carbonyls (171.58 ppm; the integration ratio is about 1 : 1 in
PhPOLY-100).
(1C, broad), 22.31 (1C, broad), 21.76 (0.4C). The H/D exchange
of the acidic acetylene H atom occurs in a few min when
PRGLPOLY-40 is dissolved in D2O at room temperature, and is
maintained in further manipulations (lyophilization and further
dissolution for hydrogenation).
Synthesis of PhPOLY-40. The procedure is the same as for
PhPOLY-100, but the quantity of ethylphenylpropiolate to be
used is only 38 ll (0.25 mmol). In this case, the product is
partially soluble in the reaction solvent; therefore, at the end
of the reaction, it is necessary to evaporate the solvent, and
then to wash repeatedly the residue with small quantities of
water to eliminate NaBr. PhPOLY-40 is soluble also in water at
pH = 6.7, and partially soluble at pH = 11. Yield = 85%. dC
(400 MHz, CD3OD): 178.3 (1C), 174 (1C), 155.9 (0.4C); 133.78
(0.8C), 130.45 (0.4C), 130.09 (0.8C), 121.84 (0.4C); 86.08 (0.4C),
84.25 (0.4C); 55.5 (1C), 49.3 (broad, 1C), 41.1 (broad,1C), 31.7
(broad,1C), 24.36 (broad,1C).
Synthesis of PRAM-DG-COOH. 2.53 g of DG-COOH
(PM = 220, 11.5 mmol) were introduced into a three-necked
recipient, connected to a gas inlet and a bubbler. Ar was
introduced, then about 10 ml of SOCl2 were dropped slowly,
while mixing. The mixture was mixed at room temperature
until no gas was detected in the bubbler, then it was connected
to a vacuum pump, and the excess of SOCl2 was removed at
room temperature. The residue was dissolved in about 40 ml of
dichloromethane under Ar atmosphere, 5.0 g of K2CO3 were
added, and the suspension was cooled to 273 K. 40 ml of a
dichloromethane solution of propargylamine (0.604 ml, 9.43
mmol) were then added in about 1 h, while vigorously stirring
the suspension. Stirring was carried on for 1 h after the end of
the addition. About 0.20 ml of water (11.1 mmol) were added
at room temperature, and the mixture was stirred for 1 h. The
suspension was filtered and PRAM-DG-COOH was extracted
by addition of about 40 ml of water to the solution. The aqueous
phase was washed twice with 40 ml of dichloromethane and
brought to pH 4.5 by adding HCl. The product was purified by
reverse phase column chromatography, gradient elution water→
water–acetonitrile 5% (Rf = 033). Yield ca. 20%. dC (400 MHz,
D2O): 174.08 (1C, pH dependent); 172.20 (1C); 79.61 (1C), 70.55
(1C, JCH = 250.6 Hz); 70.08 (1C), 69.98 (1C), 69.86 (1C), 69.69
(1C), 69.65 (1C), 69.53 (1C); 28.48 (1C). dH (400 MHz, D2O):
4.13 (2H), 4.05 (2H), 3.98 (2H), ca. 3.7 (8H); 2.65.
Synthesis of PRGL-ac. 2.33
g of glutaric anydride
(21.56 mmol) were dissolved in 100 ml of dichloromethane,
1.4 ml of propargylamine (20.4 mmol) were added, and the
mixture was refluxed for 1 h. The solvent was removed in
vacuum. The residue was loaded on an XAD 1180 chromato-
graphic column (height 40 cm), and washed with 100 ml of
water. The pure product was collected by elution with 50 ml
of methanol. Another fraction was collected immediately after
that, containing just impurities. The chromatographic curve was
obtained by measuring the UV absorbance of the fractions (215,
254 nm). Yield 81%. dC (400 MHz, D2O): 177.23 (1C), 175.01
(1C); 79.63 (1C), 71.88 (1C); 34.54 1(1C), 32.90 (1C), 28.69 (1C),
20.54 (1C). dH (400 MHz, D2O): 2.50 (1H, t, J = 2.4 Hz); 3.84
(2H, d, J = 2.4 Hz); 2.29 (2H, t), 2.20 (2H, t), 1.77 (2H, t).
Synthesis of PRGLPOLY-100. 100 mg of PRGL-ac
(0.59 mmol), 110 mg of N-hydroxysuccinimide (NHS,
0.96 mmol) and 220 mg of dicyclohexylcarbodiimide (DCC,
1.07 mmol) were dissolved in about 25 ml of acetonitrile. The
mixture was heated to 65 ◦C and stirred at this temperature
for 6 h. After this period, the mixture was left to cool to
room temperature and the solvent was evaporated in vacuum.
