI-TOF MS:

found, m=z 4453.6 ðM þ NaÞ^þ(average); calcd. for

ðM þ NaÞ^þ, 4452.0 (average). Amino acid analysis:

for ðM þ HÞ^þ, 4869.4. Amino acid analysis: Asp_2:99

-

Thr_5:43Ser_2:83Glu_1:18Gly₄Ala_2:85Val_2:51Ile_0:81Leu_5:68

Phe_0:80Lys_2:13His_0:97Arg_0:90

.

Boc-[Cys(Acm)⁴¹, Lys(Boc)^36;57]-emmprin (34-58)-

SCH₂CH₂CO-Nle-NH₂(8). Starting from Boc-Gly-

SCH₂CH₂CO-Nle-NH-resin (0.42 mmol/g, 1.2 g), the

peptide chain was elongated by an ABI 433A peptide

synthesizer, using 0.5 mmol Boc/HOBt/DCC, and Boc-

Gly-Ser(Bn)-Lys(ClZ)-Ile-Leu-Leu-Thr(Bn)-Cys(Acm)-

Ser(Bn)-Leu-Asn-Asp(OcHex)-Ser(Bn)-Ala-Thr(Bn)-

Glu(OBn)-Val-Thr(Bn)-Gly-His(Bom)-Arg(Tos)-Trp

(For)-Leu-Lys(ClZ)-Gly-SCH₂CH₂CO-Nle-NH-resin

(3.4 g) was obtained. A part of this resin (600 mg) was

treated with HF (10 ml) containing anisole (0.5 ml) and

1,4-butanedithiol (1.5 ml) at 0 ^ꢀC for 1.5 h. After HF had

been evaporated in vacuo, the crude peptide was washed

with ether (ꢁ3), extracted with 50% aqueous acetonitrile

containing 0.1% TFA and lyophilized. The crude

peptide obtained (260 mg) was puriﬁed by RPHPLC,

and [Cys(Acm)⁴¹]-emmprin (34-58)-SCH₂CH₂CO-Nle-

NH₂(4.2 mmol, 4.8% based on the Gly content of the

initial resin) was obtained. This peptide (3.7 mmol) was

Asp_4:05Thr_0:94Ser_1:75Glu_6:21Pro_3:03Gly₃Ala_1:02Val_2:92

Met_0:82Leu_3:01Tyr_0:88Phe_1:92Lys_2:97

-

.

[Asn(GlcNAc₂)⁴⁴]-emmprin (34-94)-NH₂(1). Pep-

1346

E. HAGINOYA et al.

tides 6 (2.3 mmol) and 12 (1.9 mmol) were dissolved in

DMSO (0.1 ml) containing HOOBt (15 mg, 92 mmol)

and DIEA (10 ml, 58 mmol), and AgCl (1.3 mg, 9.1 mmol)

were added. The reaction mixture was stood overnight

in the dark. The product was precipitated with ether,

washed twice with ethyl acetate and dried. The obtained

powder was treated for 10 min with TFA containing 5%

ethanedithiol (0.2 ml) to remove the Boc groups. TFA

was removed in a nitrogen stream, and the product was

precipitated with ether. The crude peptide was extracted

with 50% aqueous acetonitrile and lyophilized. The

powder was dissolved in DMSO (0.2 ml), and AgNO₃

(3.6 mg, 21 mmol) and DIEA (7.3 ml, 42 mmol) were

added. After the reaction mixture had been kept in the

dark for 2 h, DTT (44 mg, 0.29 mmol) was added. The

solution was acidiﬁed by 0.5 ^MHCl, and the product was

puriﬁed by RPHPLC to give linear [Asn(GlcNAc₂)⁴⁴]-

emmprin (34-94)-NH₂(1.2 mmol, 63% based on peptide

12). This obtained peptide (1.2 mmol) was dissolved in

DMSO (3 ml) and diluted with 0.1 ^MAcONH₄(pH 7.8,

27 ml). The solution was stirred for 2 days. After

puriﬁcation by RPHPLC, peptide 1 (0.82 mmol, 68%)

was obtained. MALDI-TOF MS: found, m=z 7131.9

ðM þ HÞ^þ(average); calcd. for ðM þ HÞ^þ, 7132.0

58 mmol). AgCl (1.3 mg, 9.1 mmol) was added, and the

reaction mixture was stood overnight in the dark. The

product was precipitated with ether, washed with ethyl

acetate and treated with TFA containing 5% ethanedi-

thiol (0.25 ml) for 10 min to remove the Boc groups.

