4.25 – 3.91 (m, 2H), 3.84 – 3.60 (m, 2H), 2.88 (d, J = 11.2 Hz,
2H), 2.70 (t, J = 11.5 Hz, 2H), 2.42 (dd, J = 14.5, 6.7 Hz, 2H),
2.18 (d, J = 6.8 Hz, 3H), 1.94 (t, J = 11.2 Hz, 2H), 1.84 – 1.55
(m, 7H), 1.47 (s, 9H), 1.37 – 1.26 (m, 2H), 1.08 (qd, J = 12.7, 4.2
Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 154.95, 136.68, 115.71,
81.16, 79.33, 64.85, 53.86, 34.75, 33.65, 32.39, 30.80, 29.36,
28.85, 28.46; HRMS (ESI): calcd for C22H39N3O4 [M+Na]+ m/z =
410.3013, found: 410.3005.
chromatography (MeOH/CH2Cl2) to afford the fully protected N-
pentenoyl aminooxy digalactose melamine dendron 15 as a clear
residue (123 mg, 75%). Rf = 0.52 (3:17, MeOH/CH2Cl2); H
1
NMR (600 MHz, CDCl3) δ 8.07 (d, J = 7.6 Hz, 4H), 7.99 (t, J =
14.7 Hz, 4H), 7.93 (d, J = 7.7 Hz, 4H), 7.77 (d, J = 7.7 Hz, 4H),
7.61 (t, J = 7.2 Hz, 2H), 7.53 (dt, J = 20.2, 7.2 Hz, 4H), 7.46 (dd,
J = 16.2, 8.7 Hz, 4H), 7.40 (dt, J = 21.7, 7.6 Hz, 10H), 7.23 (t, J
= 7.5 Hz, 4H), 5.99 (d, J = 2.1 Hz, 2H), 5.83 – 5.71 (m, 3H), 5.61
(dd, J = 10.3, 2.6 Hz, 2H), 5.00 (dd, J = 40.2, 13.5 Hz, 2H), 4.80
(d, J = 7.8 Hz, 2H), 4.73 – 4.61 (m, 8H), 4.40 (dd, J = 11.1, 6.5
Hz, 2H), 4.33 (t, J = 6.2 Hz, 2H), 3.95 – 3.81 (m, 2H), 3.81 –
3.69 (m, 2H), 3.50 – 3.17 (m, 6H), 2.77 – 2.62 (m, 9H), 2.62 –
2.41 (m, 5H), 2.41 – 2.29 (m, 4H), 2.18 – 2.11 (m, 2H), 1.97 –
1.87 (m, 4H), 1.84 – 1.64 (m, 15H), 1.62 – 1.39 (m, 4H), 1.30 –
1.02 (m, 9H); 13C NMR (151 MHz, CDCl3) δ 166.00, 165.53,
165.50, 165.28, 162.20, 161.97, 133.62, 133.44, 133.36, 133.31,
129.97, 129.71, 129.60, 129.35, 129.16, 128.99, 128.69, 128.62,
128.49, 128.28, 128.19, 117.73, 115.79, 115.71, 101.85, 71.54,
71.40, 69.83, 68.11, 62.01, 42.90, 30.05, 29.65; HRMS (ESI):
calcd for C112H128N10O22 [M+H]+ m/z = 1965.9277, found:
1965.9256.
6-chloro-N,N-[6-((1-(piperidin-4-ylmethyl)piperidin-4-
yl)methoxy)-O-2,3,4,6-tetra-O-benzoyl-β-D-
galactopyranosyl]-1,3,5-triazine-2,4-diamine
(14).
