Synthesis of 4-(Fmoc-aminoacyloxymethyl)phenoxyacetic acids for use in solid-phase peptide synthesis

Article abstract of DOI:10.1055/s-2006-942485

The synthesis of 4-(Fmoc-aminoacyloxymethyl)phenoxyacetic acids was achieved in high yield by the reaction of Fmoc-amino acids with (4-iodomethylphenoxy)acetic acid 2-oxo-2-phenylethyl ester. The removal of the temporary protecting group, phenacyl ester,

Full text of DOI:10.1055/s-2006-942485

PAPER
2789
Synthesis of 4-(Fmoc-aminoacyloxymethyl)phenoxyacetic Acids for Use in
Solid-Phase Peptide Synthesis
S
ynthesis of 4-(Fm
i
oc-am
k
o
ethyl)-pheno
s
xyacetic Acids Zinieris,a Stella Kokinaki,a Leondios Leondiadis,b Nikolas Ferderigos*a
a
Laboratory of Organic Chemistry, Chemistry Department, University of Athens, 157 71 Athens, Greece
Mass Spectrometry and Dioxine Analysis Lab, IRRP, National Centre for Scientific Research ‘Demokritos’, 153 10 Athens, Greece
Fax +30(210)7274761; E-mail: nferder@chem.uoa.gr
b
Received 7 April 2006; revised 20 April 2006
presence of moisture can severely affect loading efficien-
cies; b) reactions should be conducted using the minimum
volumes of solvent to ensure maximum reagent concen-
tration; c) after each reaction, the loading efficiency
Abstract: The synthesis of 4-(Fmoc-aminoacyloxymethyl)phen-
oxyacetic acids was achieved in high yield by the reaction of
Fmoc-amino acids with (4-iodomethylphenoxy)acetic acid 2-oxo-
2-phenylethyl ester. The removal of the temporary protecting
group, phenacyl ester, was effectively achieved by reductive cleav- should be checked and, if necessary, the reaction should
age with magnesium turnings.
be repeated with fresh reagents; and d) a strong acetylat-
ing reagent must be used in order to cap unreacted resin-
bound hydroxyl groups to prevent the growth of deletion
sequences at these sites.
Key words: solid-phase synthesis, magnesium, peptides
The solid-phase methodology proposed by Merrifield1
and fine-tuned over time is presently based on a Boc/Bn
or Fmoc/t-Bu strategy using polystyrene (PS) or PEG-PS
solid supports.2
O
Boc-A.A.O
+
OH
O
H2N
A
The anchoring of the first amino acid residue to the solid
support is one of the most critical steps in solid-phase pep-
tide synthesis. The process by which the C-terminal Na-
protected amino acid is attached to the resin and the nature
of the anchoring linkage ultimately contributes to the
yield and purity of the final product. Sites on the resin not
initially blocked by this process can be potentially acylat-
ed in subsequent steps, leading to the generation of related
C-terminally truncated by-products.
Boc-A.A.O
N
H
O
Fmoc-A.A. OH
Fmoc-A.A.O
+
HO
B
The Fmoc/t-Bu strategy has gained considerable popular-
ity over the last decade due to the use of milder reagents
and the high yields usually obtained. However, the forma-
tion of the ester bond, required when attaching the first
amino acid to the alkoxybenzyl resin in the Fmoc/t-Bu
process, is considerably more difficult than that of the cor-
responding amide bond used in the Boc/Bn strategy for
the attachment of Na-Boc-aminoacyloxymethyl acetic
acid to aminomethyl resins (Scheme1).
O
Scheme 1 A, Boc/Bn strategy; B Fmoc/t-Bu strategy; A.A., amino
acid.
An alternative method for the attachment of the C-termi-
nal Na-Fmoc-amino acid to the p-alkoxybenzyl-type resin
utilizes novel active esters of Na-Fmoc-aminoacyl-4-
oxymethylphenoxy acetic acids9 which directly acylate
amino functionalized polymers. These methods suffer
from a number of disadvantages, namely: a) from our ex-
perience and the experience of others,10 the reaction of an
active ester derivative with a protected-amino acid gives
rise to the formation of a multiplicity of by-products and
b) active esters are not the method of choice for activation
of a carboxyl function in order to achieve high loaded res-
ins. The need for an improved methodology led us to em-
ploy the new approach described here. This new approach
utilizes a phenacyl group (PAC), which has proved to be
an important reagent for protecting carboxyl functions
during orthogonal synthesis.11After insertion of the de-
sired Fmoc-amino acid, the PAC group is removed utiliz-
In the Boc/Bn approach, a controlled loading is performed
and no by-products are produced. In contrast, the p-al-
koxybenzyl ester anchoring linkage introduced by Wang
et al.3,4 and its subsequent evolution by Sheppard et al.5,6
for the Fmoc/t-Bu strategy, requires harsher conditions
which may lead to low substitution or enantiomerization,7
especially for cysteine and histidine, and formation of
dipeptide by-products.8 To ensure satisfactory results
when using this methodology, it is important that the fol-
lowing precautions are taken: a) all reagents and glass-
ware must be dried thoroughly before use, since the
SYNTHESIS 2006, No. 16, pp 2789–2793
x
x.
