Aminoacylation of nucleosides with FMOC amino acid fluorides

Full text of DOI:10.1021/jo9601674

4168
J . Org. Chem. 1996, 61, 4168-4171
The 9-fluorenylmethoxycarbonyl (FMOC) group has
been found to be an advantageous protecting group
for the aminoacylation of dinucleotides.15 Both the
dimethoxytrityl (DMT) and FMOC protecting groups are
readily removed without deacylation of an aminoacylated
nucleotide.11b,15 Therefore we have chosen to study
activated N-FMOC protected amino acids as acylating
reagents. In particular, the N-FMOC amino acid fluo-
rides show several advantages over other aminoacylation
reagents.16 These derivatives are stable crystalline
compounds, yet readily react with nucleophiles. Our
initial results using these activated amino acids to
aminoacylate nucleosides are described below.
Am in oa cyla tion of Nu cleosid es w ith F MOC
Am in o Acid F lu or id es1
J . S. Oliver* and A. Oyelere
Department of Chemistry, Brown University,
Providence, Rhode Island 02912
Received J anuary 26, 1996
In tr od u ction
Aminoacylated nucleosides and oligonucleotides are
important molecules in bioorganic chemistry. Amino-
acylated nucleotides are key intermediates in the chemi-
cal synthesis of aminoacylated tRNAs.2,3 Furthermore,
aminoacyl adenylates are intermediates in the ribosome-
mediated biosynthesis of peptides, and related adenylates
show pharmacological activity.4 Therefore, efficient
methods for the synthesis of aminoacylated nucleosides,
nucleotides, and nucleic acid derivatives are highly
desirable.
Our interest in models of ribosomal and nonribosomal
peptide synthesis on monolayer films required the syn-
thesis of a number of aminoacylated nucleoside deriva-
tives. A survey of methods for aminoacylating nucleo-
sides and nucleoside derivatives revealed a large number
of published procedures, none of which provided a high
yielding and easy route to the desired products. On the
other hand we found that the N-FMOC amino acid
fluorides used by Carpino5 for the synthesis of peptides,
are superior reagents for the aminoacylation of nucleo-
sides.
Resu lts
The synthesis of the N-FMOC amino acid fluorides 2-4
proceeded as described.16a The acid fluorides were re-
crystallized from methylene chloride/hexane and stored
as white crystals at 4 °C. We chose N-FMOC-phenyla-
lanyl fluoride (4) as the model reagent in order to verify
that a sterically demanding amino acid would be coupled.
In addition we used a similarly derivatized alanine (3)
in order to test selectivity for monoacylation in the
coupling of a small amino acid with a ribonucleoside
which contains both 2′ and 3′ free hydroxyls.
The aminoacylation is simple to perform and proceeds
in high yield (Scheme 1). A representative procedure for
aminoacylation of the nucleosides involves the addition
of an N-FMOC amino acid fluoride to a solution of the
protected nucleoside in methylene chloride and pyridine.
The solution is stirred at room temperature for 4-12 h
followed by removal of the solvent and chromatography.
Table 1 presents data on yields for representative trans-
formations. Both pyrimidine and purine bases are
compatible with this chemistry though exocyclic amines,
if present on the base, must be protected. An amino-
acylation reaction performed on 5′-DMT-2′-deoxycytidine
resulted in a complex mixture of products. Although the
material was not characterized, it was assumed that
other products resulted from acylation of N-4 as well as
the 3′ hydroxy group. The fluorenylmethyloxy group has
been used to protect bases when necessary, though other
commonly used protecting groups such as N-benzoyl or
N-isobutyryl are presumably compatible.
Ba ck gr ou n d
A number of methods for the preparation of 5′-O-, 3′-
O-, or 2′(3′)-O, or 5′-OP-aminoacyl derivatives have
appeared. Early methods focussed on the aminoacylation
of the 5′-phosphate of adenosine 5′-monophosphate by
amino acid acyl chlorides,6 thiophenol esters of amino
acids,7 or DCC-mediated couplings.8 Methods for the
aminoacylation of 2′(3′)-O have utilized amino acid
anhydrides9 or cyanomethyl esters.10 Alternatively, the
amino acid has been activated in situ by a number of
coupling agents.11 Most of these synthetic procedures
proceed in extremely low yield. The prime exception,10
while proceeding in higher yield, is limited by the nature
of the protecting groups that can be placed on the amino
acid. Although aminoacylation of nucleosides has also
been accomplished with enzymes,12 catalytic antibodies,13
and recently an RNA molecule that accelerates the
aminoacylation of its 2′(3′) terminus,14 these are not
routine synthetic methods at this time.
(11) Including 1,1′-carbonyldiimidazole: (a) Roesser, J . R.; Xu, C.;
Payne, R. C.; Surratt, C. K.; Hecht, S. M. Biochemistry 1989, 28, 5185.
(b) Robertson, S. A.; Noren, C. J .; Cahill, S. J . A.; Griffith, M. C.;
Schultz, P. G. Nucleic Acids Res. 1989, 17, 9649. (c) Baldini, G.;
Martoglio, B.; Schachenmann, A.; Zugliani, C.; Brunner, J . Biochem-
istry 1988, 27, 7951-7959. BOP-Cl (N,N-bis(2-oxooxazolidin-3-yl)-
phosphorodiamidic chloride): Herranz, R.; Pichel, J . C.; Lopez, M. T.
G.; Perez, C.; Balzarini, J .; De Clerq, E. J . Chem. Soc., Perkin Trans.
