Ring opening of a resin-bound chiral aziridine with phenol nucleophiles

Article abstract of DOI:10.1021/jo100505c

(Figure presented) An efficient and versatile solid-phase route for the preparation of aryl-alkyl ethers is described. Regioselective ring opening of a resin-bound chiral aziridine with phenolic nucleophiles constitutes the key feature of the present protocol that allows incorporation of fluorescent moieties and subsequent on-resin protecting group interconversion. Initial experiments demonstrated that a competing oligomerization may occur by concomitant attacks of transient nosylamide anions on neighboring aziridines, resulting in formation of dimeric and trimeric byproduct. Expectedly, the significance of this alternative reaction pathway was strongly dependent on resin loading, and a low loading (<0.4 mmol g^-1) was required for obtaining high yields of the desired aryl-alkyl ethers. The developed methodology allowed preparation of novel N-Fmoc-protected coumaryl amino acid building blocks, which were incorporated into peptides by solid-phase peptide synthesis.

Full text of DOI:10.1021/jo100505c

pubs.acs.org/joc
Ring Opening of a Resin-Bound Chiral Aziridine with Phenol Nucleophiles
Lars K. Ottesen, Jerzy W. Jaroszewski, and Henrik Franzyk*
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark
hf@farma.ku.dk
Received March 23, 2010
An efficient and versatile solid-phase route for the preparation of aryl-alkyl ethers is described.
Regioselective ring opening of a resin-bound chiral aziridine with phenolic nucleophiles constitutes
the key feature of the present protocol that allows incorporation of fluorescent moieties and
subsequent on-resin protecting group interconversion. Initial experiments demonstrated that a
competing oligomerization may occur by concomitant attacks of transient nosylamide anions on
neighboring aziridines, resulting in formation of dimeric and trimeric byproduct. Expectedly, the
significance of this alternative reaction pathway was strongly dependent on resin loading, and a low
loading (<0.4 mmol g-1) was required for obtaining high yields of the desired aryl-alkyl ethers. The
developed methodology allowed preparation of novel N-Fmoc-protected coumaryl amino acid
building blocks, which were incorporated into peptides by solid-phase peptide synthesis.
Introduction
of several reports concerning the corresponding solution-
phase conversions.1,10-16
Aziridines are important intermediates in organic synthesis
as their highly strained ring system readily undergoes a range
of ring-opening reactions with a variety of nucleophiles under
mild conditions.1-3 Although substantial progress in the
general exploration of aziridines has been achieved during
the past two decades, their utilization in solid-phase processes
has been poorly investigated and limited to N- and S-nucleo-
philes.4-9 In particular, solid-phase aziridinolysis with phe-
nolic nucleophiles remains unexplored despite the emergence
As a part of our ongoing research in solid-phase synthesis
(SPS) methodology involving ring opening of aziridines,7-9 we
here target functionalized aryl-alkyl ethers that are precursors
of R-amino acids featuring aromatic structural motifs, thus
contributing to the extension of the synthetic toolbox for
aziridine-based SPS. Recently, we reported on the ring opening
of resin-bound nosyl-activated aziridine-2-methanol with var-
ious 2-amino alcohols leading to chiral 2,5-disubstituted piper-
azine scaffolds.9 Now, this resin-bound aziridine is investigated
with respect to its ring opening with phenolic O-nucleophiles.
(1) Hu, X. E. Tetrahedron 2004, 60, 2701–2743.
(2) Tanner, D. Angew. Chem., Int. Ed. 1994, 33, 599–619.
(3) Sweeney, J. B. Chem. Soc. Rev. 2002, 31, 247–258.
(4) Olsen, C. A.; Franzyk, H.; Jaroszewski, J. W. Eur. J. Org. Chem. 2007,
1717–1724.
(10) Bhanu Prasad, B. A.; Sanghi, R.; Singh, V. K. Tetrahedron 2002, 58,
7355–7363.
(5) Galonic, D. P.; van der Donk, W. A.; Gin, D. Y. J. Am. Chem. Soc.
2004, 126, 12712–12713.
(11) Li, P.; Forbeck, E. M.; Evans, C. D.; Joullie, M. M. Org. Lett. 2006,
8, 5105–5107.
(6) Galonic, D. P.; Ide, N. D.; van der Donk, W. A.; Gin, D. Y. J. Am.
Chem. Soc. 2005, 127, 7359–7369.
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J. Am. Chem. Soc. 2007, 129, 14463–14469.
(7) Olsen, C. A.; Christensen, C.; Nielsen, B.; Mohamed, F. M.; Witt, M.;
Clausen, R. P.; Kristensen, J. L.; Franzyk, H.; Jaroszewski, J. W. Org. Lett.
2006, 8, 3371–3374.
(13) Rao, R. K.; Naidu, A. B.; Sekar, G. Org. Lett. 2009, 11, 1923–1926.
(14) Jamookeeah, C. E.; Beadle, C. D.; Harrity, J. P. A. Synthesis 2009,
133–137.
(8) Crestey, F.; Witt, M.; Frydenvang, K.; Stærk, D.; Jaroszewski, J. W.;
Franzyk, H. J. Org. Chem. 2008, 73, 3566–3569.
(9) Ottesen, L. K.; Olsen, C. A.; Witt, M.; Jaroszewski, J. W.; Franzyk, H.
Chem.;Eur. J. 2009, 15, 2966–2978.
(15) Morieux, P.; Stables, J. P.; Kohn, H. Bioorg. Med. Chem. 2008, 16,
8968–8975.
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Jpn. 1982, 55, 3049–3050.
DOI: 10.1021/jo100505c
r
Published on Web 07/09/2010
J. Org. Chem. 2010, 75, 4983–4991 4983
2010 American Chemical Society

Ottesen et al.
JOCArticle
The aim was to develop a versatile practical solid-phase proto-
col for the synthesis of aryl-alkyl ethers displaying amine and
alcohol/carboxylic acid functionalities.
TABLE 1. Preparation of Nosyl-Activated Resin-Bound Aziridines
3A-E
Previously, enantioselective syntheses of coumarin-bearing
amino acids have been carried out by diastereoselective alky-
lation of chiral glycine equivalents.17-19 In recent syntheses
of coumaryl amino acids, protected aspartic and glutamic
acids served as chiral starting materials that were converted to
coumarylalanines and coumarylethylglycines.20 These meth-
ods yielded optically pure amino acids; however, either multi-
ple steps were required or the products were obtained in poor
overall yields. Consequently, aziridinolysis with 7-hydroxy-
coumarin derivatives followed by on-resin protecting group
manipulation and postcleavage oxidation were envisaged to
constitute a novel four-step route to N-Fmoc-protected cou-
maryl amino acid building blocks.
resin loadingb
resin loading
of 1 (mmol g-1
loading
procedurea
of 3A-E
entry
1
)
(mmol g-1
)
1.57
0.54
1.57
1.57
1.57
2 (2 equiv)
3A (1.20)
3B (0.41)
3C (0.33)
3D (0.33)
3E (0.33)
2
3
4
5
2 (2 equiv)
MeOH (0.5 equiv)
2 (0.5 equiv)
2 (0.5 equiv)
MeOH (excess)
2 (0.5 equiv),
MeOH (0.5 equiv)
Results and Discussion
aSee Experimental Section for reaction details. bCalcd resin loading
based on weight gain.
Preparation and Loading of Activated Aziridine onto the
Solid Support. (S)-N-Nosylaziridine-2-methanol9 (2), pre-
pared from (S)-1-tritylaziridine-2-methanol,21 was loaded
onto a freshly prepared polystyrene trityl bromide (PS-TrtBr)
resin22 (1) in various ways to give resin-bound aziridines 3A-E
(Table 1). Activation by a p-nitrobenzenesulfonyl (nosyl = Ns)
group has the advantage of being a readily removable protecting
group during SPS.23,24
TABLE 2. Optimization Studies of the Ring Opening of Resin 3A with
Phenola
Optimization of Ring Opening of Resin 3A with Phenol.
Initially, an optimization of the ring opening of resin 3A was
undertaken. First, nucleophilic ring openings of resin 3A
were performed at room temperature (rt) using 10 equiv of
phenol while assessing the possible influence of base, solvent,
and additive (Table 2).
equiv of
phenol
base/
equiv
additive/
equiv
entry
solvent
yield (%)
1
2
3
4
5
6
7
8
9
10
10
10
10
10
10
10
5
DBU/10
TBD/10
DBU/10
TBD/10
toluene
toluene
DMF
DMF
DMF
DMF
DMF
DMF
DMF
none
none
none
none
none
18-Cr-6/10
18-Cr-6/1
none
8b,d
12c,d
13c,d
15c,d
50c,e
29c,e
38c,e
48c,e
18c,e
Recently, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and
1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) were reported as
suitable bases for ring opening of trisubstituted aziridines
with phenol nucleophiles under solution-phase conditions.12
However, when DBU (10 equiv) was employed in toluene,
only a very low isolated yield of the desired aryl-alkyl ether 4
was obtained after a reaction time of 16 h (Table 2, entry 1).
