4760 J . Org. Chem., Vol. 64, No. 13, 1999
Schwope et al.
Solid Su p p or t 7. Aminopropyl controlled pore glass (15 mg,
1.95 µmol amino groups) was reacted with steps A (using 5)
and B, followed by coupling with Fmoc-Gly-OH (step A) and
Fmoc removal (step C).
Solid Su p p or t 8. A sample of support 7 (5 mg, 0.6 µmol
amino groups) was reacted with 4 using step A and deprotected
with step B.
H-TGCGCA-GA-Gly-OH (9). Derivatized controlled pore
glass 8, prepared as described in the preceding paragraph, was
subjected to DNA synthesis following protocols for steps E and
F. MALDI-TOF MS for C62H80N25O39P6 [M - H]-: calcd 1985.3,
found 1985.
min. MALDI-TOF MS for C73H96N27O45P6 [M - H]-: calcd
2257.6, found 2257.
H-Lys-Gly-TGCGCA-DP -Lys-OH (32). Yield 38%. HPLC:
CH3CN gradient 0% for 5 min to 25% in 50 min, Rt ) 42.2
min. MALDI-TOF MS for C77H110N29O41P6 [M - H]-: calcd
2283.7, found 2281.
Lib r a r y H -Aa -Gly-TGCGCA-DP -Tr p -OH (33). Aa
Ala: MALDI-TOF MS for C79H101N28O41P6 [M - H]- calcd
2284.7, found 2284. Aa ) Pro: MALDI-TOF MS for C81H104
)
-
N
28O41P6 [M - H]- calcd 2311.7, found 2310. Aa ) Asp:
MALDI-TOF MS for C80H101N28O43P6 [M - H]- calcd 2328.7,
found 2327. Aa ) Met: MALDI-TOF MS for C81H105N28O41P6S
[M - H]- calcd 2344.8, found 2343. Aa ) Phe: MALDI-TOF
MS for C85H105N28O41P6 [M - H]- calcd 2360.8, found 2360.
Aa ) Tyr: MALDI-TOF MS for C85H105N28O42P6 [M - H]-
calcd 2376.8, found 2376. Aa ) Trp (30), found 2399.
H-TGCGCA-LA-Gly-OH (11). A sample of support 7 (5 mg,
0.6 µmol amino groups) was reacted with 5 using step A,
followed by steps D and B. DNA synthesis and deprotection
was performed with the protocol for steps E (manufacturer’s
protocol for 0.2 µmol scale) and F. MALDI-TOF MS for
H-P h e-Gly-TGCGCA-DP -P h e-OH (24). Yield 41%. HPLC:
CH3CN gradient 0% for 5 min to 25% in 45 min, Rt ) 47.0/
49.7 min. MALDI-TOF MS for C83H104N27O41P6 [M - H]-:
C
72H100N25O39P6 [M - H]-: calcd 2125.6, found 2126.
H-TGCGCA-DP -Gly-OH (10). Compound 10 was prepared
analogously to the synthesis of 11, above, except that 6 was
used in the first coupling reaction. Alternatively, 10 was
prepared through deprotection of solid support 25, using step
F. MALDI-TOF MS for C65H86N25O39P6 [M - H]-: calcd 2027.4,
found 2027.
Solid Su p p or t 12. Controlled pore glass (5 mg, 0.65 µmol
amino groups) was derivatized to triester support 12 by
performing three consecutive rounds of step A (with 5) and
step B, with step D prior to the final detritylation. This
decreased the loading to 0.2 µmol (31%), as measured by trityl
release.
1
calcd 2321.7, found 2320. H NMR (300 MHz, D2O, 22 °C) δ
0.93 (s, 6H, CH3-DP9), 1.59 (s, 3H, CH3-T3), 1.61 (m, 1H,
H2′-C7), [1.7-4.9 ppm range, selected resonances by residue,
2.70/2.76 (Hâ/Hâ′-Phe1), 3.70 (HR-Phe1), 3.02 (HR/HR′-
Gly2), 1.92/2.24 (H2′/H2′′-T3), 3.22/3.33 (H5′/H5′′-T3), 3.93
(H4′-T3), 4.49 (H3′-T3), 2.62/2.66 (H2′/H2′′-G4), 3.93 (H5′/
H5′′-G4/G6), 4.26 (H4′-G4/G6), 4.85 (H3′-G4), 1.91/2.29 (H2′/
H2′′-C5), 3.907 (H5′/H5′′-C5), 4.07 (H4′-C5), 4.74 (H3′-C5),
2.50/2.53 (H2′/H2′′-G6), 4.84 (H3′-G6), 2.05 (H2′′-C7), 3.90
(H4′-C7), 4.64 (H3′-C7), 2.66/2.48 (H2′/H2′′-A8), 3.96 (H5′/
H5′′-A8), 4.21 (H4′-A8), 4.83 (H3′-A8), 2.66/2.78 (Hâ/Hâ′-
Phe10), 4.34 (HR-Phe10)], 5.28 (d, J ) 7 Hz, 1H, H5-C5),
5.30 (d, J ) 6.8 Hz, 1H, H5-C7), 5.59 (m, 1H, H1′-C7), 5.62
(m, 1H, H1′-C5), 5.65 (m, 1H, H1′-T3), 5.73 (t, J ) 6.7 Hz,
1H, H1′-G6), 5.85 (dd, J ) 5.9 Hz, 9.2 Hz, 1H, H1′-G4), 6.17
(t, J ) 6.7 Hz, 1H, H1′-A8), 6.82-7.06 (m, 10H, aromatic
H-Phe1/Phe10), 7.09 (d, J ) 7.4 Hz, 1H, H6-C7), 7.15 (s, 1H,
H6-T3), 7.26 (d, J ) 7.4 Hz, 1H, H6-C5), 7.63 (s, 1H, H2-
A8), 7.78 (s, 1H, H8-G6), 7.89 (s, 1H, H8-G4), 8.15 (s, 1H,
H8-A8); exchangeable protons observed in H2O/D2O (88:12)
at 22 °C δ 6.40 (br s, 1H, H42-C5), 6.54 (br s, 1H, H42-C7),
7.45 (d, J ) 7.5 Hz, 1H, NHR-Phe10), 7.76 (NH5′-T3), 8.30
(br s, 1H, H41-C7), 8.34 (br s, 1H, H41-C5), 12.92 (br s, 1H,
NH1-G4); exchangeable proton observed in H2O/D2O (88:12)
and 100 mM NaCl at 7 °C δ 8.09 (NHR-Gly2), 12.84 (br s,
1H, NH1-G6).