1 1991, 43-48. BPOC/MST ([[2-(4-biphenylyl)isopropyl]oxy]carbonyl/
(mesitylenesulfonyl)tetrazole): (a) Kumar, G.; Celewicz, L.; Chla´dek,
S. J . Org. Chem. 1982, 47, 634. (b) Happ, E.; Scalfi-Happ, C.; Chla´dek,
S. J . Org. Chem. 1987, 52, 5387. (c) Hagen, M. D.; Scalfi-Happ, C.;
Happ, E.; Chla´dek, S. J . Org. Chem., 1988, 53, 5040. BOP/HOBT
((benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
phosphate/1-hydroxybenzotriazole): Bain, J . D.; Wacker, D. A.; Kuo,
E. E.; Lyttle, M. H.; Chamberlin, A. R. J . Org. Chem. 1991, 56, 4615.
(12) (a) Moris, F.; Gotor, V. Tetrahedron 1994, 50, 6927. (b) Montero,
J . L.; Criton, M.; Dewynter, G. F.; Imbach, J . L. Tetrahedron Lett. 1991,
32, 5357.
(1) Abbreviations used: FMOC, 9-fluorenylmethoxycarbonyl; T,
thymidine; U, uridine; AFMOC, N6-(9-fluorenylmethoxycarbonyl)adenin-
9-yl; CFMOC, N4-(9-fluorenylmethoxycarbonyl)cytos-1-yl; GFMOC, N2-
(9-fluorenylmethoxycarbonyl)guanin-9-yl.
(2) Hecht, S. M. Acc. Chem. Res. 1992, 25, 545.
(3) Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem., Int.
Ed. Engl. 1995, 34, 621-633 and references therein.
(4) Herranz, R.; Pichel, J . C.-; Lopez, M. T. G.-; Perez, C.; Balzarini,
J .; De Clercq, E. J . Chem. Soc., Perkin Trans. 1 1991, 43.
(5) Wenschuh, H.; Beyermann, M; Haber, H.; Seydel, J . K.; Krause,
E.; Bienert, M.; Carpino, L. A.; Faham, A. E.; Albericio, F. J . Org.
Chem. 1994, 60, 405.
(6) DeMoss, J . A.; Genuth, S. M.; Novelli, G. D. Proc. Nat. Acad.
Sci. USA 1956, 42, 325.
(7) Wieland, T.; Niemann, E.; Pfleiderer, G. Angew. Chem. 1956,
68, 305.
(13) J acobsen, J . R.; Prudent, J . R.; Kochersperger, L.; Yonkovich,
S; Schultz, P. G. Science 1992, 256, 365.
(14) Illangasekare, M.; Sanchez, G.; Nickles, T.; Yarus, M. Science
1995, 267, 643.
(8) Berg, P. J . Biol. Chem. 1958, 233, 608.
(9) Rammler, D. H.; Khorana, H. G. J . Am. Chem. Soc. 1963, 85,
1997.
(10) Ellman, J .; Mendel, D.; Cahill, S. A.-; Noren, C. J .; Schultz, P.
G. Methods Enzymol. 1991, 202, 301.
(15) Bain, J . D.; Wacker, D. A.; Kuo, E. E.; Lyttle, M. H.; Cham-
berlin, A. R. J . Org. Chem. 1991, 56, 4615.
(16) (a) Carpino, L. A.; Aalaee, D. S.-; Chao, H. G.; DeSelms, R. H.
J . Am. Chem. Soc. 1990, 112, 9651. (b) Carpino, L. A.; Faham, A. E. J .
Am. Chem. Soc. 1995, 117, 5401.
S0022-3263(96)00167-3 CCC: $12.00 © 1996 American Chemical Society

Notes
J . Org. Chem., Vol. 61, No. 12, 1996 4169
Sch em e 1. Syn th esis of Am in oa cyla ted Nu cleosid es
Ta ble 1. Am in oa cyla ted Nu cleosid es Syn th esized
undoubtedly be obtained by using a nucleoside with a
protecting group at the 2′ position. However the yield of
monoacylated ribonucleoside is high enough without prior
2′ protection that overall yields would suffer if protective
groups were incorporated.
amino acid
(equiv)
time
(h)
product
nucleoside
5′DMT-T
(% yielda)
Gly (2)
Ala (2)
Phe (1)
Phe (2)
Ala (2)
4
4
5a (97)
5b (97)
4
5c (71)
4
5c (96)
Con clu sion s
DMT-FMOC-C
4
5d (70)
Our present work describes a convenient method to
obtain aminoacylated derivatives of 2′-deoxy- and ribo-
nucleosides from N-protected amino acid fluorides. The
procedure is straightforward and proceeds in high yield.
12
4
5d (90)
Phe (2)
Phe (2)
5e (67)
12
4
5e (85)
DMT-FMOC-A
DMT-FMOC-G
DMT-U
5f (67)
12
12
4
5f (90)
Phe (2)
Ala (2)
Phe (1)
Phe (1.5)
Phe (2)
5g (74)
Exp er im en ta l Section
7a (57), 8a (41)
7b (55), 8b (12)
7b (60), 8b (15)
7b (51), 8b (31)
4
Pyridine and dichloromethane were refluxed over and then
distilled from CaH2. The following chemical reagents were
purchased: thymidine, uridine, cytidine, 4, 4′-dimethoxytrityl
chloride, 9-fluorenylmethyl choroformate, pyridine, N-(9-fluore-
nylmethoxycarbonyl)-L-alanine monohydrate, N-(9-fluorenyl-
methoxycarbonyl)-L-phenylalanine, and all solvents were pur-
chased from Aldrich. Cyanuric fluoride was from Alfa and was
used without further purification. Glycine was from General
Biochemicals. Trimethylchlorosilane and 2′-deoxyadenosine
were from Fluka. The 5′-O-(4,4′-dimethoxytrityl)-2′-deoxygua-
nosine was from Sigma. Analtech silica gel plates (60 F254) were
used for analytical TLC, and the spots were examined with UV
light. Column chromatography was carried out on 200-400
mesh silica gel. Melting points are reported uncorrected.