TFA was removed in a nitrogen stream, and the product

was precipitated with ether. The precipitate was puriﬁed

by RPHPLC. The obtained powder was dissolved in

DMSO (0.2 ml), and AgNO₃(1.7 mg, 10 mmol) and

DIEA (3.5 ml, 20 mmol) were added. After the reaction

mixture had been kept in the dark for 1.5 h, DTT (22 mg,

0.14 mmol) and 0.5 ^MHCl (0.3 ml) were added. The

product was puriﬁed by RPHPLC. The obtained linear

emmprin (34-94)-NH₂was dissolved DMSO (0.9 ml)

and diluted with 1% AcONH₄(pH 7.8, 6 ml). After the

solution had been stirred at room temperature for 4 days,

the product was puriﬁed by RPHPLC to give peptide 3

(0.35 mmol, 23% based on peptide 12). MALDI-TOF

MS: found, m=z 6725.1 ðM þ HÞ^þ(average); calcd. for

ðM þ HÞ^þ, 6725.6 (average). Amino acid analysis:

Asp_5:98Thr_3:45Ser_3:77Glu_6:94Pro_2:88Gly₆Ala_2:04Val_4:54

Met_1:22Ile_0:97Leu_7:19Tyr_0:80Phe_2:13Lys_4:79His_1:00Arg_0:95

-

.

Fmoc-[Lys(Boc)^63;71;75]-Emmprin (59-83)-SCH₂CH₂-

CO-Leu (14). Starting from Boc-Gly-SCH₂CH₂CO-Leu-

OCH₂-Pam-resin (0.45 mmol/g, 1.1 g), the peptide chain

was elongated with an ABI 433A peptide synthesizer by

using Boc/HOBt/DCC, and protected peptide resin,

Fmoc-Gly-Val-Val-Leu-Lys(ClZ)-Glu(OBn)-Asp

(OcHex)-Ala-Leu-Pro-Gly-Gln-Lys(ClZ)-Thr(Bn)-Glu

(OBn)-Phe-Lys(ClZ)-Val-Asp(OcHex)-Ser(Bn)-Asp

(OcHex)-Asp(OcHex)-Gln-Trp(For)-Gly-SCH₂CH₂CO-

Leu-OCH₂-Pam-resin (1.9 g), was obtained. The ﬁnal

amino acid was introduced by using Fmoc-Gly. Part of

the resin (650 mg) was treated for 1.5 h with HF (10 ml)

containing anisole (0.5 ml) and 1,4-butanedithiol (1.5

ml) at 0 ^ꢀC. HF was removed in vacuo, and the residue

was washed with ether. The peptide was extracted with

aqueous acetonitrile containing 0.1% TFA, lyophilized

and puriﬁed by RPHPLC to obtain Fmoc-emmprin (59-

83)-SCH₂CH₂CO-Leu (2.4 mmol, 1.4% based on the Gly

content of the initial resin). This peptide (2.4 mmol) was

dissolved in DMSO (0.3 ml), and Boc-OSu (18 mg,

84 mmol) and DIEA (15 ml, 86 mmol) were added. After

the resulting solution had beeen stood for 2 h, the

product was precipitated by ether, washed twice with

ethyl acetate and lyophilized from the dioxane suspen-

sion to give peptide 14 (1.7 mmol, 71%). MALDI-TOF

MS: found, m=z 3509.0 (average); calcd. for ðM þ NaÞ^þ,

(average). Amino acid analysis: Asp_5:99Thr_3:74Ser_4:47

Glu_7:33Pro_3:13Gly₆Ala_2:10Val_4:51Cys_0:26Met_0:84Ile_0:90

-

Leu_7:52Tyr_1:07Phe_1:92Lys_4:84His_1:02Arg_0:98

.