Boc
protected dipiperidine 7 (1.09 g, 1.22 mmol) was dissolved in
10% TFA/CH2Cl2 (16 mL) and stirred at rt for 1.5 h. The reaction
was concentrated under reduced pressure, excess TFA removed
by coevaporation with toluene, and run through a silica column
(MeOH/CH2Cl2) to yield deprotected galactose dipiperidine 8 in
quantitative yield. Rf = 0.14 (1:9, MeOH/CH2Cl2); 1H NMR (500
MHz, CDCl3) δ 8.07 (d, J = 7.4 Hz, 2H), 7.99 (d, J = 7.4 Hz,
2H), 7.92 (t, J = 7.4 Hz, 2H), 7.76 (d, J = 7.4 Hz, 2H), 7.59 (q, J
= 7.3 Hz, 1H), 7.54 (dd, J = 13.9, 6.5 Hz, 1H), 7.51 – 7.44 (m,
3H), 7.39 (dt, J = 21.6, 7.7 Hz, 5H), 7.23 (t, J = 7.8 Hz, 2H), 5.99
(d, J = 3.2 Hz, 1H), 5.75 (dd, J = 10.2, 8.1 Hz, 1H), 5.61 (dd, J =
10.4, 3.3 Hz, 1H), 4.80 (d, J = 7.9 Hz, 1H), 4.68 (dd, J = 11.0,
6.3 Hz, 1H), 4.43 – 4.36 (m, 1H), 4.34 (t, J = 6.3 Hz, 1H), 3.84
(s, 1H), 3.59 – 3.47 (m, 1H), 3.47 – 3.39 (m, 1H), 3.39 – 3.22 (m,
3H), 2.96 – 2.73 (m, 5H), 2.57 – 2.37 (m, 2H), 2.18 – 2.06 (m,
1H), 2.00 – 1.73 (m, 6H), 1.67 – 1.45 (m, 4H); HRMS (ESI):
calcd for C46H50N2O10 [M+H]+ m/z = 791.3538, found: 791.3546.
The resulting residue was dissolved in MeCN (18 mL) and N,N-
diisopropylethylamine (320 µL, 1.8 mmol) was added dropwise
followed by cooling to 0 ºC. Cyanuric chloride (113 mg, 0.611
mmol) was added and the mixture was allowed to warm to rt and
stirred for 2 h followed by concentration under reduced pressure.
The crude product was purified by a silica flash column
(MeOH/CH2Cl2) to afford the two-arm galactose melamine
dendron 4.14 (936 mg, 90%). Rf = 0.69 (1:9, MeOH/CH2Cl2); 1H
NMR (600 MHz, CDCl3) δ 8.07 (d, J = 7.6 Hz, 4H), 8.00 (d, J =
7.6 Hz, 4H), 7.93 (d, J = 7.6 Hz, 4H), 7.77 (d, J = 7.7 Hz, 4H),
7.61 (d, J = 7.0 Hz, 2H), 7.53 (dt, J = 24.4, 7.3 Hz, 4H), 7.48 (t, J
= 7.4 Hz, 4H), 7.40 (dt, J = 19.8, 7.7 Hz, 10H), 7.23 (t, J = 7.7
Hz, 4H), 5.99 (d, J = 2.4 Hz, 2H), 5.76 (dd, J = 9.9, 8.3 Hz, 2H),
5.61 (d, J = 9.2 Hz, 2H), 4.80 (d, J = 7.9 Hz, 2H), 4.68 (d, J = 6.2
Hz, 6H), 4.41 (dd, J = 11.2, 6.4 Hz, 2H), 4.34 (t, J = 6.3 Hz, 2H),
3.99 – 3.79 (m, 2H), 3.65 – 3.25 (m, 6H), 2.93 – 2.58 (m, 9H),
2.50 – 2.27 (m, 3H), 2.05 – 1.48 (m, 17H), 1.30 – 1.07 (m, 5H);
13C NMR (151 MHz, CDCl3) δ 165.89, 165.41, 165.37, 164.07,
133.53, 133.28, 129.85, 129.60, 129.49, 129.23, 128.88, 128.52,
128.40, 128.18, 101.72, 71.36, 69.71, 68.00, 61.93, 42.83;
HRMS (ESI): calcd for C95H98N7O20Cl [M+H]+ m/z = 1692.6628,
found: 1692.6578.
N-[1-((N-Boc-piperidin-4-yl)methyl)piperidin-4-
yl)methoxypent-4-enamide]-N,N-[6-((1-(piperidin-4-
ylmethyl)piperidin-4-yl)methoxy)-β-D-galactopyranosyl]-
melamine (16). Fully protected dendron 15 (100 mg, 0.05 mmol)
was dissolved in MeCN/H2O/CH2Cl2 (4:1:1, v:v; 3 mL) and
NaOH (1 M in NaOH, 150 µL) was added dropwise while
stirring at rt. The mixture was chilled to 0 ºC and reacted for 48 h
after which AcOH was added dropwise until the pH reached 7.