5′-Dimethoxytrityl derivatives of uridine and thymidine were
prepared following the procedure of Meier et al.17 The synthesis
of 6-N-(9-Fluorenylmethoxycarbonyl)-2′-deoxyadenosine was car-
ried out as described previously.18
4
4
a
Based on isolated pure compound.
Some interesting features of this reaction are evident.
In the case of 2′-deoxynucleosides 1a -1d the sterically
bulky amino acid phenylalanine is condensed in high
yield. The use of only 2 equiv of activated amino acid
results in a high yield of the aminoacylated product 5a -
5g. The reaction of amino acid fluorides with 5′-O-
(dimethoxytrityl)thymidine (1a ) is complete in 4 h and
isolated yields are high. The reaction of the FMOC
protected deoxynucleosides 1b-1d on the other hand is
incomplete after 4 h. Extension of the coupling time to
12 h resulted in higher yields.
In the case of ribonucleoside 6, there are two hydroxy
groups which can be aminoacylated. Aminoacylation at
either the 2′- or 3′-position gives a single monoacylated
product (7) in which the amino acid is rapidly exchanging
between the two hydroxy groups. Alternatively, both
hydroxy groups can be aminoacylated to give a diacylated
nucleoside (8). The mono- and diacylated products can
be easily separated by chromatography. For ribonucleo-
sides with free 2′ and 3′ hydroxyl groups, the yield of
mono aminoacylated product increases with ratios of
amino acid fluoride to ribose up to about 1.5. With more
than 1.5 equiv of the derivatized amino acid, the amount
of diacylated nucleosides becomes significant. Higher
yields for monoaminoacylation of the ribonucleoside could
5′-O-(Dim eth oxytr ityl)-4-N-(9-flu or en ylm eth oxycar bon yl)-
2′-d eoxycytid in e (1b). 4-N-(9-fluorenylmethoxycarbonyl)-2′-
deoxycytidine (0.5 g, 1.11 mmol) was placed in an oven-dried
round bottom flask (25 mL) and anhydrous pyridine (10 mL)
was added. 4, 4′-Dimethoxytrityl chloride (0.428 g, 1.2 mmol)
was added to the solution, and the reaction was stirred at room
temperature for 3 h. Solvent was removed from the reaction
mixture under vacuum, and the crude material was purified by
flash column chromatography (silica gel, step gradient of 30:1,
20:1, 15:1 CH2Cl2/MeOH) to give 0.55 g (65%) of a light yellow
(17) Meier, C.; Neumann, J .-M., Andre´, F.; Henin, Y.; Dinh, T. H.
J . Org. Chem. 1992, 57, 7300.
(18) Koole, L. H.; Moody, H. M.; Broeders, N. L. H. L.; Quaedflieg,
P. J . L. M.; Kuijpers, W. H. A.; van Genderen, M. H. P.; Coenen, A. J .
J . M.; van der Wal, S.; Buck, H. M. J . Org. Chem. 1989, 54, 1657.

4170 J . Org. Chem., Vol. 61, No. 12, 1996
Notes
solid. 1H-NMR (CDCl3, 250 MHz) δ 2.15 (1H, m), 2.77 (1H, m),
3.41 (2H, m), 3.72 (6H, s), 4.20-4.25 (2H, m), 4.38-4.49 (4H, m),
6.30 (1H, app t, J ) 5.7 Hz), 6.82 (4H, d, J ) 8.1 Hz), 6.99 (1H,
d, J ) 6.8 Hz), 7.16-7.41 (13H, m), 7.53 (2H, dd, J 1) 3.1 Hz,
3.1 Hz), 7.71 (2H, d, J ) 7.5 Hz), 8.23 (1H, d, J ) 7.4 Hz); 13C-
NMR (CDCl3, 100 MHz) δ 41.9, 46.5, 55.0, 62.8, 67.8, 70.8, 77.2,
86.5, 86.7, 87.2, 94.9, 113.2, 119.9, 124.9, 126.9, 127.1, 127.8,
127.8, 128.1, 129.9, 129.9, 135.3, 135.5, 141.1, 143.2, 143.3, 144.2,
144.3, 152.3, 155.2, 158.5, 162.4; HRMS (FAB, NBA/NaI) calcd
for [C45H41N3O8 + Na]+ 774.2791, found 774.2789.
5′-O-(Dim eth oxytr ityl)-6-N-(9-flu or en ylm eth oxycar bon yl)-
2′-d eoxya d en osin e (1c). 6-N-(9-Fluorenylmethoxycarbonyl)-
2′-deoxyadenosine (0.12 g, 0.25 mmol) was placed in an oven-
dried round bottom flask (15 mL). Anhydrous pyridine (3 mL)
was added by syringe. After the solid had dissolved 4, 4′-
dimethoxytrityl chloride (0.0990 g, 0.279 mmol) was added. The
reaction was stirred at room temperature for 3 h. Solvent was
removed from the reaction mixture under vacuum, and the crude
product was purified by flash column chromatography on silica
gel as for 1b to give (0.095 g, 48%) of a white powdery solid.
1H-NMR (CDCl3, 250 MHz) δ 2.54 (1H, m), 2.82 (1H, m), 3.10
(1H, br s), 3.39 (2H, m), 3.73 (6H, s), 4.17 (1H, m), 4.30 (1H, t,
J ) 6.6 Hz), 4.60 (2H, d, J ) 6.8 Hz), 4.70 (1H, br s), 6.44 (1H,
t, J ) 6.4 Hz), 6.76 (4H, d, J ) 8.7 Hz), 7.13-7.40 (13H, m),
7.62 (2H, d, J ) 7.3 Hz), 7.74 (2H, d, J ) 7.5 Hz), 8.10 (1H, s),
8.67 (1H, s), 8.81 (1H, s); 13C-NMR (CDCl3, 100 MHz) δ 40.2,
46.9, 55.2, 63.6, 67.7, 72.4, 77.2, 84.7, 86.2, 86.6, 113.2, 120.0,
122.3, 125.0, 126.9, 127.1, 127.8, 127.8, 128.1, 130.0, 135.6, 141.3,
141.3, 143.5, 144.4, 149.2, 150.8, 151.0, 152.6, 158.6; HRMS
(FAB, NBA/NaI) calcd for [C46H41N5O7 + Na]+ 798.2904, found
798.2910.