An attempted improvement by using TBD (10 equiv) led
only to a slightly higher yield (Table 2, entry 2), and changing
the solvent to DMF under otherwise similar conditions was
likewise fruitless (Table 2, entries 3 and 4). Nevertheless, we
found that these disappointing efforts to obtain aryl-alkyl
ether 4 in acceptable yields was due mainly to competing
aziridine ring-opening reactions with DBU and TBD serving
as surprisingly efficient N-nucleophiles resulting in 5 and 6,
respectively, as the main products (Scheme 1).
KOtBu/10
KOtBu/10
KOtBu/10
KOtBu/5
KOtBu/2
2
none
aConditions: resin 3A (100 mg; 1.20 mmol g-1), phenol (10 equiv),
base (10 equiv), rt, 16 h; then cleavage with TFA-CH2Cl2 (2:98), rt, 45
min. bIsolated yield after RP-HPLC purification. cDetermined by
analytical RP-HPLC (220 nm) by comparison with a reference chroma-
togram of a run with a known amount of compound 4. dUnprecedented
e
ring openings of resin 3A (see Scheme 1 for details). Oligomerization
was observed (see Scheme 2 and Table 3 for details).
Relatively few examples have so far been reported on alkyla-
tion of DBU25-29 and TBD,30,31 respectively, and either very
reactive alkylating agents (e.g., alkyl triflates) or forced reaction
conditions (typically heating to reflux at 70-110 °C for several
hours) have been utilized. Although DBU and TBU are indeed
sufficiently strong bases to effect a high degree of deprotonation
(17) Bennett, F. A.; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C.;
Lansley, A. B.; Lawrence, M. J.; Marriott, C.; Bansal, S. S. Tetrahedron
Lett. 1997, 38, 7449–7452.
(18) Bennett, F. A.; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C.;
Lansley, A. B.; Lawrence, M. J.; Marriott, C.; Bansal, S. S. Anal. Biochem.
1999, 270, 15–23.
ꢀ
(25) Shieh, W. C.; Dell, S.; Repic, O. J. Org. Chem. 2002, 67, 2188–2191.
(19) Kele, P.; Sui, G.; Huo, Q.; Leblanc, R. M. Tetrahedron: Asymmetry
2000, 11, 4959–4963.
(20) Brun, M.-P.; Bischoff, L.; Garbay, C. Angew. Chem., Int. Ed. 2004,
43, 3432–3436.
(21) Utsunomiya, I.; Fuji, M.; Sato, T.; Natsume, M. Chem. Pharm. Bull.
1993, 42, 854–860.
(26) Tolstikova, L.; Shainyan, B. Russ. J. Org. Chem. 2006, 42, 1068–1074.
(27) Kitazume, T.; Tanaka, G. J. Fluorine Chem. 2000, 106, 211–215.
(28) Sobral, A. J. F. N.; Lopes, S. H.; Rocha, G.; Antonio, M.; Silva,
M. R.; Beja, A. M. Prog. Colloid Polym. Sci. 2004, 123, 28–30.
(29) Hori, Y.; Nagano, Y.; Tanaka, K.; Taniguchi, H. Chem. Express
1986, 1, 491–494.
(22) Crestey, F.; Ottesen, L. K.; Jaroszewski, J. W.; Franzyk, H. Tetra-
hedron Lett. 2008, 49, 5890–5893.
(30) Genski, T.; Macdonald, G.; Wei, X.; Lewis, N.; Taylor, R. J. K.
ARKIVOC 2000, 266–273.
(23) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301–2302.
(24) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1998, 120, 2690–2691.
(31) Yu, K. M. K.; Curcic, I.; Gabriel, J.; Morganstewart, H.; Tsang, S. C.
J. Phys. Chem. A 2010, 114, 3863–3872.
4984 J. Org. Chem. Vol. 75, No. 15, 2010

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JOCArticle
SCHEME 1. Unprecedented Ring Opening of Resin 3A with
DBU and TBD
TABLE 3. Influence of Method of Resin Loading and Reaction Time on
Aziridine Ring Openinga
entry
resin
t (h)
4 (%)b
7 (%)b
8 (%)b
1
2
3
4
3C
3D
3E
3E
16
16
16
1
76
86
88
90
5
5
7
7
0
0
0
0
aConditions: Appropriate resin 3C-E (100 mg; 0.33 mmol g-1),
phenol (10 equiv), KOtBu (10 equiv), DMF, rt; then cleavage with
TFA-CH2Cl2 (2:98), rt, 45 min. bDetermined by analytical RP-HPLC
(220 nm) by comparison with reference chromatograms of known
amounts of compounds 4, 7, and 8.
interior of the resin beads, where a large proportion of the
reaction sites are located. This explanation was further corrobo-
rated by an additional experiment in which only 1 equiv of 18-
crown-6 was used under otherwise similar conditions. In accor-
dance with our hypothesis, this resulted in an intermediate yield
of aryl-alkyl ether 4 (Table 2, entry 7). Noteworthy, attempts to
decrease the excess of phenol and KOtBu afforded lower yields of
4 or extensive formation of byproduct (Table 2, entries 8 and 9).
During the subsequent analysis of KOtBu-mediated ring open-
ings of resin 3A with phenol, it was found that additional peaks in
the HPLC chromatogram were attributable to byproduct arising
from a competing oligomerization. In Scheme 2 we propose a
mechanism involving repeated attacks of transient nosylamide
anions on neighboring aziridines that accounts for the formation
of dimeric and trimeric byproduct 7 and 8.
To overcome this obstacle, we decided to prepare a resin-
bound aziridine on a low-loading resin (,1.20 mmol g-1). In
that case, it was anticipated that oligomerization would be
diminished as the result of an increased physical separation
of the aziridine moieties.
Evaluation of Low-Loading Resins with Phenol. Resin 3B
was prepared by attaching aziridine building block 2 to resin
1 (0.54 mmol g-1) obtained from a commercial low-loading
trityl alcohol resin (Table 1, entry 2). Subsequent ring open-
ing of resin 3B with phenol followed by cleavage afforded
aryl-alkyl ether 4 in a good yield. Notably, no detectable
amounts of oligomeric byproduct were present in the crude
(Scheme 3).
of phenols,12 these observations indicated that the nucleophilicity
of the phenolic anions were surpassed by these N-nucleophiles.
Hence, a stronger and more hindered base (KOtBu) was applied
to ensure both complete conversion of phenol into its anion and
suppression of side reactions. Encouragingly, it was found that
the use of 10 equiv of KOtBu afforded a significantly higher (but
still moderate) yield of aryl-alkyl ether 4 (Table 2, entry 5).
Notably, according to LC-MS and NMR analysis only the
desired regioisomer was present in the crude product. The struc-
ture of 4 was confirmed by 2D NMR experiments (COSY and
NOESY). Thus, correlations between the sulfonamide (NH) and
the adjacent proton (H-1) as well as between the hydroxylic and
the methylene protons (H-2) in the hydroxymethyl group were
observed. In addition, the close proximity of the H-20 and H-200/
H-600 in the aryl alkyl ether moiety (Ph-O-CH2-) was confirmed
by the presence of the corresponding correlation in the NOESY
spectrum (see Supporting Information for the spectra). Perform-
ing the ring opening in the presence of 10 equiv of 18-crown-6 in
order to increase the nucleophilicity of the phenolic anion
resulted unexpectedly in a decreased yield (Table 2, entry 6).
This observation is tentatively explained by assuming that the
entire, large potassium crown ether counterion complex must
also enter the narrow resin pores, since macroscopic separation of
charges would result in an electrostatic gradient at the resin
surface. Hence, this bulky ion pair is less capable of entering the
With the intention to identify the most favorable distribution
of the aziridine moieties on the solid support, we decided to eva-
luate different protocols leading to low-loading resins by partial
SCHEME 2. Proposed Mechanism for KOtBu-Mediated Cascade Ring Openings of Resin 3A Following Initial Attack of Phenolate
J. Org. Chem. Vol. 75, No. 15, 2010 4985

Ottesen et al.