[Asn(GlcNAc)⁴⁴]-emmprin (34-94)-NH₂(2). Peptides

7 (2.9 mmol) and 12 (3.2 mmol) were dissolved in DMSO

(0.15 ml) containing HOOBt (16 mg, 98 mmol) and

DIEA (11 ml, 63 mmol). AgCl (1.4 mg, 9.8 mmol) was

added, and the reaction mixture was stood overnight in

the dark. The product was precipitated with ether,

washed with ethyl acetate and treated with TFA

containing 5% ethanedithiol (0.25 ml) for 10 min to

remove the Boc groups. TFA was removed in a nitrogen

stream, and the product was precipitated with ether. To

the product dissolved in DMSO (0.2 ml), AgNO₃(3.8

mg, 22 mmol) and DIEA (7.8 ml, 45 mmol) were added.

After the reaction mixture had been kept in the dark for

1 h, DTT (44 mg, 0.29 mmol) and 0.5 ^MHCl (0.6 ml)

were added. The product was puriﬁed by RPHPLC to

give linear [Asn(GlcNAc)⁴⁴]-emmprin (34-94)-NH₂

(0.77 mmol, 27% based on peptide 7). The obtained

peptide (0.77 mmol) was dissolved in DMSO (1.5 ml)

and diluted with 1% AcONH₄(pH 7.8, 14 ml). After the

solution had been stirred at room temperature for 2 days,

the product was puriﬁed by RPHPLC to give peptide 2

(0.50 mmol, 65%). MALDI-TOF MS: found, m=z 6929.0

ðM þ HÞ^þ(average); calcd. for ðM þ HÞ^þ, 6928.8

3508.9 (average). Amino acid analysis: Asp_4:20Thr_0:89

Ser_0:75Glu_4:08Pro_1:07Gly₃Ala_1:00Val_2:67Leu_3:03Phe_1:00

Lys_3:13.

-

(average). Amino acid analysis: Asp_6:01Thr_3:79Ser_4:49

Glu_7:39Pro_3:25Gly₆Ala_2:07Val_4:37Met_0:84Ile_0:92Leu_7:58

-

Tyr_1:09Phe_1:94Lys_4:88His_1:03Arg_1:04

.