5′-O-(Dim eth oxytr ityl)-2-N-(9-flu or en ylm eth oxycar bon yl)-
2′-d eoxygu a n osin e (1d ). 5′-O-(Dimethoxytrityl)-2′-deoxygua-
nosine (0.12 g, 0.21 mmol) was placed in an oven-dried round
bottom flask (15 mL), and anhydrous pyridine (3 mL) was added.
Trimethylchlorosilane (0.06 mL, 0.47 mmol) was added dropwise
by syringe to the solution over a 3-min period. The reaction
mixture was stirred at room temperature for 1 h followed by
addition of 9-fluorenylmethyl choroformate (0.06 g, 0.23 mmol).
Stirring was continued for an additional 1.5 h. Hydrolysis of
excess reagent and trimethysilyl protecting groups was effected
by addition of water (2 mL) and stirring for 20 min. The solution
was poured into saturated aqueous NaHCO3 (10 mL) and
extracted with CH2Cl2 (3 × 15 mL). The organic fractions were
combined, dried over Na2SO4, and concentrated in vacuo. Flash
chromatography (silica gel, 12:1 CHCl3/MeOH) gave 65 mg (39%)
of chromatographically pure 1d as a white solid. 1H-NMR
(CDCl3, 250 MHz) δ 2.64 (2H, m), 3.43 (2H, br s), 3.61 (6H, s),
3.90 (1H, t, J ) 7.4 Hz), 4.14-4.36 (3H, m), 5.18 (1H, br s), 5.51
(1H, br s), 6.23 (1H, app t, J ) 5.5 Hz), 6.63 (4H, d, J ) 7.8 Hz),
6.97-7.50 (17H, m), 7.70 (1H, s), 9.92 (1H, br s), 11.40 (1H, br
s); 13C-NMR (CDCl3, 100 MHz) δ 40.1, 46.4, 55.1, 64.5, 68.2, 71.1,
77.2, 84.5, 86.3, 86.8, 113.0, 120.0, 120,0, 120.8, 124.8, 125.1,
126.7, 126.8, 127.0, 127.6, 127.9, 128.2, 129.9, 130.0, 135.9, 136.0,
138.2, 141.0, 141.1, 142.6, 143.1, 144.6, 146.7, 148.3, 154.1, 155.7,
158.4; HRMS (FAB, PEG600/NBA/NaOAc) calcd for [C46H41N5O8
+ Na]+ 814.2853, found 814.2864.
Rep r esen ta tive P r oced u r e for th e Syn th esis of F MOC
Am in o Acid F lu or id es. The procedure employed was as
described by Carpino et al.16 N-(9-Fluorenylmethoxycarbonyl)-
phenylalanine (0.7906 g, 2 mmol) was dissolved in dry dichlo-
romethane (15 mL) and dry pyridine (0.162 mL, 2 mmol).
Cyanuric fluoride (0.35 mL, 4 mmol) was added to the mixture,
and the reaction was refluxed for 3 h. During the reaction a
water soluble, white solid precipitated from the reaction. The
crude reaction mixture was poured into dichloromethane (30 mL)
and then extracted with cold water (3 × 15 mL). The organic
layer was dried with sodium sulfate, and the solvent was
evaporated to give a white glassy solid. The solid was recrystal-
lized from dichloromethane/hexane to give a 0.64 g (82%) of a
powdery white solid. TLC as described by Carpino in CH2Cl2/
MeOH 9:1 was used to verify that the product was free of
contaminating starting material. Mp 116-120 °C; IR (CaF2 cell)
cm-1 3427.5, 3033.3, 2955.6, 1847.0, 1728.4, 1607.9, 1509.5,
1450.9, 1400.2; 1H-NMR (CDCl3, 250 MHz) δ 3.13 (2H, m), 4.15
(1H, t, J ) 6.7 Hz), 4.39 (2H, m), 4.79 (1H, m), 5.19 (1H, d, J )
8.1 Hz), 7.09-7.75 (13H, m); 13C-NMR (CDCl3, 100 MHz) δ 36.84,
47.03, 53.43, 54.03, 67.22, 119.97, 124.88, 124.91, 127.04, 127.68,
127.75, 128.95, 129.12, 134.25, 141.28, 143.45, 143.55, 155.41,
159.87, 163.54.
Rep r esen ta tive P r oced u r e for th e Am in oa cyla tion Re-
a ction . 5′-O-(4,4′-Dim eth oxytr ityl-2′(3′)-O-(N-(9-flu or en yl-
m eth oxyca r bon yl)-L-p h en yla la n yl)ir id in e (7b) a n d 5′-O-
(4,4′-Dim eth oxytr ityl)-2′, 3′-O,O-bis(N-(9-flu or en ylm eth oxy-
ca r bon yl)-L-p h en yla la n yl)u r id in e (8b). 5′-O-Dimethoxy-tri-
tyluridine (6) (0.110 g, 0.201 mmol) was added to an oven-dried
round bottom flask (10 mL) under argon and coevaporated with
anhydrous pyridine (2 × 3 mL). Dry dichloromethane (1.5 mL)
and dry pyridine (0.33 mL) were added under argon. N-(9-
Fluorenylmethoxycarbonyl)-L-phenylalanyl fluoride (4) (0.118 g,
0.303 mmol) was added to the solution and the mixture stirred
at room temperature under argon for 4 h. The reaction was
concentrated and the crude material purified by flash chroma-
tography (silica gel, step gradient 8:1; 6:1; 3:1 CH2Cl2/acetone)
to give 0.110 g (60%) of 7b and 0.038 g (15%) of 8b as
chromatographically pure, white solids. A similar reaction
containing 6 (55 mg, 0.101 mmol) and 4 (39 mg, 0.101 mmol)
gave 51 mg (55%) of 7b and 16 mg (12%) of 8b. A reaction
containing 6 (55 mg, 0.101 mmol) and 4 (79 mg, 0.202 mmol)
gave 47 mg (51%) of 7b and 40 mg (31%) of 8b.
