Synthesis of betaglycan-type tetraosyl hexapeptide: a possible precursor regulating enzymatic elongation toward heparin.

Article abstract of DOI:10.1016/S0960-894X(02)00327-X

TETRAOSYL HEXAPEPTIDE, A PART OF THE SEQUENCE OF BETAGLYCAN: beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl-(1-->O-SerGlyTrpProAspGly (1), which was designed as a probe for glycan elongation toward heparin, was synthesized in a stere

Full text of DOI:10.1016/S0960-894X(02)00327-X

Bioorganic & Medicinal ChemistryLetters 12 (2002) 1901–1903
Synthesis of Betaglycan-type Tetraosyl Hexapeptide: A Possible
Precursor Regulating Enzymatic Elongation Toward Heparin
Jun-ichi Tamura,a,b,* Akihiro Yamaguchia and Junko Tanakaa,b
aDepartment of Environmental Sciences, Faculty of Education & Regional Sciences, Tottori University, Tottori, 680-8551 Japan
bCREST, Japan Science and Technology Corporation (JST), Japan
Received 7 March 2002; accepted 9 May2002
Abstract—Tetraosyl hexapeptide, a part of the sequence of betaglycan: b-d-GlcA-(1!3)-b-d-Gal-(1!3)-b-d-Gal-(1!4)-b-d-Xyl-
(1!O-SerGlyTrpProAspGly (1), which was designed as a probe for glycan elongation toward heparin, was synthesized in a
stereocontrolled manner. # 2002 Elsevier Science Ltd. All rights reserved.
Glycosaminoglycans (GAGs) are classified into two
categories based on the type of hexosamine residue in
the repeating region; heparin and chondroitin, which
contain a-GlcNAc and b-GalNAc, respectively. GAGs
are thought to be synthesized by the stepwise additions
of monosaccharides with the help of the corresponding
UDP-sugars and glycosyltransferases. However, the
detailed mechanisms of the first addition of the hex-
osamine have been ambiguous. This important elonga-
tion step sorts the GAG into heparin and chondroitin.
So far as we know, the factors regulating the sorting
step have been hypothesized to exist in the glycan or
core-peptide regions. The former, the tetrasaccharides
comprising the linkage regions of GAG often have sul-
fates at specific positions.1 Interestingly, the GAGs of
the heparin-type have no sulfate in the linkage region,
different from the chondroitin type.2 It seems the sulfate
might orient the GAG to the chondroitin type elonga-
tion. On the other hand, the heparin-type GAGs often
link to hydrophobic and acidic regions in the core-pep-
tide.3 Esko et al.4 demonstrated the glycan elongation
by using xylosides having hydrophobic aglycons to
produce the heparin-type GAG. Although these results
showed important evidence for the relationship between
heparin and the character of the aglycon part, the
mechanism remains unclear just in the first hexosaminyl
transfer to the tetrasaccharide of GAG.
Clarifying the sorting mechanism in detail for biomedi-
cal purposes is urgentlyneeded. These facts prompted
us to synthesize the required part of the proteoglycan.
We have selected and synthesized the tetraosyl hex-
apeptide (1) as described below. The targeted com-
pound is a part of betaglycan5 and is expected to be a
precursor for the heparin biosynthesis. Some glycosyl
peptides at the linkage region of GAG have already
been synthesized.6,7
Based on the retrosynthetic analysis, the targeted com-
pound was divided into the glycan, serylglycine and tet-
rapeptide parts, and each of them was synthesized as
follows. The glycan part was synthesized from the
reducing terminus as depicted in Scheme 1. The galac-
tosyl donor (2) was synthesized from the corresponding
p-methoxyphenyl glycoside6i in two steps: (1) CAN/
CH3CN–H2O, and (2) CCl3CN, DBU/CH2Cl2 in 79
and 75% yield, respectively. The xylosyl acceptor (3)
was converted from the known peracetate8 in three
*Corresponding author. E-mail: jtamura@fed.tottori-u.ac.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00327-X

1902
J. Tamura et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1901–1903
steps: (1) p-MPOH, TMSOTf, MSAW300/CH2Cl2, (2)
Et3N–MeOH–H2O, and (3) 2-methoxypropene, cam-
phorsulfonic acid/DMF, 60 ꢀC (48%, three steps). We
coupled 2 with 3 bya TMSOTf mediated manner at
À20 ꢀC. Removal of the isopropylidene acetal with cam-
phorsulfonic acid gave the desired disaccharide 4 in 78%
yield (from 3) without the formation of the a-glycoside.