x
x
.
2
0
0
6
Advanced online publication: 12.07.2006
DOI: 10.1055/s-2006-942485; Art ID: Z07506SS
© Georg Thieme Verlag Stuttgart · New York

2790
N. Zinieris et al.
PAPER
OH
Consequently, the phenacyl ester had to be removed with-
out affecting the ester bond between the oxygen and the
amino acid. The classic zinc dust/acetic acid18 method for
reductive cleavage of the phenacyl ester, also affects the
ester bond to the Fmoc-amino acid unit. In a recent report,
this problem was resolved by replacing zinc dust with
magnesium turnings,12 which is a milder and more conve-
nient reagent than zinc for the deprotection of the phen-
acyl temporary protecting group.
OH
i
O
O
O
O
OH
OPAC
1
2
ii
I
O
O
3
OPAC
All isolated yields were greater than 80%; the products
were of high purity (> 95%) (Table 1).
iii
Fmoc AA O
Fmoc AA O
iv
Finally, to test the ability of the synthesized derivatives 5
to bind to the aminomethyl resins,19 4-[Fmoc-(g-tert-bu-
tyl)glutamyloxymethyl]phenoxyacetic acid (5c) activated
by PyBOP,20 was reacted with aminomethyl PS-DVB res-
in (1 mmol NH2/g). The yield was quantitative, as con-
firmed by the Kaiser ninhydrine test and estimation of the
Fmoc group.21
O
O
O
O
OPAC
OH
4
5
Scheme 2 Reagents and conditions: i. Phenacyl bromide, Et3N; ii.
NaI, TMSCl, CH3CN; iii. Fmoc A.A.OH, DIPEA; iv. Mg turnings,
AcOH, DMF–MeOH.
In conclusion, we have developed a practical, controlled,
and versatile methodology for the anchoring of Na-Fmoc-
amino acids as p-alkoxybenzyl esters in solid-phase pep-
tide synthesis.
ing our recently developed methodology,12 which
tolerates the rest of the functional groups present.
Specifically, 4-[(hydroxymethyl)phenoxy]acetic acid (1)
was protected with a PAC group under standard condi-
tions (Scheme 2).13
All reagents and solvents were obtained commercially (Aldrich,
LabScan) and used without further purification. PE with a bp range
40–60 °C was used. Fmoc-amino acids were provided by CBL,
Patras, Greece. The reactions were monitored by TLC on Merck sil-
ica gel plates (60F, 254) using UV light as a visualizing agent. 1H
NMR and 13C NMR spectra were obtained on a Mercury Varian
Spectrometer (200 MHz). Chemical shifts are reported relative to
residual undeuterated solvent as the internal reference. Optical rota-
tions were recorded on a Perkin Elmer 343 Polarimeter. Elemental
analyses were performed on a Perkin Elmer Elemental Analyzer
2400. For ESI mass spectral analysis a test solution in 50% aq
MeOH was infused into an electrospray interface mass spectrome-
ter (MSQ Surveyor, Finnigan) at a flow rate of 1 mL/min. UV mea-
surements were obtained on a UV-VIS spectrophotometer Varian-
The hydroxyl group was converted to the corresponding
iodide using the chlorotrimethylsilane/sodium iodide
method14 and reacted with Fmoc-amino acids (Table 1) in
the presence of DIPEA to form the ester bond. Iodide de-
rivatives/DIPEA were preferred for the formation of the
ester bond, since no by-products were observed; in con-
trast, when we used the corresponding bromide deriva-
tives/potassium fluoride15,16 or the cesium carbonate
method,17 a low yield of the desired product was obtained.