7b: 1H NMR (CDCl3, 250 MHz) δ 3.11 (2H, m), 3.43 (2H, m),
3.74 (3H, s), 3.76 (3H, s), 3.97-4.78 (6H, m), 5.34-5.42 (2H, m),
5.75 (0.75H, d, J ) 6.2 Hz), 5.96 (0.25H, d, J ) 8.1 Hz), 6.06
(0.75H, d, J ) 4.5 Hz), 6.11 (0.25H, d, J ) 4.5 Hz), 6.83 (4H, d,
J ) 7.9 Hz), 7.12-7.38 (18H, m), 7.48 (2H, d, J ) 7.0 Hz), 7.69-
7.78 (3H, m), 9.31 (0.25H, br s), 10.04 (0.75H, br s); 13C NMR
(CDCl3, 100 MHz). δ 37.4, 38.1, 46.9, 47.1, 55.2, 55.5, 62.3, 62.6,
67.0, 67.4, 69.9, 73.4, 74.6, 77.2, 81.7, 83.8, 86.2, 87.2, 87.3, 88.2,
102.7, 102.8, 113.3, 113.4, 119.9, 124.9, 125.0, 127.0, 127.1, 127.2,
127.2, 127.3, 127.7, 127.7, 128.1, 128.2, 128.6, 128.8, 129.1, 129.2,
130.0, 130.1, 130.1, 135.0, 135.0, 135.1, 135.6, 135.8, 136.0, 139.9,
140.0, 141.3, 143.6, 143.7, 144.1, 149.7, 150.3, 150.8, 156.0, 158.7,
158.7, 158.8, 163.2, 163.8, 170.4, 170.8. HRMS (FAB, NBA/NaI)
calcd for [C54H49N3O11 + Na]+ 938.3265, found 938.3277.
8b: 1H-NMR (CDCl3, 250 MHz) δ 3.05-3.37 (6H, m), 3.76
(6H, s), 3.88-4.30 (7H, m), 4.64 (2H, m), 5.37 (1H, d, J ) 57.9
Hz), 5.58 (2H, br s), 5.78 (1H, d, J ) 8.2 Hz), 5.85 (1H, d, J )
8.2 Hz), 6.08 (1H, d, J ) 4.3 Hz), 6.85 (4H, d, J ) 8.4 Hz), 7.18-
7.44 (31H, m), 7.67 (5H, m), 9.21 (1H, br s). 13C-NMR (CDCl3,
100 MHz) δ 37.1, 38.1, 46.8, 55.1, 55.2, 55.2, 62.3, 67.0, 67.2,
71.4, 73.9, 77.2, 81.7, 86.5, 87.5, 102.9, 113.4, 119.8, 124.9, 125.1,
126.9, 127.1, 127.2, 127.3, 127.6, 128.1, 128.6, 128.6, 129.1, 130.0,
130.1, 134.7, 134.8, 135.6, 136.0, 139.8, 141.1, 143.6, 143.6, 143.8,
150.3, 155.8, 156.0, 158.8, 158.8, 162.8, 170.3, 170.9; HRMS
(FAB, PEG600/NBA/NaOAc) calcd for [C78H68N4O14 + Na]+
1307.4626, found 1307.4619,
5′-O-(4,4′-Dim eth oxytr ityl)-3′-O-(N-(9-flu or en ylm eth oxy-
ca r bon yl)glycyl)th ym id in e (5a ). Reaction of protected nu-
cleoside 1a (100 mg, 0.184 mmol) and N-(9-fluorenylmethoxy-
carbonyl)glycyl fluoride (110 mg, 0.368 mmol) for 4 h followed
by flash chromatography (silica gel, step gradient 0-1% MeOH
in CHCl3) gave 147 mg (97%) of chromatographically pure 5a
as a white solid. 1H-NMR (CDCl3, 250 MHz) δ 1.39 (3H, s), 2.47
(2H, br s), 3.47 (2H, br s), 3.77 (6H, s), 4.00 (2H, d, J ) 5.6 Hz),
4.14 (1H, br s), 4.22 (1H, t, J ) 6.9 Hz), 4.41 (2H, d, J ) 7.0 Hz),
5.48 (2H, br s), 6.44 (1H, app t), 6.83 (4H, d, J ) 8.8 Hz), 7.25-
7.39 (13H, m), 7.59 (3H, d, J ) 7.3 Hz), 7.74 (2H, d, J ) 7.3 Hz),
9.41 (1H, s); 13C-NMR (CDCl3, 100 MHz) δ 11.6, 37.8, 42.7, 47.0,
55.2, 55.2, 63.5, 67.2, 76.5, 77.2, 83.7, 84.2, 87.2, 111.7, 113.3,
120.0, 125.0, 127.0, 127.2, 127.7, 128.0, 128.0, 130.0, 130.0, 135.0,
135.1, 135.2, 141.2, 143.7, 144.1, 150.5, 156.3, 158.7, 158.7, 163.8,
169.6; HRMS (FAB, NBA/NaI) calcd for [C48H45N3O10 + Na]+
846.3003, found 846.3008.