JOCArticle
SCHEME 3. Ring Opening of Resin 3B with Phenol
SCHEME 4. Preparation of Novel Coumaryl Amino Acidsa
TABLE 4. Ring Opening of Resin 3E with an Array of Substituted
Phenolsa
entry
X
Y
product (% yield)b
1
2
3
4
5
CO2Me
CN
Cl
H
H
H
H
H
Cl
9 (71)
10 (76)
11 (81)
12 (63)
13 (76)
CH2OH
aConditions: resin 3E (200 mg; 0.33 mmol g-1), appropriate phenol
nucleophile (10 equiv), KOtBu (10 equiv), DMF, rt, 1 h; then cleavage with
TFA-CH2Cl2 (2:98), rt, 45 min. bIsolated yield after RP-HPLC purification.
aConditions: (a) resin 3E (4.0 g; 0.33 mmol g-1), appropriate 7-hydro-
xycoumarin (10 equiv), KOtBu (10 equiv), DMF, rt, 1 h. (b) 2-Mercap-
toethanol (10 equiv), DBU (5 equiv), DMF, rt, 2 h. (c) Fmoc-OSu
(5 equiv), iPr2EtN (5 equiv), THF, rt, 2 h. (d) TFA-CH2Cl2 (2:98), rt, 45
min. (e) NaOCl (5 equiv), TEMPO (1 equiv), KBr (10 mol %), 5%
NaHCO3 (aq), acetone, 0 °C, 1 h.
loading with aziridine 2 (Table 1, entries 3-5). Starting from
high-loading resin 1 (1.57 mmol g-1), aziridine building block 2
and methanol were loaded onto the solid support in various
ways to give resins 3C-E (0.33 mmol g-1). Resin 3C was ob-
tained by first loading resin 1 with 0.5 equiv of methanol for 10
min and then adding 0.5 equiv of aziridine building block 2
(Table 1, entry 3), whereas resin 3D was prepared by an initial
loading of aziridine building block 2 (0.5 equiv) onto resin 1 for
10 min followed by treatment with excess MeOH/iPr2EtN/
CH2Cl2 (5:10:85) for 5 min (Table 1, entry 4). Finally, simulta-
neous loading of a mixture of aziridine building block 2 (0.5
equiv) and methanol (0.5 equiv) onto resin 1 led to resin 3E
(Table 1, entry 5). With these differently loaded resins (all dis-
playing similar loadings of 0.33 mmol g-1) in hand, the optimal
conditions for ring opening were further investigated (Table 3).
By using resin 3C, the desired aryl-alkyl ether 4 was
obtained in 76% yield with decreased formation of byproduct
7, while byproduct 8 was not detectable (Table 3, entry 1).
Even higher yields were obtained with resins 3D and 3E
(Table 3, entries 2 and 3) indicating a more favorable distribu-
tion of the aziridine moiety on the solid support compared to
the fully loaded as well as more expensive resin 3B. Moreover,
a very short reaction time (1 h) proved adequate to achieve full
conversion of resin 3E resulting in a high yield of aryl-alkyl
ether 4 (Table 3, entry 4), and thus resin 3E was employed in
the subsequent experiments.
Ring Opening with Substituted Phenol Nucleophiles. A
collection of ortho- and para-substituted phenols was em-
ployed in the ring opening of resin 3E resulting in good to
high yields of aryl-alkyl ethers 9-13 (Table 4).
Both ortho- and para-substituted phenols were examined
in aziridine ring opening with resin 3E, and in all cases
satisfactory isolated yields of the desired aryl-alkyl ethers
9-13 were obtained. However, as expected, o-chlorophenol
afforded a lower isolated yield of aryl-alkyl ether 12 (Table 4,
entry 4) compared with that of p-chlorophenol (11; Table 4,
entry 3), indicating sterical hindrance to be of some impor-
tance. Moreover, the presence of a benzyl alcohol function-
ality proved compatible as demonstrated in the ring opening
with 2-hydroxybenzyl alcohol, which resulted exclusively in
aryl-alkyl ether 13 (Table 4, entry 5).
Preparation of Novel Fluorescent Coumaryl Amino Acids.
A number of coumarins are commercially available, and they
constitute a group of fluorescent compounds having the advan-
tage of possessing extended excitation and emission spectral
ranges, high emission quantum yield, photostability, and solu-
bility in many solvents.18,32 Therefore, coumarins have been
extensively used as fluorophores for labeling of amines, thiols,
and acids,33 thus being useful tools in chemical biology.34 As
C- and N-terminal domains of peptides often represent sites for
biological interaction, the possibility of incorporating a fluores-
cence label at a well-chosen internal position is of considerable
interest. Consequently, the use of an intact amino acid building
block as a means for introducing a fluorescent chromophore
offers two main advantages: (i) preparation by solid-phase
peptide synthesis (SPPS) is straightforward, and (ii) terminals
may be functionalized to improve other desired properties. Sub-
stituted coumarins are accessible by many pathways, including
(32) Esteves, A. P.; Rodrigues, L. M.; Silva, M. E.; Gupta, S.; Oliveira-
Campos, A. M. F.; Machalicky, O.; Mendonc-a, A. n. J. Tetrahedron 2005, 61,
8625–8632.
(33) Wendt, H.; Berger, C.; Baici, A.; Thomas, R. M.; Bosshard, H. R.
Biochemistry 1995, 34, 4097–4107.
(34) O’Kennedy, R. In Coumarins; Wiley: New York, 1997.
(35) Pechmann, H. von.; Duisburg, C. Ber. Dtsch. Chem. Ges. 1883, 16,
2119.
(36) Johnson, J. R. Org. React. 1942, 1, 210.
(37) Brufola, G.; Fringuelli, F.; Piermatti, O.; Pizzo, F. Heterocycles
1996, 43, 127.
4986 J. Org. Chem. Vol. 75, No. 15, 2010

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JOCArticle
SCHEME 5. Preparation of Coumarin-Bearing Dipeptides
17A-Ca
TABLE 5. Fluorescence Properties of Coumarin-Bearing Peptides
compound
λ
abs (nm)a
λ
em (nm)a
17A
17B
17C
319
320
326
388
381
400
aConditions: determined in HPLC-grade methanol.
SCHEME 6. Preparation of Fluorescently Labeled Octaargi-
ninea
aConditions: (a) Rink amide resin (25 mg; 1.0 mmol g-1), Fmoc-Phe-OH
(2 equiv), PyBOP (2 equiv), HOAt (2 equiv), iPr2EtN (4 equiv), DMF, rt,
2 h, then Fmoc-deprotection with 20% piperidine in DMF, rt, 2 ꢀ 10
min. (b) Appropriate N-Fmoc-protected coumaryl amino acid 16A-C
(3 equiv), DIC (3 equiv), HOAt (3 equiv), MWirradiation, 60 °C, 30 min;
then Fmoc-deprotection with 20% piperidine in DMF, rt, 2 ꢀ 10 min.
(c) Ac2O/iPr2EtN/NMP (1:2:3), rt, 1 h. (d) TFA-CH2Cl2 (95:5), rt, 1 h.
von Pechmann, Perkin, Knoevenagel, Reformatsky, and Wittig
reactions.35-39
aConditions: (a) 16B (3 equiv), DIC (3 equiv), HOAt (3 equiv), MW
irradiation, 60 °C, 30 min; then Fmoc-deprotection with 20% piperidine
in DMF, rt, 2 ꢀ 10 min. (b) Ac2O-iPr2EtN-NMP (1:2:3), rt, 1 h.
(c) TFA-TIS-H2O (95:2.5:2.5), rt, 5 h.
To illustrate the efficiency of the developed solid-phase
protocol a series of novel N-Fmoc-protected coumaryl amino
acid building blocks 16A-C were prepared (Scheme 4).
Regioselective ring opening of resin 3E with three com-
mercially available 7-hydroxycoumarin nucleophiles af-
forded resins 14A-C. Standard removal of the nosyl
group and subsequent Fmoc N-protection followed by
cleavage under mild conditions furnished alcohol inter-
mediates 15A-C in acceptable yields after column chro-
matography. Finally, the desired coumarin-bearing build-
ing blocks 16A-C were obtained upon oxidation with
TEMPO/hypochlorite, and their utility in SPPS was vali-
dated by preparation of fluorescent dipeptides 17A-C as
depicted in Scheme 5.
loadings of 0.3-0.4 mmol g-1 were sufficiently low to allow
formation of the desired aryl-alkyl ethers in high yields. The
developed solid-phase protocol was further utilized for
efficient preparation of novel N-Fmoc-protected coumaryl
amino acid building blocks. On-resin N-protecting group
exchange and a postcleavage oxidation constituted the last
steps of these syntheses. Finally, the coumaryl amino acids
were incorporated into peptides by SPPS.
The methodology presented here may readily enable pre-
paration of an array of aryl-alkyl ether building blocks for
combinatorial chemistry. In particular the corresponding un-
natural amino acids may be obtained directly with an
N-protection suitable for traditional solid-phase peptide synthe-
sis, thus contributing to the continuous extension of the diver-
sity of peptide analogues. The advantages of the protocol are
those typical for relatively short solid-phase sequences: (i) near-
quantitative conversion in the individual steps by using an
excess of reagents, (ii) simple composition of the resulting crude
product mixture, (iii) significant reduction of the number of
chromatographic purification steps, and (iv) possibility of
synthesis of many compounds in parallel.