Table 1 Fmoc-amino Acid Derivatives 4 and 5
Amino acid
Esters 4
Acids 5
20,a
20,a
Yield (%)
Mp (°C)
133–136
oil
[a]D
+6.2
–
Yield (%)
Mp (°C)
189–192
126–132
78–82
[a]D
a
Asn
Arg
Glu
Gly
Lys
Met
Phe
Ser
95
80
94
88
98
95
97
92
91
93
90
60
90
87
94
87
94
90
87
93
–10.7
–27.4
+0.4
–
b
c
84–85
101
+1.9
–
d
172–175
102–104
oil
e
105–110
85–87
123–126
oil
–3.5
+12
–4.1
–
–16.7
–
f
g
105–107
119–122
131–134
105–107
–1.21
+2.3
–20.8
–4.57
h
k
Trp
Tyr
100–104
92–94
–31.5
–4.2
l
a c = 1, DMF.
Synthesis 2006, No. 16, 2789–2793 © Thieme Stuttgart · New York

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Synthesis of 4-(Fmoc-aminoacyloxymethyl)phenoxyacetic Acids
2791
Cary 50. Mps were determined on a Büchi 535 apparatus and are
uncorrected. Column chromatography purifications were per-
formed using Merck 70–230 mesh silica gel 60.
4-[Fmoc-(NG-Pbf)arginyloxymethyl]phenoxyacetic Acid Phen-
acyl Ester (4b)
1H NMR (200 MHz, CDCl3): d = 1.39 (s, 6 H), 1.52–1.89 (m, 4 H),
2.04 (s, 3 H), 2.48 (s, 3 H), 2.56 (s, 3 H), 2.86 (s, 2 H), 2.94–3.19
(m, 2 H), 4.01–4.18 (m, 1 H), 4.22–4.39 (m, 3 H), 4.76 (s, 2 H), 4.98
(d, J = 11.7 Hz, 1 H), 5.07 (d, J = 12.2 Hz, 1 H), 5.40 (s, 2 H), 5.80
(d, J = 8.0 Hz, 1 H), 5.90 (br, 1 H), 6.20 (br, 2 H), 6.88 (d, J = 8.4
Hz, 2 H), 7.21–7.59 (m, 11 H), 7.71 (d, J = 7.4 Hz, 2 H), 7.85 (d,
J = 8.4 Hz, 2 H).
(4-Hydroxymethylphenoxy)acetic Acid 2-Oxo-2-phenylethyl
Ester (2)
To a cooled (0 °C) solution of 4-[(hydroxymethyl)phenoxy]acetic
acid (1.82 g, 10 mmol) and phenacyl bromide (2.0 g, 10 mmol) in
EtOAc (20 mL), Et3N (1.1 g, 11 mmol) was added and the reaction
mixture was left overnight at r.t. The reaction mixture was diluted
with Et2O (20 mL), and the ethereal layer was washed successively
with H2O (10 mL), 5% NaHCO3 (10 mL), and brine (10 mL). The
organic phase was dried over anhyd Na2SO4 and the solvents were
evaporated in vacuo. The residue was recrystallized from EtOAc–
PE; yield: 2.7g (90%); mp 95–97 °C.
13C NMR (75 MHz, CDCl3): d = 191.62, 172.31, 169.00, 158.91,
158.01, 156.49, 156.42, 144.08, 143.91, 141.44, 138.52, 134.42,
133.94, 133.20, 132.44, 130.53, 129.19, 128.86, 128.00, 127.34,
125.39, 124.89, 120.19, 117.72, 114.93, 86.60, 67.30, 67.05, 66.87,
66.80, 65.11, 53.93, 47.28, 43.36, 40.84, 29.88, 28.77, 25.41, 19.56,
18.22, 12.72.
1H NMR (200 MHz, CDCl3): d = 4.63 (s, 2 H), 4.85 (s, 2 H), 5.47
(s, 2 H), 6.98 (d, J = 8.6 Hz, 2 H), 7.32 (d, J = 8.6 Hz, 2 H), 7.46–
7.63 (m, 3 H), 7.91 (d, J = 7.1 Hz, 2 H).
Anal. Calcd for C51H54N4O11S: C, 65.79; H, 5.85; N, 6.02. Found:
C, 65.53; H, 5.73; N, 5.80.
13C NMR (75 MHz, CDCl3): d = 191.73, 168.89, 157.42, 134.69,
134.34, 134.06, 129.16, 128.84, 128.00, 114.93, 66.71, 65.33,
64.87.