Two hydroxyl groups of 4 were masked as the 4-
methylbenzoyl ester in 65% yield and the allyl group
was removed to yield the acceptor 5 in 97%. The same
glycosylation procedure as above with 2 and 5 exclu-
sivelyafforded the b-linked trisaccharide 6 in 86% yield.
The repetitive removal of the allyl ether at the non-
reducing terminal of 6 was effectivelyexecuted to yield 7
in 85%. We coupled the glucuronyl thio glycoside 87 to
7 bythe reported method 9 in 31% yield which should be
improved. The tetrasaccharide 910 was converted into
the imidate 10 via the corresponding hemiacetal as the
synthesis of 2 in 79% yield (two steps).
The synthesis of the peptide parts and the following
condensations are shown in Scheme 2. Starting from the
11
commerciallyavailable or known
amino acid deriva-
tives, we obtained the suitablyprotected SerGly( 11)
and TrpProAspGly( 12) sequences in high yields. The
coupling of 10 and 11 was performed in the same man-
ner as the synthesis of 4 to yield the desired glycosyl
serylglycine (13)10 in 72%. The allyl ester of 13 was
changed to the carboxylic acid in 90% yield and cou-
pled with 12 via the HBTU method to give 14 in 63%
yield. Finally, careful deprotection procedures with
TFA cocktail12 removed the tert-Bu and Boc groups,
and then saponification with NaOMe was performed to
afford the targeted compound 110 in 91% yield (two
steps). The product was purified bycolumns of LH-20,
Mono Q and Sephasil C18. The assigned structure of 1 was
1
confirmed by H NMR and FABMS. No b-elimination
product was observed.
In summary, we have synthesized for the first time the
tetraosyl hexapeptide—a partial sequence of betagly-
can—in a stereocontrolled manner. The probe 1 is
under enzymatic glycan elongation and will elucidate
the biological mechanisms for the sorting of GAG. This
tetraosyl hexapeptide will contribute to the development
of medicines composed of GAG, especiallyof heparin
and heparan sulfate.
Scheme 1. Reagents and conditions: (a) TMSOTf, MSAW300/
CH2Cl2; (b) camphorsulfonic acid/MeOH–CH2Cl2; (c) MBzCl/pyri-
dine; (d) [Ir(COD)(PMePh2)2PF6], H2/THF; (e) I2, NaHCO3/THF–
H2O; (f) AgOTf, CuBr2, n-Bu4NBr, MS4A/CH3Cl2; (g) CAN/
CH3CN–H2O; (h) CCl3CN, DBU/CH2Cl2. MP, p-MeOC6H4; MBz,
p-MeC6H4CO.
Scheme 2. Reagents and conditions: (a) HOBt, DCC/CH2Cl2, 0 ꢀC; (b) TMSOTf, MSAW300/CH2Cl2, À20 ꢀC; (c) Pd(PPh3)4, N-methylaniline/
THF; (d) HOBt, WSCDꢁ HCl/CH2Cl2, À20 ꢀC; (e) Pd/C, H2/EtOH; (f) morpholine/CH2Cl2; (g) HBTU, HOBt, iPr2EtN/DMF, À20 ꢀC; (h) TFA
cocktail,12 then NaOMe/aqMeOH. HOBt, N-hydroxybenzotriazole; WSCD, 1-ethyl-3-(30-dimethylaminopropyl)carbodiimide; HBTU, O-benzo-
triazol-1-yl-N, N, N0, N0-tetramethyluronium hexafluorophosphate.