Excess solvent was removed under reduced pressure and the
resulting residue was dissolved in ddH2O/MeCN (9:1, v:v; 1.5
mL) followed by HPLC purification. The product eluted at 14
min in a gradient of 10% to 50% MeCN in ddH2O (0.1% TFA)
over 25 min. The product peaks were collected and lyophilized to
obtain N-pentenoyl aminooxy galactose dendrimer 16 as a white
1
powder (55 mg, 95%). H NMR (500 MHz, D2O) δ 5.87 – 5.66
(m, 1H), 5.03 (dd, J = 17.8, 14.1 Hz, 2H), 4.45 (d, J = 12.7 Hz,
6H), 4.34 (d, J = 7.8 Hz, 2H), 3.88 (d, J = 2.8 Hz, 3H), 3.82 –
3.67 (m, 9H), 3.66 – 3.56 (m, 11H), 3.54 (dd, J = 10.1, 6.3 Hz,
2H), 3.47 (dd, J = 9.8, 8.0 Hz, 2H), 3.36 – 3.23 (m, 2H), 3.08 –
3.00 (m, 12H), 2.96 (t, J = 11.9 Hz, 6H), 2.36 – 2.27 (m, 2H),
2.27 – 2.19 (m, 5H), 2.09 – 1.91 (m, 10H), 1.86 – 1.80 (m, 6H),
1.59 – 1.44 (m, 6H), 1.32 – 1.23 (m, 6H); 13C NMR (126 MHz,
CDCl3) δ 172.29, 163.18, 162.90, 162.61, 162.33, 156.58,
136.35, 119.74, 117.42, 115.93, 115.10, 112.78, 102.96, 79.57,
75.09, 73.35, 72.67, 70.70, 68.54, 61.69, 60.89, 52.96, 52.83,
44.14, 33.22, 31.83, 31.62, 30.34, 28.94, 28.81, 25.70, 25.50;
HRMS (ESI): calcd for C56H96N10O14 [M+H]+ m/z = 1133.7180,
found: 1133.7173
N-[1-((N-Boc-piperidin-4-yl)methyl)piperidin-4-
N-[1-((N-Boc-piperidin-4-yl)methyl)piperidin-4-
yl)methoxypent-4-enamide]-N,N-[6-((1-(piperidin-4-
yl)methoxypent-4-enamide]-N,N-[6-((1-(piperidin-4-
ylmethyl)piperidin-4-yl)methoxy)-O-2,3,4,6-tetra-O-benzoyl-
ylmethyl)piperidin-4-yl)methoxy)-(5-acetamido-3,5-dideoxy-
D-glycero-α-D-galacto-2-nonulopyranosyl-onic acid)-(2→6)-
β-D-galactopyranosyl]-melamine (17). To N-pentenoyl
galactose dendrimer 16 (4.8 mg, 0.0042 mmol) was added N-
acetylmannosamine (2.8 mg, 0.013 mmol), sodium pyruvate (4.7
mg, 0.042 mmol), and CTP•Na (7.2 mg, 0.013 mmol) and
dissolved in ddH2O (1 mL). A concentrated stock of Tris-HCl
buffer pH 8.5 with MgCl2 was added to a final concentration of
100 mM Tris, 20 mM MgCl2. Recombinant E. coli K12 sialic
acid aldolase (1 U), N. meningitidis CMP-sialic acid synthetase
(0.5 U), and P. damsela α-2,6-sialyltransferase (0.5 U) were
added and the reaction mixture was incubated at 37 ºC for 4 h
followed by shaking at rt for 16 h. The reaction was monitored
by LCMS (5% to 50% MeCN in ddH2O over 15 min) and after
β-D-galactopyranosyl]-melamine
(15).
Boc
protected
dipiperidine 12 (95 mg, 0.232 mmol) was dissolved in 10%
TFA/CH2Cl2 (2.5 mL) and stirred at rt for 1 h. The reaction was
concentrated under reduced pressure and excess TFA removed by
coevaporation with toluene to yield deprotected N-
methoxypentenamide dipiperidine 13. The resulting residue was
dissolved in THF/MeCN (1:1, v:v; 4 mL) and the two-arm
galactose melamine dendron 14 (140 mg, 0.083 mmol) was
added
followed
by
dropwise
addition
of
N,N-
diisopropylethylamine (87 µL, 0.50 mmol). The reaction was
heated at 60 ºC for 7 days upon which the solvent was removed
under reduced pressure and purified by silica column