[Supplementary Information]
A novel approach for the parallel synthesis of glycopeptides
by
combining
solid-phase
peptide
synthesis
and
dendrimer-supported enzymatic modifications
Takahiko Matsushita,1 Seiji Handa,1 Kentaro Naruchi,2 Fayna Garcia-Martin,1 Hiroshi
Hinou,1,2 and Shin-Ichiro Nishimura*1,2
1
Graduate School of Life Science, Hokkaido University, N22, W11, Kita-ku, Sapporo
001-0021, Japan.
2
Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12. Kita-ku, Sapporo
001-0021, Japan
Corresponding author: Shin-Ichiro Nishimura, E-mail: shin@sci.hokudai.ac.jp; Fax:
+81-11-706-9042; Tel: +81-11-706-9043
S1
Materials and Methods
All solid-phase reactions for glycopeptide synthesis were performed manually in a
polypropylene tube equipped with a filter (LibraTube; Hipep Laboratories). Coupling
reactions of Fmoc- amino acid derivatives and Fmoc removal reactions were conducted
under microwave irradiation (0-120 w) at 50°C, and acetyl capping reactions after
coupling were carried out by conventional manner at ambient temperature.
Microwave-assisted solid-phase reactions were performed manually using a single
mode microwave reactor (IDX Corp., GreenMotif I). The microwave reactor was a
customized reaction cavity for installation of LibraTube and introduced an additional
function of mechanically shaking for the reaction cavity in our laboratories.
Bis-Boc-aminooxyacetyl-G7 PAMAM dendrimer (1).
To a solution of 5 wt% G7 PAMAM dendrimer (ethylenediamine core) in methanol
(400 l, 0.058 mol, 29.7 mol of amino group) were added bis-Boc-aminooxyacetic
acid N-hydroxysuccinimide ester (115 mg, 297 mol) and N-methylmorpholine (3.3 l,
29.7 mol) and stirred at ambient temperature for 24 h. The crude product was purified
by size-exclusion chromatography (Sephadex LH-20, GE Healthcare Bio-Science)
eluted with MeOH to give 1 (8.5 mg, 57%). 600 MHz 1H-NMR spectrum of 1
(methanol-d4, 300 K) is shown in Figure S4.
Aminooxy-functionalized G7 PAMAM dendrimer (2).
4M HCl (1.88 ml) was added to dendrimer 1 (8.5 mg, 16.9 mol peripheral amino
group) and stirred at ambient temperature for 12h. After quenching with 4M NaOH (1.8
ml) and 1M NaOH (50 l) to adjust at pH 7.0, water (7.53 ml) was added to prepare a
stock solution of 2 (theoretically 1.5 mM aminooxy group). The 600 MHz 1H-NMR
spectrum of 2 (D2O, 300 K) is shown in Figure S5.
Solid-phase synthesis of molecular shuttle (model glycopeptide) (3).
The solid-phase synthesis of molecular shuttle carrying model glycopeptide 3 was
carried out using TentaGel S RAM resin (Leading: 0.26 mmol/g, 100 mg, 26 mol). To
the pre-swollen resin in DMF for 30 min at ambient temperature was added 20%
S2
piperidine in DMF (3 ml) and shaken under microwave irradiation (0-120W) at 50°C
for 6 min. After washing with DMF (5×3 ml), Fmoc-amino acid (130 μmol, 5.0 equiv)
activated with HBTU (130 μmol, 5 equiv), HOBt (130 μmol, 5 equiv), and DIPEA (260
μmol, 10 equiv) in DMF (1.5 mL) was coupled to the resin under microwave irradiation
(0-120W) at 50°C for 12 min. For incorporation of Fmoc-glycosylated amino acid to the
resin, Fmoc-Ser(Ac3GlcNAc)-OH (75 μmol, 1.5 equiv) was used in the presence of
HBTU (75 μmol, 1.5 equiv), HOBt (75 μmol, 1.5 equiv), and DIPEA (150 μmol, 3
equiv) in DMF (1.5 mL) under microwave irradiation (0-120W) at 50 °C for 20 min.
After filtration and washing with DMF (5×3 mL), unreacted amino group on the resin
were capped with acetyl group by treatment of acetic anhydride (4.75%, v/v), DIPEA
(2.25%, v/v), and HOBt (13 mM) in DMF (4 mL). After shaking under microwave
irradiation (0-120W) at 50 °C for 3 min, the resin was filtered and washed with DMF
(5×3 mL). Fmoc-removal, coupling, and capping procedures were carried out repeatedly.