Absorption (λabs) and fluorescence (λem) wavelength maxima
were measured for 17A-C (Table 5). The coumarin-labeled
dipeptides exhibited both higher absorption and emission
maxima than the natural fluorescent amino acids tryptophan
(λabs=278 nm, λem=352 nm) and tyrosine (λabs=274 nm, λem
=
303 nm).
Finally, coumaryl amino acid 16B was used for N-terminal
labeling of octaarginine (Scheme 6), a well-known cell-penetrat-
ing peptide (CPP) often used as a reference compound in cellular
uptake studies relating to CPP-mediated drug delivery.40
Conclusion
Experimental Section
In the present work, an efficient solid-phase protocol for
the preparation of aryl-alkyl ethers via regioselective ring
opening of a resin-bound chiral aziridine with phenolic
nucleophiles has been developed. Thorough studies revealed
a strong correlation between the degree of resin loading and
formation of oligomeric byproduct. It was found that resin
General. Starting materials were purchased from commercial
suppliers and used without further purification. Trityl alcohol
polystyrene resins with loadings of 1.75 and 0.60 mmol g-1 were
obtained from IRIS Biotech and Matrix Innovation, respectively.
Rink-amide-(aminomethyl)-polystyrene resin (1.0 mmol g-1) was
from Fluka. DMF dried over 4 A molecular sieves (H2O < 0.01%)
was from Fluka, and anhydrous CH2Cl2 was distilled from P2O5
˚
and kept over 4 A molecular sieves. Water for reversed-phase
˚
(38) Shriner, R. L. Org. React. 1942, 1, 1.
(39) Yavari, I.; Hekmat-Shoar, R.; Zonouzi, A. Tetrahedron Lett. 1998,
39, 2391–2392.
(40) Rothbard, J. B.; Jessop, T. C.; Wender, P. A. Adv. Drug Delivery Rev.
2005, 57, 495–504.
HPLC was filtered through a 0.22 μm membrane filter (Millipak40,
Millipore). Other solvents were analytical or HPLC grade and were
used as received. Microwave-assisted coupling of 16A-C in the
J. Org. Chem. Vol. 75, No. 15, 2010 4987

Ottesen et al.
JOCArticle
syntheses of 17A-C was carried out in an Initiator single mode
microwave cavity producing controlled irradiation at 2.45 GHz
(Biotage AB, Uppsala). The reactions were run in sealed vessels
(0.5-2.0 mL), and magnetic stirring was used. Variable power was
employed to reach the temperature desired (within 1-2 min) and
then to maintain it during the period of time programmed. Tem-
perature was monitored by an IR sensor focused on a point on the
reactor vial; the sensor was calibrated to internal solution reaction
temperature by the manufacturer. Preparation of resin-bound
octaarginine was performed on a CEM Liberty microwave peptide
synthesizer, and the final introduction of 17B was performed
manually as above. Analytical DAD-HPLC was performed by
using a Phenomenex Luna (150 mm ꢀ 4.6 mm; C18(2); particle size
3μm) column eluted at a rate of 0.8 mL/min. Injection volumes were
20 μL of a 1 mg/mL solution, and separations were performed at
40 °C. Solvent mixtures A (H2O-MeCN-TFA 95:5:0.1) and B
(MeCN-H2O-TFA 95:5:0.1) were used in a gradient rising
linearly from 0% to 100% of B during 30 min. Preparative HPLC
separations were carried out on a Phenomenex Luna (250 mm ꢀ
21.2 mm; C18; particle size 5 μm) column. Solvent mixtures A
and washed with MeOH/iPr2EtN/CH2Cl2 (5:10:85; 100 mL for
5 min). The resin was drained and washed with DMF, MeOH,
and CH2Cl2 (each 3 ꢀ 100 mL for 5 min). The resin was dried
under vacuum to afford resin 3E (16.45 g; calcd resin loading
based on weight gain: 0.33 mmol g-1).
General Procedure A (GP A): Ring Opening of Resin-Bound
Aziridine with Phenols. A solution of the appropriate phenol (10
equiv) and KOtBu (74 mg, 0.66 mmol; 10 equiv) in anhydrous
DMF (1 mL) was added to resin 3E (200 mg; 0.33 mmol g-1; in a
10 mL syringe equipped with a PP filter). The resin was agitated
at rt for 1 h under N2 and then drained and washed with DMF,
MeOH, and CH2Cl2 (each 3 ꢀ 5 mL for 5 min). The product was
cleaved with 2% TFA-CH2Cl2 (1.5 mL for 30 min, then 1.5 mL
for 15 min), and finally the resin was washed with CH2Cl2
(2 mL). The eluates were pooled and the solvents were removed
in vacuo. The crude material was purified by preparative
reversed-phase HPLC.
(R)-N-(1-Hydroxy-3-phenoxypropan-2-yl)-4-nitrobenzenesul-
fonamide (4). Preparation according to GP A using phenol (62
mg, 0.66 mmol). Yield: 21 mg (90%) as a colorless syrup. [R]20
D
þ11.1 (c 0.44, DMF). 1H NMR (DMSO-d6, 300 MHz): δ 8.37
(d, J = 7.0 Hz, 1H), 8.33-8.29 (m, 2H), 8.06-8.03 (m, 2H),
7.20-7.15 (m, 2H), 6.88-6.83 (m, 1H), 6.64-6.61 (m, 2H), 4.97
(t, J = 5.5 Hz, 1H, OH), 3.95-3.83 (m, 2H), 3.53-3.40 (m, 3H).
13C NMR (DMSO-d6, 75 MHz): δ 157.7, 149.1, 147.3, 129.2
(2C), 127.9 (2C), 124.2 (2C), 120.6, 114.1 (2C), 66.7, 61.1, 55.0.
vmax (KBr) cm-1 3420, 3177, 2884, 1526, 1351, 1245, 1157.
HRMS (m/z): [M þ H]þ calcd for C15H16N2O6S, 353.0807;
found, 353.0797.
(H2O-MeCN-TFA 95:5:0.1) and
B (MeCN-H2O-TFA
95:5:0.1) were used in a gradient rising linearly from 0% to 100%
of B during 30 min. Vacuum liquid chromatography (VLC) was
performed using Merck silica gel 60 (particle size 0.015-0.040 mm).
1H NMR and 13C NMR spectra were recorded at 300 and 75 MHz,
respectively, using CDCl3, CD3OD or DMSO-d6 as solvents and
1
TMS as internal standard. H NMR and 13C NMR spectra of
compounds 17A-C were recorded at 600 and 150 MHz, respec-
tively. Multiplicities of 1H NMR signals are reported as follows: s,
singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; ddd,
doublet of doublet of doublets; t, triplet; m, multiplet. All target
compounds were characterized by HRMS.
Preparation of Resins 3A and 3B. The resins were prepared
using a previously reported procedure,9 except that resin 3B was
prepared from a low-loading (0.60 mmol g-1) polystyrene trityl
alcohol resin.
(S)-1-[3-Hydroxy-2-(4-nitrophenylsulfonamido)propyl]-2,3,4,
6,7,8,9,10-octahydropyrimido[1,2-a]azepin-1-ium (5). Prepara-
tion according to GP A using phenol (155 mg, 1.20 mmol).
Note: resin 3A (100 mg; 1.20 mmol g-1) and DBU (179 μL, 1.20
mmol) were used instead of resin 3E and KOtBu, respectively.
Yield: 38 mg (77%) as a pale yellow oil. [R]20 þ28.9 (c 0.54,
D
1
DMF). H NMR (CD3OD, 300 MHz): δ 8.43-8.40 (m, 2H),
8.13-8.10 (m, 2H), 3.76-3.57 (m, 9H), 3.32-3.27 (m, 2H;
hidden in part under the solvent peak), 3.01-2.96 (m, 2H),
2.23-2.15 (m, 2H), 1.93-1.77 (m, 6H). 13C NMR (CD3OD, 75
MHz): δ 169.0, 151.4, 148.2, 129.2 (2C), 125.4 (2C), 61.9, 56.3,
55.9, 55.7 (2C), 50.4, 29.6 (2C), 26.9, 24.3, 20.8. HRMS (m/z):
[M þ H]þ calcd for C18H27N4O5Sþ, 411.1697; found, 411.1674.
(S)-N-[1-(2,3,4,6,7,8-Hexahydro-1H-pyrimido[1,2-a]pyrimi-
din-1-yl)-3-hydroxypropan-2-yl]-4-nitrobenzenesulfonamide (6).
Preparation according to GP A using phenol (155 mg, 1.20
mmol). Note: resin 3A (100 mg; 1.20 mmol g-1) and TBD (167
mg, 1.20 mmol) were used instead of resin 3E and KOtBu,
Preparation of Resin 3C. A solution of MeOH (32 μL, 0.79
mmol) and iPr2EtN (0.69 mL, 3.95 mmol) in anhydrous CH2Cl2
(5 mL) was added to a freshly prepared trityl bromide resin 1
(1.00 g; 1.57 mmol g-1) preswollen in anhydrous CH2Cl2 (10
mL; in a 25 mL syringe equipped with a PP filter) under N2 at rt.