The residue was then redissolved in a few ml of anhydrous
dichloromethane and filtered upon celite. The solution was dried
again, yielding a slightly yellow solid residue of reactive ester.
This was dissolved again in 10 ml of acetonitrile, and added to
about 20 ml of acetonitrile solution of poly(lysine) (obtained
by neutralizing the commercial poly(lysine) hydrobromide with
tetrabutylammonium hydroxide in water up to pH = 11). The
mixture was stirred at room temperature for 48 h: during this
period a white precipitate of PRGLPOLY-100 is formed. The
solid was isolated, washed once with acetonitrile and once with
a few ml of water, and then dried in vacuum. It is soluble in
DMSO. Yield 60%. dC (400 MHz, DMSO-d6): 171.77 (3C);
81.40 (1C), 72.83 (1C); 52.60 (broad, 1C); 38.52 (broad, 1C),
34.97 (1C), 34.60 (1C), 31.70 (broad,1C), 28.96 (broad,1C),
27.88 (1C), 23.01 (broad,1C), 21.49 (1C). The evaluation of
the percentage of substituted arms (for both PRGLPOLY-100
and PRGLPOLY-40) was achieved by integration of the 13C
NMR signal attributed to the chain CH groups (52.60 ppm)
with respect to the signal attributed to the triple bond carbon
atom (81.40 ppm), in the absence of NOE (the integration ratio
is about 1 : 1 in PRGLPOLY-100).
Synthesis of PRDGPOLY-40. 67 mg of PRAM-DG-COOH
(0.27 mmol), 57 mg of N-hydroxysuccinimide (NHS, 0.50 mmol)
and 98 mg of dicyclohexylcarbodiimide (DCC, 0.47 mmol) were
dissolved in about 20 ml of distilled acetonitrile. The mixture was
◦
heated to 70 C and stirred at this temperature for 24 h. After
this period, the mixture was left to cool to room temperature
and filtered. The solution was then added to about 20 ml of an
acetonitrile solution of poly(lysine) (obtained by neutralizing
100 mg of commercial poly(lysine) hydrobromide (0.04 mmol)
with tetrabutylammonium hydroxide in water up to pH = 11).
The mixture was stirred at room temperature for 3 d: during
this period a white precipitate of PRDGPOLY-40 is formed.
The solid was isolated and washed once with acetonitrile, and
then dried in vacuum. The product was purified by means of
a de-salting process, eluting it on a Sephadex G10 column
with water. The H/D exchange of the acidic acetylene H atom
occurs in a few min when PRDGPOLY-40 is dissolved in D2O at
room temperature, and is maintained in further manipulations
(lyophilization and further dissolution for hydrogenation). Yield
50%. dC (400 MHz, D2O): 173.68 (1C, broad), 172.62; 78.31
(0.4 C, t, JCD = 10 Hz), 72.23 (0.4 C, d, JCD = 70 Hz); 70.00
(0.4 C, t), 69.5 (2 C); 53.62 (1C, broad); 39.21 (1.4C, broad),
30.65 (1C, broad), 26.88 (1C, broad), 22.05 (1C, broad). The
evaluation of the percentage of substituted arms was achieved
by integration of the 13C NMR signal attributed to the chain CH
groups (53.62 ppm) with respect to the signal attributed to the
triple bond carbon atom (78.31 ppm), in the absence of NOE.
Synthesis of PRGLPOLY-40. The procedure is the same
described for PRGLPOLY-100, but the following quantities were
used: 37 mg of monopropargylamide of glutaric acid, 41 mg of
NHS, 82 mg of DCC in about 12 ml of acetonitrile. In this
case, the product is water-soluble, therefore for the purification
a de-salting process was carried out, eluting the product on
a Sephadex G10 column with water. Yield 55%.dC 400 MHz,
D2O): 175.53 (0.8C), 173.84 (1C, broad); 79.26 (0.4C), 71.69
(0.4C, t, JCD = 70 Hz); 53.72 (1C, broad); 39.54 (1C, broad),
34.97(0.4C), 34.71 (0.4C), 30.69 (1C, broad), 28.80 (0.4C), 28.00
References
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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 4 8 – 3 9 5 4
3 9 5 3