[Cys(Acm)⁸⁷, Lys(Boc)^108;111]-Emmprin (84-118)-

NH₂(15). Starting from Fmoc-Rink amide-MBHA-resin

(0.73 mmol/g, 0.34 g), the peptide chain was elongated

with an ABI 433A peptide synthesizer by using the

FastMoc protocol, and the protected peptide resin,

Emmprin (34-94)-NH₂(3). Peptides 8 (1.8 mmol) and

12 (1.5 mmol) were dissolved in DMSO (0.1 ml) con-

taining HOOBt (15 mg, 92 mmol) and DIEA (10 ml,

Synthesis of Glycoprotein Carrying Chitobiose

1347

Fmoc-Glu(OBu^t)-Tyr(Bu^t)-Ser(Bu^t)-Cys(Acm)-Val-Phe-

Leu-Pro-Glu(OBu^t)-Pro-Met-Gly-Thr(Bu^t)-Ala-Asn(Trt)-

Ile-Gln(Trt)-Leu-His(Trt)-Gly-Pro-Pro-Arg(Pbf)-Val-

Lys(Boc)-Ala-Val-Lys(Boc)-Ser(Bu^t)-Ser(Bu^t)-Glu(OBu^t)-

His(Trt)-Ile-Asn(Trt)-Glu(OBu^t)-NH-resin (1.87 g), was

obtained. Part of the resin (930 mg) was treated with

Reagent K (9 ml) for 2 h. TFA was removed in a

nitrogen stream, and the peptide was precipitated with

ether. The residue was dissolved in aqueous acetonitrile

and puriﬁed by RPHPLC to obtain Fmoc-[Cys(Acm)⁸⁷]-

emmprin (84-118)-NH₂(13 mmol, 11% based on the

amino group of the initial resin). To a solution of this

peptide (13 mmol) in DMSO (0.4 ml), Boc-OSu (26 mg,

120 mmol) and DIEA (21 ml, 120 mmol) were added, and

the mixture was stood for 1 h. The peptide was

precipitated by ether, washed with ethyl acetate and

lyophilized from the dioxane suspension. The peptide

was dissolved in DMSO (0.4 ml) containing 5% piper-

idine, and the solution was kept for 30 min. The product,

precipitated by ether, was puriﬁed by RPHPLC, and

peptide 15 was obtained (11 mmol, 85%). MALDI-TOF

MS: found, m=z 4148.3 ðM þ HÞ^þ; calcd. for ðM þ HÞ^þ,

overnight in 10 m^MAcONH₄containing 6 M guanidine

HCl (pH 8.0, 5 ml). The product was puriﬁed by

RPHPLC, and peptide 4 (83 nmol, 7.5% based on

peptide 14) was obtained. MALDI-TOF MS: found, m=z

9696.5 (average); calcd. for ðM þ HÞ^þ, m=z 9696.9

(average). Amino acid analysis: Asp_8:05Thr_4:55Ser_6:36

-

Glu_10:03Pro_5:08Gly_8:29Ala_4:33Val_6:28Met_0:90Ile_2:70Leu₈-

Tyr_1:10Phe_2:03Lys_6:70His_2:85Arg_2:00

.

Emmprin (22-118)-NH₂(5). Peptides 14 (160 nmol)

and 15 (240 nmol) were condensed in the same manner

as that described for peptide 4. Obtained crude peptides

16 and 13 (48 nmol) were dissolved in DMSO (20 ml)

containing HOOBt (1.0 mg, 6.1 mmol) and DIEA (0.7 ml,

4.0 mmol). To the solution, AgCl (0.1 mg, 0.7 mmol) was

added, and the resulting mixture was stirred overnight in

the dark. Ether was added to form a precipitate, which

was washed twice with the same solvent and dried in

vacuo. The powder was treated with 10% ethanedithiol

TFA (0.1 ml) for 10 min. TFA was removed in an N₂

stream, and the product was precipitated with ether and

dried in vacuo. The product was extracted with 50%

aqueous acetonitrile containing 0.1% TFA and lyophi-

lized. The obtained powder was dissolved in DMSO

(40 ml) containing DIEA (0.4 ml, 2.3 mmol), and AgNO₃

(0.17 mg, 1.0 mmol) was added. After the solution had

been kept in the dark for 2 h, DTT (1.5 mg, 9.7 mmol)

and 0.5 ^MHCl (20 ml) were added, and the product was

puriﬁed in a G3000PW_XLcolumn (7:8 mm ꢁ 300 mm,

Tosoh, Japan), using 50% aqueous acetonitrile contain-

ing 0.1% TFA at a ﬂow rate of 0.5 ml/min, to give

peptide 18 (20 nmol). The peptide was dissolved in 5 ml

of a 6 ^Mguanidine HCl solution and dropped into 0.01 M

sodium phosphate (pH 7.0, 400 ml) for 2 days. The

precipitated peptide was again dissolved in 6 ^Mguani-

dine hydrochloride (500 ml) containing DTT (1.0 mg,

6.5 mmol) and kept at 37 ^ꢀC for 30 min. The solution was

applied to the G3000PW_XLcolumn, and peptide 18 was

recovered. The peptide was dissolved in 0.1 ^MAcONH₄,

pH 8.0, 200 ml), and the solution was gently vortexed for

2 days. This solution was applied to the G3000PW_XL

column and the fraction containing peptide 5 was

collected (3.4 nmol). MALDI-TOF MS: found, m=z

10899.8 (average); calcd. for ðM þ HÞ^þ, m=z 10900.2

4148.1. Amino acid analysis: Asp_1:98Thr_0:84Ser_1:81

Glu_4:75Pro_4:41Gly₂Ala_1:98Val_2:87Met_0:95Ile_1:87Leu_1:99

-

Tyr_1:02Phe_0:97Lys_1:94His_1:96Arg_1:01

.