4-[Fmoc-(g-tert-butyl)glutamyloxymethyl]phenoxyacetic Acid
Phenacyl Ester (4c)
1H NMR (200 MHz, CDCl3): d = 1.44 (s, 9 H), 1.81–2.11 (m, 2 H),
2.21–2.39 (m, 2 H), 4.08–4.19 (m, 1 H), 4.26–4.50 (m, 3 H), 4.75
(s, 2 H), 5.09 (s, 2 H), 5.38 (s, 2 H), 5.73 (d, J = 8.1 Hz, 1 H), 6.92
(d, J = 8.6 Hz, 2 H), 7.23–7.58 (m, 11 H), 7.72 (d, J = 7.4 Hz, 2 H),
7.84 (d, J = 8.5 Hz, 2 H).
Anal. Calcd for C17H16O5: C, 67.99; H, 5.37. Found: C, 67.67; H,
5.25.
(4-Iodomethylphenoxy)acetic Acid 2-Oxo-2-phenylethyl Ester
(3)
13C NMR (CDCl3): d = 191.39, 172.19, 172.12, 168.52, 158.17,
156.25, 144.21, 143.96, 141.49, 134.22, 134.11, 130.36, 129.10,
128.77, 127.99, 127.94, 127.32, 125.40, 125.35, 120.20, 115.03,
80.91, 67.24, 67.12, 66.68, 65.18, 53.86, 47.36, 31.64, 28.32, 27.61.
To a solution of 1 (2.5 g, 8.3 mmol) and NaI (1.25 g, 8.3 mmol) in
MeCN (10 mL) covered with aluminum foil was slowly added
TMSCl (0.9 g, 8.3 mmol) with continuous stirring. After 2 h the re-
action was complete (monitored by TLC), the reaction mixture was
taken up in Et2O (100 mL) and washed successively with H2O (10
mL), a 10% solution of Na2S2O3 (25 mL), brine (20 mL), dried over
Na2SO4, and concentrated in vacuo to dryness. The residue was pu-
rified by recrystallization from EtOAc–PE (1:4); yield: 3.0 g (90%);
mp 116–117 °C.
Anal. Calcd for C41H41NO10: C, 69.58; H, 5.84; N, 1.98. Found: C,
69.32; H, 5.82; N, 1.67.
4-(Fmoc-glycyloxymethyl)phenoxyacetic Acid Phenacyl Ester
(4d)
1H NMR (200 MHz, DMSO-d6): d = 3.84 (d, J = 5.9 Hz, 1 H), 4.18–
4.34 (m, 3 H), 4.95 (s, 2 H), 5.08, (s, 2 H), 5.62 (s, 2 H), 6.97 (d,
J = 8.6 Hz, 2 H), 7.27 –7.79 (m, 11 H), 7.86 (d, J = 7.0 Hz, 2 H),
7.97 (d, J = 7.1 Hz, 2 H).
1H NMR (200 MHz, CDCl3): d = 4.45 (s, 1 H), 4.83 (s, 1 H), 5.46
(s, 2 H), 6.91 (d, J = 8.8 Hz, 2 H), 7.34 (d, J = 8.8 Hz, 2 H), 7.45–
7.63 (m, 3 H), 7.91 (d, J = 7.0 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 191.46, 168.61, 157.55, 134.35,
13C NMR (75 MHz, DMSO-d6): d = 192.95, 170.83, 169.04,
158.15, 157.25, 144.47, 141.43, 134.71, 134.33, 130.59, 129.57,
129.32, 128.50, 128.31, 127.74, 125.86, 120.76, 115.14, 67.63,
66.50, 66.35, 65.12, 47.28, 42.89.
134.10, 132.78, 130.40, 129.19, 128.00, 115.29, 66.75, 65.35, 6.27.
Anal. Calcd for C17H15IO4: C, 49.78; H, 3.69. Found: C, 49.82; H,
3.52.
Anal. Calcd for C34H29NO8: C, 70.46; H, 5.04; N, 2.42. Found: C,
69.82; H, 5.22; N, 2.27.
4-(Fmoc-asparaginyloxymethyl)phenoxyacetic Acid Phenacyl
Ester (4a); Typical Procedure
To a stirred and cooled (0 °C) solution of Fmoc-asparagine (2.6 g,
6 mmol) and DIPEA (0.61 g 5.5 mmol) under an Ar atmosphere was
added 3 (2.0 g, 10 mmol), the resulting solution was left overnight
at 0 °C. The mixture was taken up in EtOAc (70 mL) and washed
successively with H2O (10 mL), a 5% solution of NaHCO3 (25 mL),
a 10% solution of Na2S2O3 (25 mL), brine (10 mL), and dried over
Na2SO4. The solvent was evaporated under reduced pressure and
the residue was purified by column chromatography (CHCl3–
MeOH, 9:1).