The resin was agitated for 10 min under N2. The resin was
drained, and a solution of 2 (204 mg, 0.79 mmol) and iPr2EtN
(0.69 mL, 3.95 mmol) in anhydrous CH2Cl2 (5 mL) was added.
The resin was agitated for an additional 10 min under N2. The
resin was drained and washed with MeOH/iPr2EtN/CH2Cl2
(5:10:85, 5 mL for 5 min). The resin was drained and washed
with DMF, MeOH, and CH2Cl2 (each 3 ꢀ 5 mL for 5 min). The
resin was dried under vacuum to afford resin 3C (1.09 g; calcd
resin loading based on weight gain: 0.33 mmol g-1).
Preparation of Resin 3D. A solution of 2 (204 mg, 0.79 mmol)
and iPr2EtN (0.69 mL, 3.95 mmol) in anhydrous CH2Cl2 (5 mL)
was added to a freshly prepared trityl bromide resin 1 (1.00 g;
1.57 mmol g-1) preswollen in anhydrous CH2Cl2 (10 mL; in a 25
mL syringe equipped with a PP filter) under N2 at rt. The resin
was agitated for 10 min under N2. The resin was drained and
washed with MeOH/iPr2EtN/CH2Cl2 (5:10:85, 5 mL for 5 min).
The resin was drained and washed with DMF, MeOH, and
CH2Cl2 (each 3 ꢀ 5 mL for 5 min). The resin was dried under
vacuum to afford resin 3D (1.09 g; calcd resin loading based on
weight gain: 0.33 mmol g-1).
respectively. Yield: 40.0 mg (84%) as a pale yellow oil. [R]20
D
þ21.7 (c 0.59, DMF). 1H NMR (CD3OD, 300 MHz): δ 8.41-
8.38 (m, 2H), 8.12-8.09 (m, 2H), 3.75-3.67 (m, 1H), 3.53-3.46
(m, 3H), 3.42-3.53 (m, 7H), 3.32-3.30 (m, 2H; hidden in part
under the solvent peak), 2.12-1.99 (m, 4H). 13C NMR
(CD3OD, 75 MHz): δ 152.6, 151.3, 148.5, 129.2 (2C), 125.3
(2C), 61.9, 54.5, 52.3, 49.2 (2C) (hidden in part under the solvent
peak), 40.0 (2C), 21.9, 21.7. HRMS (m/z): [M þ H]þ calcd for
C16H23N5O5S, 398.1498; found, 398.1481.
N-[(S)-3-Hydroxy-2(4-nitrophenylsulfonamido)propyl]-N-[(R)-
1-hydroxy-3-phenoxypropan-2yl]-4-nitrobenzenesulfonamide (7).
Preparation according to GP A using phenol (115 mg, 1.20
mmol) and KOtBu (135 mg, 1.20 mmol). Note: resin 3A (100 mg;
1.20 mmol g-1) was used instead of resin 3E. Yield: 27 mg (37%) as
a colorless syrup. [R]20D þ45.9 (c 0.56, DMF). 1H NMR (CD3OD,
300 MHz): δ 8.36-8.33 (m, 2H), 8.25-8.22 (m, 2H), 8.14-8.11
(m, 2H), 8.05-8.02 (m, 2H), 7.14-7.09 (m, 2H), 6.87-6.82 (m,
1H), 6.54-6.51 (m, 2H), 4.08-4.01 (m, 3H), 3.83-3.79 (m, 1H),
3.70-3.47 (m, 5H), 3.34-3.27 (m, 1H; hidden in part under the
solvent peak). 13C NMR (CD3OD, 75 MHz): δ 158.7, 151.2, 151.1,
148.4, 147.0, 130.2 (2C), 129.8 (2C), 129.4 (2C), 125.2 (2C), 125.1
Large-Scale Preparation of Resin 3E. A solution of 2 (3.10 g,
11.80 mmol), MeOH (0.95 mL, 11.80 mmol), and iPr2EtN (12.3
mL, 70.80 mmol) in anhydrous CH2Cl2 (100 mL) was added to a
freshly prepared trityl bromide resin 1 (15.00 g; 1.57 mmol g-1
)
preswollen in anhydrous CH2Cl2 (100 mL; in a 250 mL reaction
vessel equipped with a filter). The resin was agitated for 10 min
using a 180° variable-speed flask shaker. The resin was drained
4988 J. Org. Chem. Vol. 75, No. 15, 2010

Ottesen et al.
JOCArticle
(2C), 122.2, 115.1 (2C), 66.9, 62.3, 62.1, 61.8, 56.7, 48.4 (hidden in
part under the solvent peak). HRMS (m/z): [M þ H]þ calcd for
C24H26N4O11S2, 611.1118; found, 611.1126.
1529, 1347, 1248, 1152, 1051. HRMS (m/z): [M þ H]þ calcd for
C15H15ClN2O6S, 387.0418; found, 387.0392.
(R)-N-[1-Hydroxy-3-[2-(hydroxymethyl)phenoxy]propan-2-
yl]-4-nitrobenzenesulfonamide (13). Preparation according to GP
A using 2-hydroxybenzyl alcohol (82 mg, 0.66 mmol). Yield: 19 mg
(76%) as a pale yellow syrup. [R]20D þ10.7 (c0.41, DMF). 1HNMR
(DMSO-d6, 300 MHz): δ 8.33 (d, J = 7.0 Hz, 1H, NH), 8.31-8.28
(m, 2H), 8.06-8.03 (m, 2H), 7.28 (dd, J = 1.0, 7.5 Hz, 1H), 7.10
(ddd, J=1.0, 7.5, 8.5 Hz, 1H), 6.87 (ddd, J=1.0, 7.5, 8.5 Hz, 1H),
6.73 (dd, J=1.0, 8.5 Hz, 1H), 5.05-4.89 (m, 2H, OH), 4.33 (d, J=
14.5 Hz, 1H), 4.24 (d, J = 14.5 Hz, 1H), 3.89 (d, J = 4.5 Hz, 2H),
3.53-3.44 (m, 3H). 13C NMR (DMSO-d6, 75 MHz):δ154.5, 149.0,
147.1, 130.5, 127.7 (2C), 127.2, 126.6, 124.3 (2C), 120.1, 110.5, 66.4,
60.7, 57.7, 54.8. vmax (KBr) cm-1 3326, 2876, 1530, 1350, 1243, 1162.
HRMS (m/z): [M þ Na] calcd for C16H18N2O7S, 405.0732; found,
405.0745.
General Procedure B (GP B): Preparation of Coumaryl Amino
Alcohols. A solution of the appropriate 7-hydroxycoumarin (10
equiv) and KOtBu (1.50 g, 13.20 mmol; 10 equiv) in anhydrous
DMF (40 mL) was added to resin 3E (4.00 g; 0.33 mmol g-1; in a 60
mL syringe equipped with a PP filter). The resin was agitated at rt
for 1 h under N2 and then drained and washed with DMF, MeOH,
and CH2Cl2 (each 3 ꢀ 40 mL, for 5 min). Then a solution of
2-mercaptoethanol (0.90 mL, 13.20 mmol; 10 equiv) and DBU
(1.00 mL, 6.60 mmol; 5 equiv) in anhydrous DMF (40 mL) was
added to the resin. After gentle agitation at rt for 1.5 h under N2, the
resin was drained and then treated again with 2-mercaptoethanol
(0.90 mL, 13.60 mmol; 10 equiv) and DBU (1.00 mL, 6.60 mmol; 5
equiv) in anhydrous DMF (40 mL) for 30 min under N2. The resin
was drained and washed with DMF, MeOH, and CH2Cl2 (each3ꢀ
40 mL, for 5 min). Fmoc-OSu (2.20 g, 6.60 mmol; 5 equiv) in
anhydrous THF (30 mL) was added to the resin preswollen in
anhydrous THF (10 mL) and iPr2EtN (1.20 mL, 6.60 mmol; 5
equiv). The resin was gently stirred at rt for 2 h under N2. The resin
was drained and washed with DMF, MeOH, and CH2Cl2 (each3ꢀ
40 mL, for 5 min). The product was cleaved with 2% TFA-CH2Cl2
(40 mL for 30 min, then 40 mL for 15 min), and finally the resin was
eluted with CH2Cl2 (40 mL). The eluates were pooled, and the
solvents were removed in vacuo. The crude material was purified by
VLC (heptane/EtOAc 4:1 as eluent).
