Synthesis of the extracellular Ig domain I of Emmprin carrying a chitobiose unit

Article abstract of DOI:10.1016/S0040-4039(01)00342-2

The extracellular Ig domain I of Emmprin (34-94) carrying a chitobiose unit at Asn⁴⁴ was chemically synthesized. Boc-Asn with a benzyl-protected chitobiose unit was synthesized and used for the preparation of peptide thioester with the sequence of Emmprin (34-58) by Boc strategy. C-Terminal peptide amide (59-94) was also prepared by the solid-phase method. These segments were condensed by activation of the thioester group by silver ions to obtain a protected form of Emmprin (34-94)-NH₂. After deprotection and air oxidation, the desired Emmprin (34-94)-NH₂ with chitobiose was successfully obtained.

Full text of DOI:10.1016/S0040-4039(01)00342-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 3001–3004
Synthesis of the extracellular Ig domain I of Emmprin carrying
a chitobiose unit
Hironobu Hojo,a,b,* Jun Watabe,a Yoshiaki Nakahara,a,b,c Yuko Nakahara,b,c Yukishige Ito,b,c
Kazuki Nabeshimad and Bryan P. Toolee
aDepartment of Industrial Chemistry, Tokai University, Kanagawa 259-1292, Japan
bThe Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan
cCREST, Japan Science and Technology Corporation (JST), Japan
dDepartment of Pathology, Miyazaki Medical College, Miyazaki 889-1692, Japan
eDepartment of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Ave., Boston,
MA 02111, USA
Received 15 January 2001; revised 19 February 2001; accepted 23 February 2001
Abstract—The extracellular Ig domain I of Emmprin (34-94) carrying a chitobiose unit at Asn44 was chemically synthesized.
Boc-Asn with a benzyl-protected chitobiose unit was synthesized and used for the preparation of peptide thioester with the
sequence of Emmprin (34-58) by Boc strategy. C-Terminal peptide amide (59-94) was also prepared by the solid-phase method.
These segments were condensed by activation of the thioester group by silver ions to obtain a protected form of Emmprin
(34-94)-NH2. After deprotection and air oxidation, the desired Emmprin (34-94)-NH2 with chitobiose was successfully obtained.
© 2001 Elsevier Science Ltd. All rights reserved.
Emmprin is a glycoprotein located on the surface of
human tumor cells.1 It belongs to a member of the
immunogloblin superfamily and stimulates nearby
fibroblasts to produce matrix metalloproteinases. Sev-
eral experiments have shown that the extracellular
immunoglobulin (Ig) domain I (34-94), which possesses
an N-glycosylation site at Asn44, plays an essential role
in metalloproteinase stimulatory activity (Fig. 1). To
analyze the biological function of Emmprin at a molec-
ular level, therefore, the Ig domain with a distinct
carbohydrate structure at Asn44 has to be synthesized.
We have previously established synthetic routes for
glycosylated amino acids, such as Ser(Neu5Ac-Gal-Gal-
NAc) and Asn(Man3-GlcNAc-GlcNAc) derivatives,
which were used for the syntheses of several glycopep-
tides by the Fmoc solid-phase method.2 In these synthe-
ses, construction of the oligosaccharide portions was
realized using benzyl-protected sugars, which retain
higher reactivity than the generally used acyl-protected
sugars. Thus, the deprotection of the carbohydrate
portion was carried out at the final stage of the synthe-
sis by catalytic hydrogenation or 1 M trimethylsilyl
Figure 1. The structure of the first Ig domain (34-94) of Emmprin carrying a chitobiose unit at Asn44. The arrow indicates the
site of segment coupling.
Keywords: thioesters; glycopeptides; condensations.
* Corresponding author. Fax: +81-463-50-2075; e-mail: hojo@keyaki.cc.u-tokai.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00342-2

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H. Hojo et al. / Tetrahedron Letters 42 (2001) 3001–3004
triflate in trifluoroacetic acid (TFA). These treatments
are advantageous over the base treatment required for
the removal of acyl-protecting groups in that they are
free of the danger of b-elimination and racemization.