Emmprin (34-118)-NH₂(4). Peptides 14 (1.1 mmol)

and 15 (1.6 mmol) were dissolved in DMSO (85 ml)

containing HOOBt (7.7 mg, 47 mmol) and DIEA (5.5 ml,

32 mmol). AgCl (0.7 mg, 4.9 mmol) was added, and the

reaction mixture was stirred overnight in the dark. DTT

(15 mg, 97 mmol) was added, and the peptide was

precipitated by distilled water containing 0.1% TFA.

The precipitate was dried in vacuo and dissolved in

DMSO (0.1 ml) containing 5% piperidine and DTT

(500 mg, 3.2 mmol). After the solution had been stood

for 1 h at room temperature, the product was precipitat-

ed by ether and successively washed with acetonitrile

and 0.1% TFA. Lyophilized crude [Cys(Acm)⁸⁷,

Lys(Boc)^{63;71;75;108;111}]-emmprin (59-118)-NH₂16 and

peptide 6 (1.1 mmol) were dissolved in DMSO (0.1 ml)

containing HOOBt (5.4 mg, 33 mmol) and DIEA (3.8 ml,

22 mmol). To the solution, AgCl (0.47 mg, 3.3 mmol) was

added, and resulting mixture was stirred overnight in the

dark. Ether was added to form a precipitate, which was

washed twice with the same solvent and dried in vacuo.

The powder was treated with 10% ethanedithiol TFA

(0.2 ml) for 10 min. TFA was removed in an N₂stream,

and the product was precipitated with ether and dried in

vacuo. The product was extracted with 50% aqueous

acetonitrile containing 0.1% TFA and lyophilized. The

obtained powder was dissolved in DMSO (0.1 ml)

containing DIEA (1.9 ml, 11 mmol), and AgNO₃(0.93

mg, 5.5 mmol) was added. After the solution had been

kept in the dark for 1 h, DTT (10 mg, 65 mmol) and 0.5 M

HCl (0.1 ml) were added to the solution, and the product

was puriﬁed by RPHPLC to give peptide 17. The

obtained reduced form of the peptide was air-oxidized

(average). Amino acid analysis: Asp_9:34Thr_7:36Ser_6:32

Glu_11:35Pro_4:77Gly_9:28Ala₆Val_8:45Met_0:63Ile_2:91Leu_9:34

-

Tyr_1:37Phe_3:02Lys_7:47His_2:87Arg_1:73

.

CD spectral measurement. Each peptide was dis-

solved in a 10 m^Msodium phosphate buﬀer (pH 7.0) at a

concentration of 0.5 to 1 mg/ml. The CD spectrum was

recorded between 205 nm and 260 nm by a J-720

instrument (Jasco, Tokyo, Japan) at room temperature,

using a 1-mm-path-length cell.

Acknowledgments

This work was supported in part by grant-aid for

1348

E. HAGINOYA et al.

synthesis of peptide thioester carrying N-linked core

pentasaccharide through modiﬁed Fmoc thioester prep-

aration: synthesis of an N-glycosylated Ig domain of

emmprin. Tetrahedron Lett., 44, 2961–2964 (2003).

15) Warren, J. D., Miller, J. S., Keding, S. J., and

Danishefsky, S. J., Toward fully synthetic glycoproteins

by ultimately convergent routes: a solution to a long-

standing problem. J. Am. Chem. Soc., 126, 6576–6578

(2004).

16) Botti, P., Villain, M., Manganiello, S., and Gaertner, H.,

Native chemical ligation through in situ O to S acyl shift.

Org. Lett., 6, 4861–4864 (2004).

17) Macmillan, D., and Bertozzi, C. R., Modular assembly of

glycoproteins: towards the synthesis of GlyCAM-1 by

using expressed protein ligation. Angew. Chem. Int. Ed.,

43, 1355–1359 (2004).