At the final step of glycopeptide elongation on the resin, extra Fmoc- Glu(OtBu)-OH,
Fmoc-Phe-OH, and 5-oxohexanoic acid were assembled under the same coupling
condition of Fmoc amino acids. Upon completion of the synthesis, the
glycopeptidyl-resin was treated with 90% aqueous TFA (3 ml) at ambient temperature
for 1 h, and the resin was filtered off. The resin was washed with 90% aqueous TFA
(2×1 ml) and the combined filtrate was concentrated by a flow of nitrogen gas. The
crude glycopeptide was precipitated using cold tert-butylmethylether. After collecting
the precipitate by centrifugation (3,000×g, 15 min, 4°C) and removal of supernatant, the
dried precipitate was dissolved in 50% acetonitrile aqueous (4 ml) and lyophilized. Next,
to a solution of the lyophilized material in methanol (2 ml) was added 1 M NaOH in
methanol (1:1, v/v) to adjust pH to 12.5 and kept at room temperature for 40 min. After
quenching with acetic acid, solvent was removed under reduced pressure. The crude
glycopeptide was purified by preparative RP-HPLC (tR = 25.4 min) to give 3 (14.2 mg,
8.8 mol) in 34% overall yield. The RP-HPLC purification condition was described
below: column, Inertsil ODS-3 (250×20 mmI.D.); eluent A, water containing 0.1%
TFA; eluent B, acetonitrile containing 0.1% TFA; Eluent (A/B = 98/2) was employed
and kept over 10 min, then the ratio of eluent B was increased lineally from 2% to 10%
over 40 min with a flow rate of 5.0 mL/min. Preparative RP-HPLC and
MALDI-TOFMS of 3 were shown in Figure S1. MALDI-TOFMS: C70H111N18O25
[M+H]+ calcd (m/z) 1603.797, found (m/z) 1603.565.
S3
(A)
concentration of eluent B (%)
3
time (min)
1603.565
Intens . [a .u.]
(B)
5000
3 [M+H]+
4000
1641.636
3000
2000
2356.480
2233.138
2029.813
2119.387
1895.467
1700.783
1546.504
1441.912
1271.483
1117.343
955.237
839.015
777.501
684.592
597.299
538.014
396.184
346.589
1000
0
500
750
1000
1250
1500
1750
2000
2250
2500
m /z
Figure S1. (A) Preparative RP-HPLC of crude glycopeptide 3. (B) MALDI-TOFMS of
the fraction corresponding to the peak at 25.48 min shown in (A).
S4
BLase-catalyzed enzymatic cleavage for molecular shuttle (model glycopeptides) 3
(Scheme S1).
To the stock solution of 3 in water (4 mM, 35 l) were added 500 mM ammonium
acetate buffer, pH 6.8 (14 l), 17.4 gml-1 BLase (1.6 l) and water (89 l). After
standing at room temperature, 10 l of the reaction mixtures after 0, 1, 2, 3, 6 and 12 h
were analyzed by RP-HPLC (Figure S2). The analytical RP-HPLC condition was
described below: column, Inertsil ODS-3 (φ4.6x250 mm); eluent A, 25 mM ammonium
acetate buffer, pH 5.0; eluent B, 10% eluent A in acetonitrile. Eluent (A/B = 95/5) was
employed and the ratio of eluent B was increased lineally from 5% to 40% over 40 min
and from 40% to 90% over 2 min, and kept 90% over 2min; then the ratio of eluent B
was decreased lineally from 90% to 5% over 1 min and kept 5% over 14 min with a
flow rate of 1.0 mL/min.
S5
Scheme S1. BLase-catalyzed enzymatic cleavage for molecular shuttle (model
glycopeptides) 3.
3
5
26
time (min)
Figure S2. RP-HPLC stack plot of reaction mixtures after 0, 1, 2, 3, 6 and 12 h of
BLase-catalyzed enzymatic cleavage for 3. The peaks at 17 min, 24 min, and 36 min are
correspondeding to 5, 26, and 3.
S6
Figure S3. SEC-HPLC stack plots of the reaction mixtures after 0, 1, 2, 3, 6, 12, 24 and
48 h of conjugation reaction between dendrimer 2 and molecular shuttle 3 under
different pH condition (pH 3.5, 4.0, 4.5, 5.0 and 5.5). The SEC-HPLC condition is as
indicated bellow; column: YMC-Pack Diol-200 column, 500×8.0 mm I.D., flow rate:
0.7 ml/min, eluent: 50 mM sodium phosphate buffer, 0.3 M NaCl (pH 7.0).
S7
10
9
3
8
buffer
7
1.0 eq
0.8 eq
0.6 eq
0.4 eq
0.2 eq
6
time (min)
Figure S4. SEC-HPLC stack plot of conjugation reaction after 48 h for preparing
glycopeptide-dendrimer conjugates 6, 7, 8, 9 and 10 using 0.2, 0.4, 0.6, 0.8 and 1.0
equivalent molecular shuttle 3 to 1.0 equivalent aminooxy group on dendrimer 2,
respectively. The analytical SEC-HPLC condition is as indicated bellow: column;
YMC-Pack Diol-200 column, 500×8.0 mmI.D., flow rate; 0.7 ml/min, eluent; 50 mM
sodium phosphate buffer, 0.3 M NaCl (pH 7.0).
S8
Figure S5. 600 MHz 1H-NMR spectrum of 1 in methanol-d4 at 300 K.
Figure S6. 600 MHz 1H-NMR spectrum of 2 in D2O at 300 K.
S9
S10