5′-O-(4,4′-Dim eth oxytr ityl)-3′-O-(N-(9-flu or en ylm eth oxy-
ca r bon yl)-L-a la n yl)th ym id in e (5b). Reaction of protected
nucleoside 1a (100 mg, 0.184 mmol) and N-(9-fluorenylmethoxy-
carbonyl)-L-alanyl fluoride (116 mg, 0.368 mmol) for 4 h followed
by flash chromatography (silica gel, step gradient 0-1% MeOH
in CHCl3) gave 149 mg (97%) of chromatographically pure 5b
as a white solid. 1H-NMR (CDCl3, 250 MHz) δ 1.39-1.42 (6H,
m), 2.47 (2H, m), 3.47 (2H, br s), 3.77 (6H, s), 4.10 (1H, br s),
4.22 (1H, t, J ) 6.8 Hz), 4.39 (3H, m), 5.39 (1H, d, J ) 7.5 Hz),
5.52 (1H, br s), 6.44 (1H, app t), 6.83 (4H, d, J ) 8.9 Hz), 7.23-
7.41 (13H, m), 7.59 (3H, br s), 7.75 (2H, d, J ) 7.4 Hz), 9.10
(1H, s); 13C-NMR (CDCl3, 100 MHz) δ 11.6, 18.2, 37.7, 47.1, 49.6,
55.2, 55.2, 63.5, 67.0, 76.2, 77.2, 84.0, 84.3, 87.2, 111.7, 113.3,

Notes
J . Org. Chem., Vol. 61, No. 12, 1996 4171
120.0, 125.0, 127.0, 127.2, 127.7, 128.0, 128.0, 130.0, 135.1, 135.2,
135.2, 141.3, 143.7, 143.8, 144.1, 150.4, 155.6, 158.8, 163.6, 172.6;
HRMS (FAB, NBA/NaI) calcd for [C49H47N3O10 + Na]+ 860.3159,
found 860.3162.
) 7.2 Hz, 7.2 Hz), 3.05 (3H, m), 3.43 (2H, m), 3.75(6H, s), 4.05
(1H, br s), 4.21 (1H, t, J ) 6.7 Hz), 4.31 (1H, t, J ) 6.6 Hz),
4.36-4.50 (2H, m), 4.60-4.68 (3H, m), 5.33 (1H, d, J ) 8.1 Hz),
5.48 (1H, br s), 6.26 (1H, app t), 6.78 (4H, d, J ) 8.8 Hz), 6.94-
7.41 (22H, m), 7.55-7.64 (4H, m), 7.75 (4H, d, J ) 7.4 Hz), 8.05
(1H, s), 8.52 (1H, s), 8.6(1H, s); 13C-NMR (CDCl3, 100 MHz) δ
37.5, 38.4, 46.9, 47.1, 54.9, 55.2, 63.4, 67.0, 67.7, 76.5, 77.2, 84.1,
84.5, 86.7, 113.1, 113.2, 120.0, 122.4, 125.0, 127.0, 127.0, 127.1,
127.3, 127.7, 127.8, 127.9, 128.1, 128.7, 129.2, 129.9, 130.0, 135.4,
135.4, 141.1, 141.3, 143.5, 143.6, 143.7, 144.3, 149.3, 151.0, 151.0,
152.7, 155.5, 158.6, 171.0; HRMS (FAB, NBA/NaI) calcd for
[C70H60N6O10 + Na]+ 1167.4269, found 1167.4286.
5′-O-(4,4′-Dim eth oxytr ityl)-2-N-(9-flu or en ylm eth oxyca r -
b on yl)-2′-d eoxy-3′-O-(N-(9-flu or en ylm et h oxyca r b on yl)-L-
p h en yla la n yl)gu a n osin e (5g). Reaction of protected nucleo-
side 1d (50 mg, 0.064 mmol) and N-(9-fluorenylmethoxycarbonyl)-
L-phenylalanyl fluoride (4) (50 mg, 0.128 mmol) for 12 h followed
by flash chromatography (silica gel, step gradient 0-1% MeOH
in CHCl3) gave 54 mg (74%) of chromatographically pure 5g as
a white solid. 1H-NMR (CDCl3, 250 MHz) δ 2.40 (1H, m), 2.83
(1H, m), 3.08 (2H, m), 3.27 (2H, br s), 3.74 (6H, s), 3.94 (1H, br
s), 4.20 (2H, m), 4.39-4.65 (5H, m), 5.37 (2H, m), 5.92 (1H, m),
6.79 (4H, m), 7.09-7.55 (27H, m), 7.67-7.76 (5H, m), 11.20 (1H,
s); 13C-NMR (CDCl3, 100 MHz) δ 37.4, 38.3, 46.6, 47.1, 55.0, 55.2,
63.5, 67.2, 68.4, 76.3, 77.2, 83.7, 84.0, 86.7, 113.3, 120.0, 120.2,
121.5, 124.8, 124.9, 125.0, 127.0, 127.1, 127.3, 127.3, 127.8, 128.0,
128.1, 128.7, 129.2, 130.0, 135.4, 135.5, 136.9, 141.3, 141.3, 142.8,
142.8, 143.6, 143.6, 144.3, 146.2, 148.1, 153.2, 155.3, 155.7, 158.7,
171.1; HRMS (FAB, NBA/triethanolamine/NaI) calcd for
[C70H60N6O11 + Na]+ 1183.4218, found 1183.4229.