N-[(S)-3-Hydroxy-2-[4-nitrophenylsulfonamido]propyl]-N-[(S)-
1-hydroxy-3-[N-[(R)-1-hydroxy-3-phenoxypropan-2-yl]-4-nitrophe-
nylsulfonamido]propan-2-yl]-4-nitrobenzenesulfonamide (8). Pre-
paration according to GP A using phenol (115 mg, 1.20 mmol)
and KOtBu (135 mg, 1.20 mmol). Note: resin 3A (100 mg; 1.20
mmol g-1) was used instead of resin 3E. Yield: 2 mg (2%) as a
colorless syrup. [R]20D þ4.6 (c 0.26, DMF). 1H NMR (CD3OD,
300 MHz): δ 8.40-8.37 (m, 2H), 8.30-8.27 (m, 2H), 8.21-8.18
(m, 2H), 8.14-8.12 (m, 4H), 8.10-8.05 (m, 2H), 7.15-7.10 (m,
2H), 6.87-6.82 (m, 1H), 6.67-6.64 (m, 2H), 4.30-4.21 (m, 1H),
4.13-4.04 (m, 3H), 4.00-3.87 (m, 3H), 3.83-3.75 (m, 4H),
3.63-3.51 (m, 2H), 3.40-3.36 (m, 2H). 13C NMR (CD3OD, 75
MHz): δ 158.7, 151.3 (2C), 150.9, 148.4, 147.0, 145.8, 130.1 (4C),
129.7 (2C), 129.6 (2C), 125.3 (2C), 125.2 (2C), 125.0 (2C), 122.2,
115.3 (2C), 67.0, 63.2, 62.5, 61.7, 60.5, 59.7, 58.1, 48.4 (hidden in
part under the solvent peak), 48.0 (hidden in part under the sol-
vent peak). HRMS (m/z): [M þ H]þ calcd for C33H36N6O16S3,
869.1428; found, 869.1375.
(R)-Methyl-4-[3-hydroxy-2-(4-nitrophenylsulfonamido)propoxy]-
benzoate (9). Preparation according to GP A using methyl-
4-hydroxybenzoate (100 mg, 0.66 mmol). Yield: 19 mg (71%) as
a colorless syrup. [R]20D þ26.7 (c 0.36, DMF). 1H NMR (DMSO-
d6, 300 MHz): δ 8.42-8.40 (br s, 1H, NH), 8.32-8.29 (m, 2H),
8.05-8.02 (m, 2H), 7.80-7.78 (m, 2H), 6.75-6.72 (m, 2H), 5.01 (t,
J = 5.5 Hz, 1H, OH), 4.04-3.93 (m, 2H), 3.80 (s, 3H), 3.54-3.41
(m, 3H). 13C NMR (DMSO-d6, 75 MHz): δ 165.5, 161.5, 149.1,
147.2, 130.9 (2C), 127.8 (2C), 124.2 (2C), 121.8, 114.1 (2C), 67.1,
61.0, 54.7, 51.8. vmax (KBr) cm-1 3433, 3147, 2906, 1730, 1529,
1348, 1244, 1169. HRMS (m/z): [M þ H]þ calcd for C17H18N2O8S,
411.0862; found, 411.0890.
(R)-N-[1-(4-Cyanophenoxy)-3-hydroxypropan-2-yl]-4-nitro-
benzenesulfonamide (10). Preparation according to GP A using
4-cyanophenol (79 mg, 0.66 mmol). Yield: 19 mg (76%) as a
colorless syrup. [R]20D þ27.1 (c 0.41, DMF). 1H NMR (DMSO-
d6, 300 MHz): δ 8.38 (d, J = 7.0 Hz, 1H, NH), 8.32-8.29 (m,
2H), 8.05-8.02 (m, 2H), 7.67-7.64 (m, 2H), 6.83-6.80 (m, 2H),
4.04-3.95 (m, 2H), 3.80-3.62 (m, 1H), 3.58-3.38 (m, 3H). 13
C
NMR (DMSO-d6, 75 MHz): δ 161.1, 149.1, 147.1, 133.9 (2C),
127.8 (2C), 124.2 (2C), 118.9, 115.1 (2C), 102.9, 67.2, 60.9, 54.6.
(R)-(9H-Fluoren-9-yl)methyl 1-Hydroxy-3-(2-oxo-2H-chro-
men-7-yloxy)propan-2-ylcarbamate (15A). Preparation accord-
ing to GP B using 7-hydroxycoumarin (2.1 g, 13.2 mmol). Yield:
v
max (KBr) cm-1 3422, 3140, 2901, 2224, 1529, 1350, 1258, 1165.
HRMS (m/z): [M þ Na]þ calcd for C16H15N3O6S, 400.0579;
154 mg (25%; overall yield in 3 steps) as a white foam. [R]20
D
found, 400.0578.
þ15.2 (c 0.50, DMF). 1H NMR (CDCl3, 300 MHz): δ 7.75-7.73
(m, 2H), 7.62-7.57 (m, 3H), 7.41-7.29 (m, 5H), 6.84-6.80 (m,
2H), 6.24 (d, J = 9.5 Hz, 1H), 5.52-5.44 (br s, 1H, NH),
4.59-4.44 (m, 2H), 4.22-4.09 (m, 3H), 3.94-3.66 (m, 2H),
3.23-3.14 (m, 2H). 13C NMR (CDCl3, 75 MHz): δ 161.4, 161.3,
156.4, 155.6, 143.7 (2C), 143.5 (2C), 141.3, 128.9, 127.8 (2C),
127.1 (2C), 125.0 (2C), 120.1 (2C), 113.3, 112.9, 112.7, 101.8,
67.1 (2C), 61.8, 51.7, 47.3. HRMS (m/z): [M þ Na] calcd for
C27H23NO6, 480.1423; found, 480.1423.
(R)-N-[1-(4-Chlorophenoxy)-3-hydroxypropan-2-yl]-4-nitro-
benzenesulfonamide (11). Preparation according to GP A using
4-chlorophenol (84 mg, 0.66 mmol). Yield: 21 mg (81%) as a
colorless syrup. [R]20D þ22.1 (c 0.28, DMF). 1H NMR (DMSO-
d6, 300 MHz): δ 8.38 (d, J = 7.5 Hz, 1H, NH), 8.33-8.30 (m,
2H), 8.05-8.02 (m, 2H), 7.22-7.19 (m, 2H), 6.68-6.65 (m, 2H),
4.98 (t, J = 5.5 Hz, 1H, OH), 3.94-3.84 (m, 2H), 3.54-3.39 (m,
3H). 13C NMR (DMSO-d6, 75 MHz): δ 156.5 (2C), 149.1, 147.2,
128.9 (2C), 127.8 (2C), 124.2 (2C), 115.8 (2C), 67.1, 60.9, 54.8.
(R)-(9H-Fluoren-9-yl)methyl 1-Hydroxy-3-(4-methyl-2-oxo-
2H-chromen-7-yloxy)propan-2-ylcarbamate (15B). Preparation
according to GP B using 7-hydroxy-4-methylcoumarin (2.3 g,
13.2 mmol). Yield: 122 mg (20%; overall yield in 3 steps) as a
white foam. [R]20D þ12.6 (c 0.31, DMF). 1H NMR (DMSO-d6,
300 MHz): δ 7.92-7.86 (m, 2H), 7.72-7.66 (m, 3H), 7.46-7.28
(m, 5H), 7.02-6.96 (m, 2H), 6.22 (s, 1H), 4.40-4.30 (m, 2H),
4.28-4.20 (m, 3H), 4.19-4.06 (m, 2H), 3.54-3.48 (m, 2H), 2.40
(s, 3H). 13C NMR (DMSO-d6, 75 MHz): δ 162.3, 160.9, 156.7,
155.5, 154.2, 144.7 (2C), 141.5 (2C), 128.4 (2C), 127.9 (2C),
127.3, 126.0 (2C), 120.9 (2C), 114.1, 113.3, 112.0, 102.2, 68.4,
66.3, 61.2, 53.2, 47.7, 19.1. HRMS (m/z): [M þ Na] calcd for
C28H25NO6, 494.1580; found, 494.1596.
v
max (KBr) cm-1 3426, 3145, 2907, 1529, 1344, 1248, 1153, 1090.
HRMS (m/z): [M þ H]þ calcd for C15H15ClN2O6S, 387.0418;
found, 387.0391.
(R)-N-[1-(2-Chlorophenoxy)-3-hydroxypropan-2-yl]-4-nitroben-
zenesulfonamide (12). Preparation according to GP A using
2-chlorophenol (85 mg, 0.66 mmol). Yield: 16 mg (63%) as a
colorless syrup. [R]20D þ11.4 (c 0.51, DMF). 1H NMR (DMSO-
d6, 300 MHz): δ 8.40 (d, J=6.5 Hz, 1H, NH), 8.31-8.28 (m, 2H),
8.06-8.03 (m, 2H), 7.31 (dd, J=1.5, 8.0 Hz, 1H), 7.22 (ddd,
J=1.5, 7.0, 8.0 Hz, 1H), 6.99 (dd, J=1.5, 8.0 Hz, 1H), 6.89 (ddd,
J = 1.5, 7.0, 8.0 Hz, 1H), 5.05-4.89 (br s, 1H, OH), 4.06-3.95
(m, 2H), 3.53-3.43 (m, 3H). 13C NMR (DMSO-d6, 75 MHz): δ
153.1, 149.0, 146.9, 129.7, 128.1, 127.8 (2C), 124.2 (2C), 121.5,
121.1, 113.5, 67.6, 60.8, 54.7. vmax (KBr) cm-13428, 3133, 2917,
(R)-(9H-Fluoren-9-yl)methyl 1-Hydroxy-3-(4-phenyl-2-oxo-
2H-chromen-7-yloxy)propan-2-ylcarbamate (15C). Preparation
J. Org. Chem. Vol. 75, No. 15, 2010 4989

Ottesen et al.