On the other hand, benzyl groups are readily removed
by HF. In the case of glycopeptides with acid-stable
carbohydrate chains, the Boc strategy would be a better
choice, as the peptide and carbohydrate portion can be
simultaneously deprotected by HF treatment. However,
few reports have appeared on glycopeptide synthesis by
the Boc strategy. In addition, all of them are on gly-
copeptides carrying a monosaccharide.3 To examine the
applicability of the Boc strategy to the synthesis of
glycopeptide with a longer carbohydrate chain, a Boc-
Asn derivative carrying a benzyl-protected chitobiose
was prepared. Based on the result of HF treatment of
natural glycoproteins, the chitobiose on Asn seems to
be stable to this reagent.4 This unit was used for the
synthesis of a peptide thioester with a sequence of
Emmprin (34-58) and then condensed with the Emm-
prin (59-94) segment to obtain the Ig domain I by the
thioester method.5
Boc-Asn with a chitobiose unit was prepared as shown
in Fig. 2. Starting from a tetra-O-acetyl derivative of
glucosamine 1, the fluoride 3 was synthesized. This
compound was then coupled with another glucosamine
unit to give disaccharide 4. After removal of the
phthaloyl group and acetylation, compound 5 was
obtained. Then the azido group of 5 was selectively
reduced in the presence of benzyl groups by catalytic
hydrogenation using ethylenediamine poisoned Pd/C.6
The obtained amine was directly used for the coupling
with Boc-Asp-OAll by 1-ethyl-3-(3-dimethylamino-
propyl)-carbodiimide (WSCI) without further purifica-
tion. Removal of the allyl group by Pd(PPh3)4 and
N-methylaniline gave the desired product 7.7
The glycosylated Asn 7 was then used for the prepara-
tion of Emmprin (34-94) by the thioester method.5
Emmprin (34-94) was divided at Gly58-Gly59 and the
solid-phase synthesis of an N-terminal peptide thioester
with a chitobiose unit and C-terminal peptide was
carried out. The N-terminal segment was synthesized as
shown in Fig. 3. Starting from Boc-Gly-SCH2CH2CO-
AcO
AcO
AcO
AcO
Ph
O
O
O
C,D,E,F,G
45%
H
81%
A,B
56%
O
AcO
O
AcO
OAc
OTBDPS
BnO
F
NPhth
NPhth
1
3
2
NPhth
Ph
O
Ph
O
O
BnO
O
I
J
O
O
BnO
O
BnO
O
O
85%
BnO
49%
O
BnO
N3
BnO
NPhth
N3
NPhth
NHAc
4
5
NHAc
Ph
O
Ph
O
O
BnO
O
O
BnO
O
O
O
O
N C
H
BnO
O
O
K
84%
H
BnO
O
BnO
BnO
N C
NHAc
NHAc
NHAc
Boc-NH COOAll
NHAc
Boc-NH COOH
6
7
Figure 2. Synthetic route for chitobiose-linked Asn unit 7. (A) NH2NH2–AcOH/DMF. (B) t-Butyldiphenylsilyl chloride,
imidazole, 4-dimethylaminopyridine/DMF. (C) t-BuOK/MeOH, THF. (D) Benzaldehyde dimethylacetal, camphorsulfonic acid/
CH3CN. (E) Benzyl bromide, NaH/DMF. (F) Tetrabutylammonium fluoride, AcOH/THF. (G) Dimethylaminosulfur trifluoride/
THF. (H) 3,6-Di-O-benzyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl azide, Cp2HfCl2, AgClO4/CH2Cl2. (I) 1: Ethylenediamine/
BuOH. 2: (CH3CO)2O/MeOH. (J) 1: H2, Pd/C(en)/CH2Cl2, MeOH. 2: Boc-Asp-OAll, WSCI/CH2Cl2. (K) Pd(PPh3)4, N-
methylaniline/THF.
Figure 3. Synthetic route for peptides 11 and 12.

H. Hojo et al. / Tetrahedron Letters 42 (2001) 3001–3004
3003
Figure 4. Synthetic route for peptide [Asn(R)44]-Emmprin (34-94)-NH2.
Figure 5. HPLC elution profiles of the peptides; (A) crude peptide 14, (B) crude peptide 16, (C) purified peptide 16, (D) crude
peptide 18. Elution conditions: column, Mightysil RP-4 GP (4.6×150 mm) at a flow rate of 1 ml min−1; eluent, aqueous acetonitrile
containing 0.1% TFA.
conditions is required. Peptide 10 was used for Emm-
Leu-OCH2-Pam-resin, the sequence of Emmprin (34-
58) was synthesized by an ABI 433A peptide synthe-
sizer (Applied Biosystems Inc., Foster City, CA) using
system software version 2.03. The capping module ‘g’
of this protocol was omitted, as the TFA treatment
after coupling of compound 7 would remove the ben-
zylidene group of the chitobiose moiety, which causes
the acetylation of 4,6-hydroxyl groups during the cap-
ping cycle. The side chain functional groups of Arg and
Trp were protected by the Mts group to achieve the
final deprotection using both HF and 1 M triflic acid
(TfOH)–TFA. The chitobiose-linked Asn unit 7 was
introduced manually using the 1,3-dicyclohexylcarbodi-
imide (DCC)-1-hydroxybenzotriazole (HOBt) method.