5′-O-(4,4′-Dim eth oxytr ityl)-3′-O-(N-(9-flu or en ylm eth oxy-
ca r bon yl)-L-p h en yla la n yl)th ym id in e (5c). Reaction of pro-
tected nucleoside 1a (100 mg, 0.184 mmol) and N-(9-fluorenyl-
methoxycarbonyl)-L-phenylalanyl fluoride (72 mg, 0.184 mmol)
for 4 h followed by flash chromatography (silica gel, step gradient
0-1% MeOH in CHCl3) gave 119 mg (71%) of chromatographi-
cally pure 5c. An identical reaction containing 2 equiv of N-(9-
fluorenylmethoxycarbonyl)-L-phenylalanyl fluoride (144 mg, 0.368
mmol) gave 161 mg (96%) of chromatographically pure 5c as a
white solid. 1H-NMR (CDCl3, 250 MHz) δ 1.37 (3H, s), 2.32-
2.43 (2H, br s), 3.10 (2H, m), 3.39 (2H, m), 3.77 (6H, s), 3.83
(1H, s), 4.20 (1H, t, J ) 6.7 Hz), 4.41 (2H, m), 4.63 (1H, m), 5.38
(2H, d, J ) 7.5 Hz), 6.31 (1H, app. t), 6.84 (4H, d, J ) 8.9 Hz),
7.00-7.42 (18H, m), 7.54-7.57 (3H, br s), 7.75 (2H, d, J ) 7.3
Hz), 9.17 (1H, s); 13C-NMR (CDCl3, 100 MHz) δ 11.6, 37.7, 38.3,
47.1, 54.8, 55.2, 63.5, 67.0, 76.5, 77.2, 83.6, 84.2, 87.2, 111.6,
113.3, 120.0, 125.0, 127.0, 127.2, 127.3, 127.7, 128.0, 128.0, 128.7,
129.1, 130.0, 135.0, 135.2, 135.2, 135.3, 141.3, 143.6, 143.8, 144.1,
150.4, 155.5, 158.8, 158.8, 163.7, 171.3; HRMS (FAB, NBA/NaI)
calcd for [C55H51N3O10 + Na]+ 936.3472, found 936.3473.
5′-O-(4,4′-Dim eth oxytr ityl)-4-N-(9-flu or en ylm eth oxyca r -
b on yl)-2′-d eoxy-3′-O-(N-(9-flu or en ylm et h oxyca r b on yl)-L-
a la n yl)cytid in e (5d ). Reaction of protected nucleoside 1b (100
mg, 0.133 mmol) and N-(9-fluorenylmethoxycarbonyl)-L-alanyl
fluoride (83 mg, 0.26 mmol) for 12 h followed by flash chroma-
tography (silica gel, step gradient 0-3% MeOH in CHCl3) gave
125 mg (90%) of chromatographically pure 5d as a white solid.
An identical reaction which was allowed to react for 4 h yielded
97 mg (70%) of 5d . 1H-NMR (CDCl3, 250 MHz) δ 1.40 (3H, d, J
) 7.1 Hz), 2.32 (1H, m), 2.81 (1H, m), 3.45 (2H, br s), 3.76 (6H,
s), 3.19-3.28 (3H, m), 4.38-4.47 (5H, m), 5.37-5.44 (2H, m),
6.29 (1H, t, J ) 6.2 Hz), 6.83 (4H, d, J ) 7.9 Hz), 6.95 (1H, d, J
) 7.2 Hz), 7.25-7.40 (17H, m), 7.57 (4H, d, J ) 7.0 Hz), 7.75
(4H, d, J ) 7.3 Hz), 8.07 (2H, br s); 13C-NMR (CDCl3, 100 MHz)
δ 18.2, 39.2, 46.6, 47.1, 49.6, 55.2, 55.2, 62.9, 67.0, 68.0, 75.2,
77.2, 84.5, 87.0, 87.1, 94.6, 113.3, 119.9, 120.1, 124.9, 125.0,
127.0, 127.2, 127.7, 128.0, 128.0, 129.9, 130.0, 135.0, 135.2, 141.3,
143.1, 143.2, 143.7, 143.8, 144.0, 144.1, 152.1, 154.7, 155.6, 158.7,
162.2, 172.4; HRMS (FAB, NBA/NaI/2-nitrophenyl octyl ether)
calcd for [C63H56N4O11 + Na]+ 1067.3843, found 1067.3832.
5′-O-(4,4′-Dim eth oxytr ityl)-4-N-(9-flu or en ylm eth oxyca r -
b on yl)-2′-d eoxy-3′-O-(N-(9-flu or en ylm et h oxyca r b on yl)-L-
p h en yla la n yl)cytid in e (5e). Reaction of protected nucleoside
1b (100 mg, 0.133 mmol) and N-(9-fluorenylmethoxycarbonyl)-
L-phenylalanyl fluoride (101 mg, 0.26 mmol) for 12 h followed
by flash chromatography (silica gel, step gradient 0-3% MeOH
in CHCl3) gave 126 mg (85%) of chromatographically pure 5e
as a white solid. An identical reaction which was allowed to
5′-O -(4,4′-D im e t h o x y t r it y l)-2′(3′)-O -(N -(9-flu o r e n y l-
m et h oxyca r b on yl)-L-a la n yl)u r id in e (7a ) a n d 5′-O-(4,4′-
Dim eth oxytr ityl)-2′, 3′-O,O-bis(N-(9-flu or en ylm eth oxyca r -
bon yl)-L-a la n yl)u r id in e (8a ). Reaction of protected nucleoside
6 (110 mg, 0.202 mmol) and N-(9-fluorenylmethoxycarbonyl)-L-
alanyl fluoride (3) (127 mg, 0.405 mmol) for 4 h followed by flash
chromatography (silica gel, step gradient 14-25% acetone in
CH2Cl2) gave 97.4 mg (58%) of 7a and 94.1 mg (41%) of 8a as
chromatographically pure, white solids.