JOCArticle
according to GP B using 7-hydroxy-4-phenylcoumarin (3.1 g,
13.2 mmol). Yield: 95 mg (14% overall yield) as a white foam.
2 equiv), PyBOP (26 mg, 0.05 mmol; 2 equiv), HOAt (6 mg,
0.05 mmol; 2 equiv), and iPr2EtN (18 μL, 0.10 mmol; 4 equiv) in
anhydrous DMF (0.50 mL) was added to Fmoc-deprotected Rink
amide resin (25 mg; 1.00 mmol g-1) preswollen in anhydrous DMF
(0.50 mL) in a Teflon reactor. The resin was gently agitated at rt for 2
h, drained, and washed with DMF, MeOH, and CH2Cl2 (each3ꢀ 5
mL, for 5 min). The resin was then treated with a mixture of
Ac2O-iPr2EtN-NMP (1:2:3; 2 mL) for 10 min, drained, and
washed with DMF, MeOH, and CH2Cl2 (each 3 ꢀ 5 mL for 5
min). Next, the resin was treated with 20% piperidine-DMF (5 mL;
2 ꢀ 10 min), drained, and washed with DMF, MeOH, and CH2Cl2
(each 3 ꢀ 5 mL for 5 min). A solution of the appropriate N-Fmoc-
protected coumaryl amino acid (0.075 mmol; 3 equiv), DIC (13 μL,
0.075 mmol; 3 equiv), and HOAt (10 mg, 0.075 mmol; 3 equiv) in
anhydrous DMF (1 mL) was added to the resin in a sealed
microwave reactor (0.5-2 mL) in which the mixture was heated
to 60 °C for 30 min under MW irradiation (Biotage). The resin was
transferred to a Teflon reactor, drained, and washed with DMF,
MeOH, and CH2Cl2 (each 3 ꢀ 5 mL, for 5 min). After treatment
with 20% piperidine-DMF (2 ꢀ 10 min), the resin was drained
and washed with DMF, MeOH, and CH2Cl2 (each 3 ꢀ 5 mL, for
5 min). The resin was treated with a solution of Ac2O-iPr2EtN-
NMP (1:2:3; 2 mL) for 1 h at rt, drained, and washed with DMF,
MeOH and CH2Cl2 (each 3 ꢀ 5 mL for 5 min). The product was
cleaved with 95% TFA-CH2Cl2 (2 mL for 1 h), and then the resin
was eluted with CH2Cl2 and MeOH (each 2 mL upon shaking for
5 min). Finally, the resin was eluted with TFA (2 mL upon shaking
for 5 min). The eluates were pooled, and the solvents were removed
in vacuo. The crude material was purified by preparative reversed-
phase HPLC.
1
[R]20 þ16.2 (c 0.37, DMF). H NMR (CDCl3, 300 MHz): δ
D
7.74-7.72 (m, 2H), 7.59-7.56 (m, 2H), 7.50-7.48 (m, 3H),
7.39-7.25 (m, 7H), 6.87-6.76 (m, 2H), 6.17 (s, 1H), 5.57 (d, J =
6.0 Hz, 1H, NH), 4.44-4.42 (m, 2H), 4.21-4.14 (m 4H), 3.98-
3.77 (m, 2H), 2.87-2.64 (br s, 1H, OH). 13C NMR (CDCl3, 75
MHz): δ 161.5, 161.3, 156.4, 155.8, 155.7, 143.7 (2C), 141.3 (2C),
135.3, 129.7, 128.9 (2C), 128.4 (2C), 128.0, 127.8 (2C), 127.1
(2C), 125.0 (2C), 120.0 (2C), 112.8, 112.4, 112.0, 102.1, 67.0
(2C), 61.7, 51.8, 47.3. HRMS (m/z): [M þ Na]þ calcd for
C33H27NO6, 556.1736; found, 556.1755.
General Procedure C (GP C): Preparation of Coumaryl Amino
Acids. To a stirred heterogeneous mixture of the appropriate
coumaryl amino alcohol (1 equiv) in acetone (12 mL) and aqueous
5% NaHCO3 (3 mL) were added KBr (0.1 equiv) and TEMPO
(1 equiv) at 0 °C. An aqueous NaOCl solution (1.7 M; 5 equiv) was
then added dropwise during 15 min, and the mixture was stirred
vigorously at 0 °C for 1 h. Acetone was removed in vacuo, and the
resulting aqueous mixture was acidified to pH 5-6 with 10% citric
acid. After extraction of the aqueous layer with EtOAc (3 ꢀ
15 mL), the combined organic layers were dried (Na2SO4), filtered,
and concentrated in vacuo. The crude material was purified by
VLC (heptane/EtOAc/HOAc 50:50:1 as eluent).
(S)-2[[(9H-Fluoren-9-yl)methoxy]carbonylamino]-3-(2-oxo-2H-
chromen-7-yloxy)propanoic Acid (16A). Preparation according
to GP C using 15A (150 mg, 0.33 mmol), KBr (4 mg, 0.03 mmol),
TEMPO (52 mg, 0.33 mmol), and NaOCl (∼1.7 M; 0.97 mL,
1.65 mmol). Yield: 131 mg (85%) as a white foam. [R]20D þ25.2
1
(c 0.50, DMF). H NMR (DMSO-d6, 300 MHz): δ 8.00-7.94
(m, 2H), 7.87-7.84 (m, 2H), 7.74-7.72 (m, 2H), 7.61 (d, J = 8.5
Hz, 1H), 7.42-7.37 (m, 2H), 7.33-7.26 (m, 2H), 7.00-6.93 (m,
2H), 4.56-4.50 (m, 1H), 4.40-4.32 (m, 4H), 4.25-4.20 (m, 1H).
13C NMR (DMSO-d6, 75 MHz): δ 170.8, 161.1, 160.1, 156.1,
155.1, 144.1, 143.6 (2C), 140.6 (2C), 129.4, 127.6 (2C), 127.0
(2C), 125.2 (2C), 120.1 (2C), 112.7 (2C), 112.6, 101.4, 68.0, 66.0,
53.9, 46.7. HRMS (m/z): [M þ Na] calcd for C27H21NO7,
494.1216; found, 494.1198.
(S)-2[[(9H-Fluoren-9-yl)methoxy]carbonylamino]-3-(4-methyl-
2-oxo-2H-chromen-7-yloxy)propanoic Acid (16B). Preparation
according to GP C using 15B (262 mg, 0.56 mmol), KBr (7 mg,
0.06 mmol), TEMPO (88 mg, 0.56 mmol), and NaOCl (∼1.7 M;
1.60 mL, 2.80 mmol). Yield: 251 mg (93%) as a white foam.
[R]20D þ12.6 (c 0.31, DMF). 1H NMR (DMSO-d6, 300 MHz): δ
7.95 (d, J = 8.0 Hz, 1H, NH), 7.89-7.87 (m, 2H), 7.74-7.66 (m,
3H), 7.42-7.40 (m, 2H), 7.33-7.28 (m, 2H), 6.99-6.95 (m, 2H),
6.21 (s, 1H), 4.49-4.41 (m, 1H), 4.38-4.30 (m, 4H), 4.24-4.20
(m, 1H), 2.38 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): δ 170.7,
161.0, 159.9, 155.9, 154.4, 153.2, 143.6 (2C), 140.5 (2C), 127.5
(2C), 126.9 (2C), 126.4, 125.1 (2C), 119.1 (2C), 113.3, 112.3,
111.3, 101.4, 67.9, 65.8, 53.6, 46.6, 18.2. HRMS (m/z): [M þ H]þ
calcd for C28H23NO7, 486.1553; found, 486.1568.