After completion of the peptide chain assembly, the
protected peptide resin was treated with anhydrous HF
or 1 M TfOH–TFA. Both treatments successfully gave
the desired peptide 9. The yield of peptide 9 by HF and
1 M TfOH–TFA treatment was 12 and 14%, respec-
tively, based on the compound 7 used for the prepara-
tion of resin 8. However, the glycosidic linkage between
GlcNAc-GlcNAc seems to be not completely stable to
these treatments, since GlcNAc-truncated peptide 10
was also obtained in 7.5 and 6% yields. To reduce this
side reaction, further optimization of the deprotection
prin (34-94)-NH2 with one GlcNAc unit.
The amino groups of peptides 9 and 10 were protected
with Boc groups using N-(t-butoxycarbonyloxy)-
succinimide (Boc-OSu) to obtain peptides 11 and
12 in yields of 73 and 92%, respectively.7 The C-termi-
nal segment 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(Acm)-
Val-Phe-Leu-Pro-Glu-Pro-Met-NH2 (13) was prepared
via a peptide segment synthesized on Fmoc-Rink amide
MBHA-resin (Calbiochem-Novabiochem, Switzerland)
using FastMoc protocol. The yield was 8.9% based on
the content of the amino groups on the initial resin.
The condensation of the segments was carried out
following the procedure of Kawakami et al. as shown
in Fig. 4.5b For example, the preparation of peptide 18
was briefly described. Peptides 11 and 13 were dissolved
in DMSO containing 3,4-dihydro-4-oxo-3-hydroxy-
1,2,3-benzotriazine (HOOBt) and N,N-diisopropylethyl-
amine (DIEA). Then, AgCl was added to initiate the
coupling reaction. As shown in Fig. 5, the reaction was
almost completed within 4 h without significant side

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H. Hojo et al. / Tetrahedron Letters 42 (2001) 3001–3004
reactions. The product was successively treated with
TFA and AgNO3 in the presence of DIEA to remove
all protecting groups. The crude peptide was purified by
HPLC to obtain the linear [Asn(GlcNAc-GlcNAc)44]-
Emmprin (34-94)-NH2 16 in a yield of 77%. To form a
disulfide bond between Cys41 and Cys87, the peptide
was dissolved in DMSO and the solution was diluted
by 1% AcONH4 (pH 7.8) to 20% DMSO solution.
After the reaction mixture was gently stirred in the
presence of air overnight, the final product 18 was
obtained as shown in Fig. 5D. The yield of peptide 18
was 50% from 16. In the same manner, peptide 19 was
obtained in 27% yield based on peptide 13 used for
segment coupling. In conclusion, the Boc-Asn deriva-
tive with a benzyl-protected chitobiose moiety was pre-
pared and successfully applied to the synthesis of the Ig
domain I of Emmprin. The collagenase stimulation
activity of the peptides 18 and 19 is now being studied.
Soc. Jpn. 1995, 68, 330–336; (b) Kawakami, T.; Yoshimura,
S.; Aimoto, S. Tetrahedron Lett. 1998, 39, 7901–7904; (c)
Aimoto, S. Biopolymers (Peptide Science) 1999, 51, 247–265.
6. Sajiki, H.; Hattori, K.; Hirota, K. J. Org. Chem. 1998, 63,
7990–7992.