7a : 1H-NMR (CDCl3, 250 MHz) δ 1.43 (3H, m), 3.45 (2H, m),
3.75 (3H, s), 3.76 (3H, s), 4.00-4.50 (5H, m), 4.63 (1H, m), 5.40
(2H, m), 5.88 (0.25H, d, J ) 7.4 Hz), 5.98 (0.75H, d, J ) 6.3 Hz),
6.09 (0.75H, d, J ) 4.5 Hz), 6.15(0.25H, d, J ) 4.5 Hz), 6.82
(4H, d, J ) 8.7 Hz), 7.17-7.36 (13H, m), 7.53 (2H, m), 7.69-
7.85 (3H, m), 9.45 (0.25H, br s), 10.06 (0.75H, br s); 13C-NMR
(CDCl3, 100 MHz) δ 17.4, 18.4, 46.9, 47.1, 49.9, 50.2, 55.2, 55.2,
62.2, 67.0, 67.4, 69.8, 72.8, 74.6, 77.2, 81.6, 83.7, 86.5, 87.2, 87.3,
88.7, 102.7, 109.6, 113.3, 113.4, 119.9, 124.9, 125.0, 127.0, 127.1,
127.2, 127.7, 127.7, 128.0, 128.1, 130.0, 130.1, 134.9, 135.0, 135.2,
140.0, 141.2, 141.3, 143.7, 143.8, 144.0, 144.1, 150.4, 150.8, 156.1,
156.9, 158.7, 163.3, 163.9, 171.4, 172.0; HRMS (FAB, NBA/NaI)
calcd for [C48H45N3O11 + Na]+ 862.2952, found 862.2961.
8a : 1H-NMR (CDCl3, 250 MHz) δ 1.40 (3H, d, J ) 7.0 Hz),
1.46 (3H, d, J ) 6.8 Hz), 3.43 (2H, br s), 3.76 (6H, s), 4.09-4.43
(9H, m), 5.36 (1H, d, J ) 7.7 Hz), 5.54 (1H, m), 5.68-5.77 (2H,
m), 6.00 (1H, d, J ) 7.7 Hz), 6.17 (1H, d, J ) 4.9 Hz), 6.83 (4H,
d, J ) 8.6 Hz), 7.19-7.37 (17H, m), 7.49 (4H, m), 7.67 (5H, m),
9.24 (1H, br s); 13C-NMR (CDCl3, 100 MHz) δ 17.7, 18.0, 46.9,
49.6, 49.6, 55.2, 55.2, 62.1, 67.1, 71.0, 74.1, 77.2, 81.7, 86.7, 87.4,
103.0, 113.4, 119.8, 125.0, 127.0, 127.3, 127.6, 127.6, 128.1, 130.0,
130.1, 134.7, 134.8, 139.6, 141.2, 141.2, 143.6, 143.7, 143.8, 150.3,
155.9, 156.0, 158.8, 158.8, 162.8, 171.5, 171.9; HRMS (FAB,
NBA/NaI) calcd for [C66H60N3O14 + Na]+ 1155.4004, found
1155.3983.
1
react for 4 h yielded 100 mg (67%) of 5e; H-NMR (CDCl3, 250
MHz) δ 2.24 (1H, m), 2.77 (1H, m), 3.08 (2H, d, J ) 6.2 Hz),
3.38 (2H, br s), 3.74 (6H, s), 4.01 (1H, br s), 4.21 (2H, m), 4.42
(4H, m), 4.65 (1H, m), 5.31 (1H, d, J ) 5.0 Hz), 5.48 (1H, d, J )
8.0 Hz), 6.31 (1H, t, J ) 6.3 Hz), 6.81 (4H, d, J ) 7.6 Hz), 6.97
(1H, d, J ) 7.2 Hz), 7.07-7.45 (22H, m), 7.56 (4H, d, J ) 7.2
Hz), 7.74 (4H, d, J ) 7.4 Hz), 8.04 (4H, d, J ) 7.2 Hz); 13C-NMR
(CDCl3, 100 MHz) δ 38.2, 39.2, 46.6, 47.1, 54.8, 55.1, 63.0, 67.0,
67.9, 75.8, 77.2 84.3, 87.1, 94.6, 113.3, 119.9, 120.1, 123.7, 124.9,
125.0, 127.0, 127.1, 127.7, 127.9, 128.0, 128.0, 128.6, 129.1, 129.2,
129.9, 130.0, 135.0, 135.2, 135.5, 135.9, 141.3, 143.2, 143.2, 143.7,
143.8, 144.0, 149.7, 152.2, 154.7, 155.5, 158.7, 162.3, 171.0.
HRMS (FAB, NBA/NaI/2-nitrophenyl octyl ether) calcd for
[C69H60N4O11 + Na]+ 1143.4156, found 1143.4153.
5′-O-(4,4′-Dim eth oxytr ityl)-6-N-(9-flu or en ylm eth oxyca r -
b on yl)-2′-d eoxy-3′-O-(N-(9-flu or en ylm et h oxyca r b on yl)-L-
p h en yla la n yl)a d en osin e (5f). Reaction of protected nucleo-
side 1c (120 mg, 0.155 mmol) and N-(9-fluorenylmethoxycarbonyl)-
L-phenylalanyl fluoride (4) (181 mg, 0.465 mmol) for 12 h
followed by flash chromatography (silica gel, step gradient 0-1%
MeOH in CHCl3) gave 160 mg (90%) of chromatographically pure
5f as a white solid. An identical reaction containing 1c (85 mg,
0.110 mmol) and 4 (85 mg, 0.219 mmol) yielded 84 mg (67%) of
5f. A similar reaction of 1c (54 mg, 0.0696 mmol) and 4 (54
mg, 0.139 mmol) which was allowed to react for 4 h yielded 40.3
mg (51%) of 5f. 1H-NMR (CDCl3, 250 MHz) δ 2.56 (1H, dd, J 1
Ack n ow led gm en t. This work was supported in part
by the National Science Foundation under grant num-
ber MCB-9508066 and by Brown University.
Su p p or tin g In for m a tion Ava ila ble: Proton and carbon
NMR spectra are available for all compounds described in the
experimental section (31 pages). This material is contained
in libraries on microfiche, immediately follows this article in
the microfilm version of the journal, and can be ordered from
the ACS; see any current masthead page for ordering
information.
J O9601674