(S)-2[[(9H-Fluoren-9-yl)methoxy]carbonylamino]-3-(2-oxo-4-
phenyl-2H-chromen-7-yloxy)propanoic Acid (16C). Preparation
according to GP C using 15C (85 mg, 0.16 mmol), KBr (2 mg, 0.02
mmol), TEMPO (25 mg, 0.16 mmol), and NaOCl (∼1.7 M; 0.60
mL, 0.80 mmol). Yield: 63 mg (72%) as a white foam. [R]20D þ15.0
(c 0.58, DMF). 1H NMR (DMSO-d6, 300 MHz): δ 7.98 (d, J = 8.5
Hz, 1H, NH), 7.89-7.87 (m, 2H), 7.73-7.71 (m, 2H), 7.56-7.48
(m, 5H), 7.42-7.27 (m, 5H), 7.11 (d, J = 2.5 Hz, 1H), 6.95 (dd, J =
2.5, 9.0 Hz, 1H), 6.24 (s, 1H), 4.51-4.41 (m, 1H), 4.40-4.30 (m,
4H), 4.23-4.19 (m, 1H). 13C NMR (DMSO-d6, 75 MHz): δ 170.7,
161.1, 159.8, 156.0, 155.2, 154.9, 143.6 (2C), 140.6 (2C), 134.8, 129.6,
128.8 (2C), 128.3 (2C), 127.8, 127.5 (2C), 126.9 (2C), 125.2 (2C),
120.0 (2C), 112.8, 112.2, 111.5, 101.9, 68.0, 65.8, 53.6, 46.6. HRMS
(m/z): [M þ H]þ calcd for C33H25NO7, 548.1709; found, 548.1705.
General Procedure D (GP D): Preparation of Coumarin-Bear-
ing Dipeptides. A solution of Fmoc-Phe-OH (20 mg, 0.05 mmol;
(S )-2-Acetamido-N-[(S)-1-amino-1-oxo-3-phenylpropan-2-yl]-
3-(2-oxo-2H-chromen-7-yloxy)propanamide (17A). Preparation ac-
cording to GP D using 16A (35 mg, 0.075 mmol). Yield: 7 mg (64%)
as a white foam. [R]20D -9.4 (c 0.50, DMF). 1H NMR (DMSO-d6,
600 MHz): δ8.32 (d, J= 8.0 Hz, 1H), 8.14 (d, J= 8.0 Hz, 1H), 8.00
(d, J = 9.0 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.32 (br s, 1H), 7.24-
7.18(m, 5H), 7.16(brs, 1H), 6.95(d, J= 2.5 Hz, 1H), 6.90 (dd, J=
2.5, 8.5 Hz, 1H), 6.31 (d, J= 9.0 Hz, 1H), 4.67-4.64 (m, 1H), 4.47-
4.43 (m, 1H), 4.19-4.15 (m, 2H), 3.05 (dd, J = 5.0, 14.0 Hz, 1H),
2.86 (dd, J = 9.0, 14.0 Hz, 1H), 1.87 (s, 3H). 13C NMR (DMSO-d6,
150 MHz): δ 172.4, 168.8, 168.6, 161.1, 160.1, 155.1, 144.2, 137.7,
129.4, 129.1 (2C), 127.9 (2C), 126.1, 112.6 (3C), 101.4, 68.0, 53.8,
52.3, 37.1, 22.4. HRMS (m/z): [M þ Na]þ calcd for C23H23N3O6,
460.1485; found, 460.1500.
(S )-2-Acetamido-N-[(S)-1-amino-1-oxo-3-phenylpropan-2-yl]-
3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamide (17B). Pre-
paration according to GP D using 16B (36 mg, 0.075 mmol). Yield:
9 mg (79%) as a white foam. [R]20D -9.3 (c 0.50, DMF). 1H NMR
(DMSO-d6, 600 MHz): δ 8.33 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 8.5
Hz, 1H), 7.69 (d, J = 9.0 Hz, 1H), 7.33 (br s, 1H), 7.25-7.17 (m,
5H), 7.16 (br s, 1H), 6.93 (dd, J = 2.5, 8.0 Hz, 1H), 6.91 (d, J = 2.5
Hz, 1H), 6.23 (s, 1H), 4.67-4.64 (m, 1H), 4.47-4.43 (m, 1H),
4.19-4.14 (m, 2H), 3.05 (dd, J = 5.0, 14.0 Hz, 1H), 2.86 (dd, J =
9.5, 14.0 Hz, 1H), 2.40 (s, 3H), 1.87 (s, 3H). 13C NMR (DMSO-d6,
150 MHz): δ 172.4, 169.8, 168.6, 161.0, 160.0, 154.5, 153.3, 137.7,
129.1 (2C), 127.9 (2C), 126.4, 126.1, 113.3, 112.3, 111.3, 101.4, 68.0,
53.8, 52.3, 37.1, 22.4, 18.0. HRMS (m/z): [M þ Na]þ calcd for
C24H25N3O6, 474.1641; found, 474.1653.
(S )-2-Acetamido-N-[(S)-1-amino-1-oxo-3-phenylpropan-2-yl]-
3-(2-oxo-4-phenyl-2H-chromen-7-yloxy)propanamide (17C). Pre-
paration according to GP D using 16C (41 mg, 0.075 mmol).
Yield: 9 mg (70%) as a white foam. [R]20D -9.0 (c 0.10, DMF). 1H
NMR (DMSO-d6, 600 MHz): δ 8.33 (d, J = 7.0 Hz, 1H), 8.14 (d,
J=8.5 Hz, 1H), 7.58-7.52 (m, 5H), 7.36 (d, J=9.0 Hz, 1H), 7.32
(br s, 1H), 7.25-7.17 (m, 5H), 7.15 (br s, 1H), 7.06 (d, J = 2.5 Hz,
1H), 6.90 (dd, J = 2.5, 9.0 Hz, 1H), 6.26 (s, 1H), 4.68-4.65 (m,
1H), 4.47-4.43 (m, 1H), 4.21-4.16 (m, 2H), 3.05 (dd, J=5.0, 14.5
Hz, 1H), 2.85 (dd, J = 9.0, 14.5 Hz, 1H), 1.87 (s, 3H). 13C NMR
4990 J. Org. Chem. Vol. 75, No. 15, 2010

Ottesen et al.
JOCArticle
(DMSO-d6, 75 MHz): δ 172.3, 169.7, 168.5, 161.2, 159.9, 155.2,
155.0, 137.7, 134.9, 129.6, 129.1 (2C), 128.8 (2C), 128.3 (2C), 127.9
(2C), 127.8, 126.1, 112.7, 112.0, 111.5, 101.9, 68.1, 53.8, 52.2, 37.2,
22.4. HRMS (m/z): [M þ H]þ calcd for C29H27N3O6, 514.1978;
found, 514.1996.
2 mL upon shaking for 5 min). Finally, the resin was eluted with
TFA (2 mL upon shaking for 5 min). The eluates were pooled, the
solvents were removed in vacuo, and the crude material was purified
by preparative reversed-phase HPLC. Yield: 10 mg (26%; overall
yield in 9 steps) as a white foam. 1H NMR (CD3OD, 300 MHz): δ
7.73 (d, J = 9.5 Hz, 1H), 7.04-7.01 (m, 2H), 6.22 (s, 1H),
4.60-4.57 (m, 1H), 4.43-4.41 (m, 2H), 4.34-4.22 (m, 8H), 3.23-
3.19 (m, 16H), 2.46 (s, 3H), 2.09 (s, 3H), 1.87-1.70 (m, 32H).
HRMS (m/z): [M þ 3H/3]þ calcd for C29H27N3O6, 518.6383;
found, 518.6381.
Preparation of Coumarin-Labeled Octaarginine (18). Resin-
bound octaarginine (100 mg, 0.025 mmol) was prepared with a
Rink amide linker by using Fmoc chemistry on a CEM Liberty
Microwave Peptide Synthesizer. Loading as well as chain elonga-
tion were performed as double-couplings each with Fmoc-Arg(Pbf)-
OH (5 equiv), TBTU (5 equiv), and iPr2EtN (5 equiv) at 60 °C for
15 min. Fmoc-deprotection was carried out with 20% piperidine-
DMF. Compound 16B (30 mg, 0.075 mmol) was introduced by
using a coupling with DIC (12 μL, 0.075 mmol) and HOAt (10 mg,
0.075 mmol) in anhydrous DMF (2 mL) under microwave irradia-
tion (Biotage) at 60 °C for 30 min in a sealed microwave reactor
(0.5-2 mL). Final Fmoc-deprotection was performed at rt with
20% piperidine-DMF (2 ꢀ 10 min), while acetylation (Ac2O-
iPr2EtN-NMP; 1:2:3 in volume) was performed at rt for 1 h. The
product was cleaved with TFA-TIS-H2O (2 mL; 95:2.5:2.5) at rt
for 5 h, and then the resin was eluted with CH2Cl2 and MeOH (each
Acknowledgment. Technical assistance of Mrs. Birgitte
Simonsen, Department of Medicinal Chemistry, and Mrs.
Maria Michaelsen, Department of Pharmaceutics and Analy-
tical Chemistry, University of Copenhagen, is gratefully ack-
nowledged. We thank the Lundbeck Foundation for a grant.
Supporting Information Available: Procedures and full
characterization of new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 15, 2010 4991