18) Hojo, H., Matsumoto, Y., Nakahara, Y., Ito, E., Suzuki,

Y., Suzuki, M., Suzuki, A., and Nakahara, Y., Chemical

synthesis of 23-kDa glycoprotein by repetitive segment

condensation: a synthesis of MUC2 basal motif carrying

multiple O-GalNAc moieties. J. Am. Chem. Soc., 127,

13720–13725 (2005).

19) Marcaurelle, L. A., Mizoue, L. S., Wilken, J., Oldham,

L., Kent, S. B. H., Handel, T. M., and Bertozzi, C. R.,

Chemical synthesis of lymphotactin: a glycosylated

chemokine with a C-terminal mucin-like domain. Chem.

Eur. J., 7, 1129–1132 (2001).

20) Lavielle, S., Ling, N. C., and Guillemin, R. C., Solid-

phase synthesis of two glycopeptides containing the

amino acid sequence 5 to 9 of somatostatin. Carbohydr.

Res., 89, 221–228 (1981).

21) Teshima, T., Urabe, T., Yamamoto, T., Isaka, S.,

Kumagaye, K., Nakajima, K., and Shiba, T., Solid-phase

synthesis of the antibacterial glycopeptide, formaecin,

using Boc-strategy. In ‘‘Peptide Science 1998,’’ ed.

Kondo, M., Protein Research Foundation, Osaka,

pp. 157–160 (1999).

scientiﬁc research (no. 16580093) from the Ministry of

Education, Culture, Sport, Sciences and Technology of

Japan. We thank Tokai University for grant-aid for high-

technology research. We also thank the Japan Society

for the promotion of Science for grant-aid for creative

scientiﬁc research (no. 17GS0420).

References

1) Hojo, H., and Aimoto, S., Polypeptide synthesis using

the S-alkyl thioester of a partially protected peptide

segment. Synthesis of the DNA-binding domain of c-

Myb protein (142-193)-NH₂. Bull. Chem. Soc. Jpn., 64,

111–117 (1991).

2) Aimoto, S., Polypeptide synthesis by the thioester

method. Biopolymers (Peptide Science), 51, 247–265

(1999).

3) Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S.

B. H., Synthesis of proteins by native chemical ligation.

Science, 266, 776–779 (1994).

4) Dawson, P. E., and Kent, S. B. H., Synthesis of native

proteins by chemical ligation. Ann. Rev. Biochem., 69,

923–960 (2000).

5) Futaki, S., Sogawa, K., Maruyama, J., Asahara, T.,

Niwa, M., and Hojo, H., Preparation of peptide thioesters

using Fmoc-solid-phase peptide synthesis and its appli-

cation to the construction of a template-assembled

synthetic protein (TASP). Tetrahedron Lett., 38, 6237–

6240 (1997).

6) Li, X., Kawakami, T., and Aimoto, S., Direct preparation

of peptide thioesters using an Fmoc solid-phase method.

Tetrahedron Lett., 39, 8669–8672 (1998).

7) Shin, Y., Winans, K. A., Backes, B. J., Kent, S. B. H.,

Ellman, J. A., and Bertozzi, C. R., Fmoc-based synthesis

of peptide-^ꢂthioesters: application to the total chemical

synthesis of a glycoprotein by native chemical ligation.

J. Am. Chem. Soc., 121, 11684–11689 (1999).

8) Alsina, J., Yokumu, T. S., Albericio, F., and Barany, G.,

Backbone amide linker (BAL) strategy for Nꢂ-9-ﬂuo-

renylmethoxycarbonyl (Fmoc) solid-phase synthesis of

unprotected peptide p-nitroanilides and thioesters. J.

Org. Chem., 64, 8761–8769 (1999).

22) Mizuno, M., Haneda, K., Iguchi, R., Muramoto, I.,

Kawakami, T., Aimoto, S., Yamamoto, K., and Inazu,

T., Synthesis of a glycopeptide containing oligosaccha-

rides: chemoenzymatic synthesis of eel calcitonin ana-

logues having natural N-linked oligosaccharides. J. Am.