J. Tamura et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1901–1903
1903
Acknowledgements
22), 5.02 (dd, J1,2=8.1 Hz, J2,3=10.2 Hz, H-23), 4.91 (d, H-14),
4.51 (d, H-12), 4.37 (d, H-13), 4.26 (d, J4,5=10.0 Hz, H-54),
4.21 (dd, J4,5e=3.9 Hz, Jgem=12.2 Hz, H-5e1), 4.16 (dd,
J5,6b=5.9 Hz, Jgem=11.7 Hz, H-6b3), 4.03 (dd, J5,6a=6.3 Hz,
H-6a3), 3.93 (m, H-41), 3.85–3.81 (m, H-6b2, 33), 3.79–3.73 (m,
H-32, 52, 33, 53), 3.76 (s, MeOPh), 3.70 (s, COOMe), 3.61 (dd,
J4,5a=5.6 Hz, H-5a1), 3.51 (dd, J5,6a=7.1 Hz, Jgem=11.5 Hz,
H-6a2), 2.40, 2.37, 2.37, 2.36, 2.30 (5s, 5MePh), 2.16, 2.09,
2.02, 1.93, 1.89, 1.65 (6s, 6Ac). 13: dH (500 MHz, CDCl3) 7.80–
7.62 (Ph), 7.12–7.00 (Ph), 6.82 [m, NH(Gly)], 5.80 (m, ¼CH),
5.72 (dd, J2,3=9.0 Hz, J3,4=9.8 Hz, H-34), 5.57 (t, H-44), 5.49
(t, J2,3=J3,4=7.8 Hz, H-31), 5.40 (d, J3,4=3.2 Hz, H-43), 5.36
[m, NH(Ser)], 5.28 (dd, J1,2=7.1 Hz, H-24), 5.20 (m, ¼CH2),
5.14 (d, J3,4=1.7 Hz, H-42), 5.12 (d, J1,2=6.1 Hz, H-21), 4.95
(dd, J1,2=8.1 Hz, J2,3=7.8 Hz, H-22), 4.92 (dd, J1,2=7.8 Hz,
J2,3=10.5 Hz, H-23), 4.84 (d, H-14), 4.65 (d, H-11), 4.46 (m,
OCH2), 4.37 (d, H-12), 4.29 (d, H-13), 4.19 (d, H-54), 4.15 (m,
Sera), 4.09 (dd, J5,6b=6.1 Hz, Jgem=11.0 Hz, H-6b3), 4.06 (dd,
J4,5e=5.6 Hz, Jgem=13.2 Hz, H-5e1), 3.97 (dd, J5,6a=5.9 Hz,
H-6a3), 3.89 (m, H-41), 3.89 (dd, Ja,NH=5.4 Hz,
Jgem=18.5 Hz, Glyaa), 3.79 (dd, Glyab), 3.76 (dd, H-33), 3.73
(dd, J5,6b=5.9 Hz, Jgem=11.2 Hz, H-6b2), 3.68 (brt, J=6.1 Hz,
H-53), 3.63 (m, H-32), 3.63 (s, COOMe), 3.57 (brt, J=6.1 Hz,
H-52), 3.48 (m, Serb), 3.45 (d, H-5a1, 6a2), 2.29, 2.28 (2s,
4 MBz), 2.22 (s, MBz), 2.07, 2.01, 1.94, 1.85, 1.83, 1.59 (6s,
6Ac), 1.36 (s, tert-Bu). 14: dH (500 MHz, CDCl3) 8.2–8.6 (Ph),
7.45 [d, J=8.3 Hz, NH(Asp)], 7.3–7.0 (Ph), 7.18 [m, NH(Gly)],
6.94 [br, NH(Gly)], 6.78 [br, NH(Trp)], 5.80 (t,
J2,3=J3,4=9.4 Hz, H-34), 5.64 (brt, J=9.6 Hz, H-44), 5.53 (dd,
J2,3=7.8 Hz, J3,4=8.3 Hz, H-31), 5.47 (d, J3,4=2.8 Hz, H-43),
5.37 [m, NH(Ser)], 5.36 (dd, J1,2=7.1 Hz, H-24), 5.20 (d,
J3,4=3.2 Hz, H-42), 5.17 (d, J1,2=6.2 Hz, H-21), 5.06 (m,
Trpa), 4.99 (m, 2H, H-22, 23), 4.92 (d, H-14), 4.79 (m, Aspa),
4.66 (d, H-11), 4.49 (t, J=6.4 Hz, Proa), 4.40 (d, J1,2=8.0 Hz,
H-13or2), 4.37 (d, J1,2=8.0 Hz, H-12or3), 4.28 (m, Sera), 4.27
(d, J4,5=9.9 Hz, H-54), 4.16 (dd, J5,6b=5.7 Hz, Jgem=11.6 Hz,
H-6b3), 4.06 (m, H-5e1), 4.03 (dd, J5,6a=5.3 Hz, H-6a3), 4.00
(m, Glya), 3.92 (m, H-41), 3.83 (dd, J2,3=10.3 Hz, H-33), 3.81
(m, Glyb), 3.8.5 (m, 2H, H-62), 3.76 (brt, J=6.6 Hz, H-53), 3.75
(m, Gly), 3.70 (s, COOMe), 3.69 (m, H-32), 3.68 (m, Progb),