Solid Su p p or ts 13-17, 18-22, 23, a n d 25-28. A sample
of support 12 (0.5-0.7 µmol of terminal hydroxyl groups),
prepared as described in the preceding paragraph, was twice
subjected to step A with the respective Fmoc-amino acid
building block, followed by step C and steps A (twice, with 6)
and B. DNA synthesis (step E) was performed with the DNA
synthesis program for 1.0 µmol syntheses. Solid support 23
was prepared on a 4 µmol scale to provide sufficient material
for NMR spectroscopy. Release of the dimethoxytrityl cation
during DNA syntheses indicated between 81% and 90% overall
yield (97-98.3% per step) for the chain assembly.
H -Aa -Gly-TGCGCA-DP -Gly-OH (24, 29-33). The
amino-terminal glass supports, prepared as described in the
preceding protocol, were subjected to step A with Fmoc-Gly-
OH (repeated once), Fmoc removal with step C, and two
consecutive couplings A with the appropriate Fmoc-amino
acid building block. For 24, this was performed at a 4-fold
scale. For library 33, a mixture of Fmoc-Ala-OH (3.5 mg, 11
µmol), Fmoc-Pro-OH (7.1 mg, 21 µmol), Fmoc-Asp(Bn)-OH (5.1
mg, 11 µmol), Fmoc-Met-OH (4.8 mg, 13 µmol), Fmoc-Phe-OH
(3.5 mg, 9 µmol), Fmoc-Tyr-OH (3.9 mg, 10 µmol), and Fmoc-
Trp-OH (4.5 mg, 10 µmol) was used with the activation agents
as given in the general protocol for step A. The last coupling
step was followed by deprotection according to step F. For 32,
allyloxycarbonyl groups were removed by exposure of 28 to
PPh3 (1.5 mg, 23 µmol), Pd[PPh3]4 (7.5 mg, 20.5 µmol), and
diethylammonium hydrogen carbonate (7.5 mg, 56 µmol) in
CH2Cl2 (0.7 mL) for 2.5 h at room temperature under light
protection, followed by washing with CH3CN (10 mL). Yields
of hybrids were determined from the integration of the product
peaks in the HPLC trace of the crude. For lipophilic sequences,
no full recovery from RP18 columns was achieved, leading to
lowered yields.
H-P h e-TGGTTGAC-DP -P h e-OH (35). Starting from sup-
port 22 (0.1 µmol free hydroxyl groups), 34 was prepared with
the DNA synthesis program for 0.2 µmol scale analogously to
the preparation of 25-28. A single coupling step (A) with
Fmoc-Phe-OH was followed by treating 20% of the protected
hybrid with NH4OH according to protocol F . MALDI-TOF MS
for C102H127N30O54P8 [M - H]-: calcd 2902.1, found 2906.
Nu clea se Sta bility Assa y. The exonuclease stability assay
was performed similarly to those described previously.22,20
Specifically, a solution of 35 (300 pmol) in water (1.3 µL) was
mixed with a solution of the unmodified DNA control octamer
5′-TGGTTGAC-3′ (300 pmol) in water (1.7 µL), followed by
addition of additional water (7 µL) and 0.5 M NH4OAc buffer
pH 6.0 (1.8 µL). The sample was annealed at 55 °C, and the
nuclease reaction was performed at 37 °C with phosphodi-
esterase I, type VII (EC 3.1.4.1, 0.5 units). The unmodified
DNA decamer 5′-CGCATTAGCA-3′ was used as internal
standard. Data acquisition16 and analysis22,20 were performed
as previously described.
H-Gly-Gly-TGCGCA-DP -Gly-OH (29). Yield 84%. HPLC:
CH3CN gradient 0% for 5 min to 25% in 45 min, Rt ) 39.2
min. MALDI-TOF MS for C69H92N27O41P6 [M - H]-: calcd
2142.5, found 2139.
Ack n ow led gm en t. The authors are grateful to
David Sarracino for help with the acquisition of MALDI
spectra, Charles Tetzlaff for computer support and help
with DNA syntheses, and Dr. Dwayne Daniels and
Robert Altman for helpful discussions. I.S. was sup-
ported by a postdoctoral fellowship from the Deutsche
H-Tr p -Gly-TGCGCA-DP -Tr p -OH (30). Yield 69%. HPLC:
CH3CN gradient 0% for 5 min to 25% in 45 min, Rt ) 47.1/
49.8 min. MALDI-TOF MS for C87H106N29O41P6 [M - H]-:
calcd 2399.8, found 2402.
H-Asp -Gly-TGCGCA-DP -Asp -OH (31). Yield 93%. HPLC:
CH3CN gradient 0% for 5 min to 22% in 45 min, Rt ) 36.2