7. Selected physical data are given below.
5: Calcd for C44H49N5O10: C, 65.41; H, 6.11; N, 8.67. Found:
C, 65.20; H, 6.11; N, 8.59. 1H-NMR: 6.13 (d, 1H, J 7.9 Hz,
NH
H-1a), 4.87 (d, 1H, J 12.0 Hz, PhCH2
Hz, NHAc), 4.70 (d, 1H, J 11.9 Hz, PhCH
J 11.2 Hz, PhCH2), 4.63 (d, 1H, J 12.2 Hz, PhCH2
(d, 1H, J 11.7 Hz, PhCH2), 4.41 (d, 1H, J 8.3 Hz, H-1b),
6
Ac), 5.53 (s, 1H, PhCH
6
(O)2), 4.88 (d, 1H, J 7.0 Hz,
), 4.78 (d, 1H, J 8.6
2), 4.65 (d, 2H,
), 4.47
6
6
6
6
6
6
4.22 (dd, 1H, J 5.0, 10.6 Hz, H-6b), 3.97 (brt, 1H, J 6.0 Hz,
H-3b), 3.29–3.25 (m, 1H, H-5b), 1.94 (s, 3H, Ac), 1.80 (s,
3H, Ac). 7: Calcd for C53H64N4O15·H2O: C, 62.71; H, 6.45;
1
N, 5.52. Found: C, 62.43; H, 6.44; N, 5.66. H NMR: 5.52
(s, 1H, PhCH
4.50–4.43 (m, 3H, PhCH
6
(O)2), 4.88–4.79 (m, 3H, PhCH2
6 ×2, H-1a),
6
2, H-1b, Asn b-H), 1.89 (s, 3H, Ac),
1.84 (s, 3H, Ac), 1.43 (s, 9H, t-Bu). 11: MALDI-TOF MS:
found: m/z 3665.2 (M+H)+, calcd for (M+H)+: 3664.8.
Amino acid analysis: Asp2.01Thr2.83Ser2.68Glu1.04Gly3-
References
1. Nabeshima, K.; Kataoka, H.; Koono, M.; Toole, B. P. In
Collagenases; Hoeffler, W., Ed.; R.G. Landes Company:
Texas, 1999; pp. 91–113.
2. (a) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T.
Carbohydr. Res. 1998, 309, 287–296; (b) Guo, Z.-W.;
Nakahara, Y.; Nakahara, Y.; Ogawa, T. Bioorg. Med.
Chem. 1997, 5, 1917–1924.
3. (a) Lavielle, S.; Ling, N. C.; Guillemin, R. C. Carbohydr.
Res. 1981, 89, 221–228; (b) Mizuno, M.; Haneda, K.; Iguchi,
R.; Muramoto, I.; Kawakami, T.; Aimoto, S.; Yamamoto,
K.; Inazu, T. J. Am. Chem. Soc. 1999, 121, 284–290; (c)
Teshima, T.; Urabe, T.; Yamamoto, T.; Isaka, S.; Kuma-
gaye, K.; Nakajima, K.; Shiba, T. In Peptide Science 1998;
Kondo, M., Ed.; Protein Research Foundation: Osaka,
1999; pp. 157–160.
Ala1.03Val1.251/2Cys0.14Ile0.86Leu4.98Lys1.96His1.02Arg1.00
.
12: MALDI-TOF MS: found: m/z 3461.8 (M+H)+, calcd for
(M+H)+: 3461.8. Amino acid analysis: Asp1.92Thr2.69
Ser2.59Glu1.00Gly3Ala1.01Val1.271/2Cys0.14Ile0.89 Leu4.98
Lys1.94His0.97Arg0.99. 13: MALDI-TOF MS: found: m/z
4453.6 (M+Na)+ (average), calcd for (M+Na)+: 4452.0
(average). Amino acid analysis: Asp4.05Thr0.94Ser1.75
Glu6.21Pro3.03Gly3Ala1.02Val2.921/2Cys0.06Met0.82Leu3.01
Tyr0.88Phe1.92Lys2.97. 18: MALDI-TOF MS: found: m/z
7131.9 (M+H)+ (average), calcd for (M+H)+: 7132.0 (aver-
age). Amino acid analysis: Asp5.99Thr3.74Ser4.47Glu7.33
Pro3.13Gly6Ala2.10Val4.511/2Cys0.52Met0.84Ile0.90Leu7.52Tyr1.07
Phe1.92Trp0.74Lys4.84His1.02 Arg0.98. 19: MALDI-TOF MS:
found: m/z 6929.0 (M+H)+ (average), calcd for (M+H)+:
6928.8. Amino acid analysis: Asp6.01Thr3.79Ser4.49Glu7.39
Pro3.25Gly6Ala2.07Val4.371/2Cys0.75Met0.84Ile0.92Leu7.58
4. Mort, A. J.; Lamport, D. T. A. Anal. Biochem. 1977, 82,
289–309.
5. (a) Hojo, H.; Yoshimura, S.; Go, M.; Aimoto, S. Bull. Chem.
Tyr1.09Phe1.94Trp0.79Lys4.88His1.03Arg1.04.
.
.