Chem. Soc., 121, 284–290 (1999).

9) Ingenito, R., Bianchi, E., Fattori, D., and Pessi, A., Solid

phase synthesis of peptide C-terminal thioesters by

Fmoc/t-Bu chemistry. J. Am. Chem. Soc., 121, 11369–

11374 (1999).

10) Clippingdale, A. B., Barrow, C. J., and Wade, J. D.,

Peptide thioester preparation by Fmoc solid phase

peptide synthesis for use in native chemical ligation. J.

Peptide Sci., 6, 225–234 (2000).

11) Swinnen, D., and Hilvert, D., Facile Fmoc-compatible

solid-phase synthesis of peptide C-terminal thioesters.

Org. Lett., 2, 2439–2442 (2000).

12) Mezo, A. R., Cheng, R. P., and Imperiali, B., Oligome-

rization of uniquely folded mini-protein motifs: develop-

ment of a homotrimeric ꢁꢁꢂ peptide. J. Am. Chem. Soc.,

123, 3885–3891 (2001).

23) Hojo, H., Watabe, J., Nakahara, Y., Nakahara, Y., Ito,

Y., Nabeshima, K., and Toole, B. P., Synthesis of

the extracellular Ig domain I of emmprin carrying a

chitobiose unit. Tetrahedron Lett., 42, 3001–3004

(2001).

24) Ellis, S. M., Nabeshima, K., and Biswas, C., Monoclonal

antibody preparation and puriﬁcation of a tumor cell

collagenase-stimulatory factor. Cancer Res., 49, 3385–

3391 (1989).

25) Nabeshima, K., Kataoka, H., Koono, M., and Toole, B.

P., Activation and induction of collagenases. In ‘‘Colla-

genases,’’ ed. Hoeﬄer, W., R. G. Landes Company,

Texas, pp. 91–113 (1999).

26) Mort, A. J., and Lamport, D. T., Anhydrous hydrogen

ﬂuoride deglycosylates glycoproteins. Anal. Biochem.,

82, 289–309 (1977).

13) Brask, J., Albericio, F., and Jensen, K. J., Fmoc solid-

phase synthesis of peptide thioesters by masking as

trithioortho esters. Org. Lett., 5, 2951–2953 (2003).

14) Hojo, H., Haginoya, E., Matsumoto, Y., Nakahara, Y.,

Nabeshima, K., Toole, B. P., and Watanabe, Y., The ﬁrst

27) Kerekgyarto, J., Agoston, K., Batta, G., Kamerling, J. P.,

and Vliegenthart, J. F. G., Synthesis of fully and partially

benzylated glycosyl azides via thioalkyl glycosides as

precursors for the preparation of N-glycopeptides.

Synthesis of Glycoprotein Carrying Chitobiose

1349

Tetrahedron Lett., 39, 7189–7192 (1998).

28) Tam, J. P., Heath, W. F., and Merriﬁeld, R. B., S_N2

deprotection of synthetic peptides with a low concen-

tration of HF in dimethyl sulﬁde: evidence and applica-

tion in peptide synthesis. J. Am. Chem. Soc., 105, 6442–

6455 (1983).

method which minimizes side reactions following Fmoc

solid phase peptide synthesis. Int. J. Peptide Protein

Res., 36, 255–266 (1990).

30) Tsunenaga, M., Goto, Y., Kawata, Y., and Hamaguchi,

K., Unfolding and refolding of a type ꢄ immunoglobulin

light chain and its variable and constant fragments.

Biochemistry, 26, 6044–6051 (1987).

29) King, D. S., Fields, C. G., and Fields, G. B., A cleavage

Article Doi

Synthesis of a glycosylated peptide thioester by the Boc strategy and its application to segment condensation

DOI: 10.1271/bbb.50621

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Article abstract of DOI:10.1271/bbb.50621

Full text of DOI:10.1271/bbb.50621

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