3.61 (brt, J=7.1 Hz, H-52), 3.55 (m, Serb), 3.47 (dd,
J4,5a=8.0 Hz, Jgem=12.1 Hz, H-5a1), 3.37 (m, Proga), 3.24,
3.15 (m, Trpb), 2.90 (dd, Ja,bb=4.8 Hz, Jgem=17.0 Hz,
Aspba), 2.62 (dd, Ja,ba=6.0 Hz, Aspbb), 2.36, 2.35, 2.34 (3s,
4 MBz), 2.29 (s, MBz), 2.15 (s, 2Ac), 2.09, 2.02, 1.93, 1.91 (4s,
each 3H, 4Ac), 1.66, 1.44, 1.40, 1.39 (4s, each 9H, 4tert-Bu). 1:
dH (500 MHz, D2O) (selected) 7.71, 7.59, 7.53, 7.27, 7.20 (m,
Ph), 4.96 (t, J=6.9 Hz, Trpa), 4.75 (dd, J=6.0, 7.0 Hz, Aspa),
4.70 (d, J1,2=7.9 Hz, H-14), 4.66 (dd, J=5.8, 7.8 Hz, Aspa0),
4.59 (d, J1,2=7.9 Hz, H-13), 4.57 (m, Trpa0), 4.56 (d,
J1,2=7.5 Hz, H-12), 4.46 (d, J1,2=7.8 Hz, H-11), 4.42 (d,
J1,2=7.3 Hz, H-101), 4.41 (m, Proa, Sera), 4.33 (dd, J=5.0,
11.0 Hz, Serba), 4.30 (t, J=4.1 Hz, Sera0), 4.24 (m, Serba0),
4.14 (s, H-42or3), 4.10 (s, H-43or2), 4.10 (m, Serbb), 4.04 (m,
Serbb0), 3.78 (m, H-32or3), 3.73 (m, H-22, 23, Proda), 3.63 (m,
H-33or2), 3.42 (m, H-24), 3.37 (m, H-21, Prodb), 3.33 (m, H-
The authors thank Dr. S. Kurono for the mass spectral
measurements. This work was supported bya Grant-in-
Aid for Scientific Research (C) 12660098 from the
Ministryof Education, Science, Culture and Sport of
Japan.
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10. 1H NMR data for keycompounds are given below, values
of dH were measured at 25 ꢀC. Chemical shifts are expressed in
ppm downfield from the signal for internal Me4Si for solutions
in CDCl3, and for the solutions in D2O, in ppm downfield
from the signal for Me4Si, byreference to internal tert-BuOH
(1.23). Signal assignment such as 13 stands for a proton at C-1
of sugar residue 3 from the reducing terminal. 9: dH (500 MHz,
CDCl3) 7.97–7.93 (Ph), 7.81–7.70 (Ph), 7.23–7.07 (Ph), 6.96
(m, MeOPh), 6.81 (m, MeOPh), 5.80 (t, J2,3=J3,4=9.3 Hz, H-
34), 5.65 (t, J2,3=J3,4=6.1 Hz, H-31), 5.64 (brt, J=9.8 Hz, H-
44), 5.47 (d, J3,4=3.5 Hz, H-43), 5.41 (dd, J1,2=4.9 Hz, H-21),
5.37 (dd, J1,2=7.6 Hz, H-24), 5.29 (d, H-11), 5.22 (d,
J3,4=3.4 Hz, H-42), 5.08 (dd, J1,2=8.1 Hz, J2,3=10.0 Hz, H-
0
21 ), 3.31 (m, Trpba), 3.30 (m, Prodb0), 3.14 (m, Trpbb), 2.94
(dd, Jgem=19.1 Hz, Aspba), 2.90 (dd, Jgem=19.1 Hz, Aspba0),
2.79 (dd, Aspbb), 2.77 (dd, Aspbb0), 2.22 (m, Proba), 1.92 (m,
Probb, Prog), 1.64, 1.44 (m, Prob0, Proga0), 0.97 (m, Progb0);
FAB-MS (positive) m/z: 1316.3 (calcd for C50H69N7Na3O30
1316.38, [M+H]+), 1338.4 (calcd for C50H68N7Na4O30
1338.36, [M+Na]+).
11. Waldmann, H.; Kunz, H. Liebigs Ann. Chem. 1983, 1712.
12. TFA/thioanisol/phenol/H2O/1,2-ethanedithiol/triiso-pro-
pylsilane/CH2Cl2=40.7:2.5:2.5:2.5:1.3:0.5:50.