1H NMR (200 MHz, DMSO-d6): d = 2.52–2.64 (m, 2 H), 4.14–4.34
(m, 4 H), 4.34–4.50 (m, 1 H), 4.91 (s, 2 H), 5.03 (s, 2 H), 5.60 (s, 2
H), 6.91 (d, J = 8.7 Hz, 2 H), 7.20–7.74 (m, 11 H), 7.86 (d, J = 7.1
Hz, 2 H), 7.95 (d, J = 7.1 Hz, 2 H).
4-[Fmoc-(e-Boc)-lysyloxymethyl]phenoxyacetic Acid Phenacyl
Ester (4e)
1H NMR (200 MHz, CDCl3): d = 1.42 (s, 9 H), 1.57–2.12 (m, 6 H),
2.93–3.14 (m, 2 H), 4,12–4.22 (m, 1 H), 4.29–4.45 (m, 3 H), 4.60
(br, 1 H), 4.80 (s, 2 H), 5.06 (d, J = 11.8 Hz, 1 H), 5.15 (d, J = 11.8
Hz, 1 H), 5.44 (s, 2 H), 6.95 (d, J = 8.5 Hz, 2 H), 7.25–7.50 (m, 9
H), 7.58 (d, J = 7.3 Hz, 2 H), 7.75 (d, J = 7.2 Hz, 2 H), 7.88 (d, J =
8.6 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 191.46, 172.54, 168.64, 158.16,
156.29, 156.21, 144.17, 143.96, 141.50, 134.33, 134.08, 130.48,
129.16, 128.83, 127.98, 127.93, 127.30, 125.35, 120.19, 115.02,
67.23, 67.03, 66.72, 65.24, 54.05, 47.37, 40.24, 32.28, 29.80, 28.65,
22.52.
13C NMR (75 MHz, DMSO-d6): d = 193.03, 172.28, 171.45,
169.04, 158.02, 156.52, 144.44, 141.37, 134.75, 134.35, 130.18,
129.61, 129.38, 128.51, 128.31, 127.77, 125.89, 120.79, 115.09,
67.66, 66.45, 65.12, 51.39, 47.24, 37.31.
Anal. Calcd for C43H46N2O10: C, 68.78; H, 6.18; N, 3.73. Found: C,
68.66; H, 6.35; N, 3.57.
4-(Fmoc-methionyloxymethyl)phenoxyacetic Acid Phenacyl
Ester (4f)
Anal. Calcd for C36H32N2O8: C, 69.67; H, 5.20; N, 4.51. Found: C,
69.42; H, 5.45; N, 4.70.
1H NMR (200 MHz, CDCl3): d = 2.01 (s, 3 H), 1.98–2.22 (m, 2 H),
2.30–2.53 (m, 2 H), 4.06–4.60 (m, 4 H), 4.77 (s, 2 H), 5.03–5.17 (m,
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N. Zinieris et al.
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2 H), 5.40 (s, 2 H), 5.68 (d, J = 7.9 Hz, 1 H), 6.94 (d, J = 8.5 Hz, 2
H), 7.20–7.60 (m, 11 H), 7.74 (d, J = 7.3 Hz, 2 H), 7.86 (d, J = 8.4
Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 191.46, 172.11, 168.57, 158.20,
156.17, 144.15, 143.95, 141.51, 134.28, 134.08, 130.46, 129.14,
128.74, 127.33, 125.33, 120.23, 115.06, 67.19, 66.73, 65.21, 53.50,
47.38, 31.98, 30.06, 15.64.
Anal. Calcd for C45H43N2O9: C, 72.86; H, 5.84; N, 1.89. Found: C,
73.15; H 5.75; N, 2.07.
4-(Fmoc-asparaginyloxymethyl)phenoxyacetic Acid (5a); Typi-
cal Procedure
To a solution of 4-(Fmoc-asparaginyloxymethyl)phenoxy acetic
acid phenacyl ester (4a; 1.27 g, 2 mmol) in MeOH–DMF (8:2, 20
mL), AcOH (24 mmol, 1.5 mL) and Mg turnings (0.32 g, 14 mmol)
were added. After stirring for 120 min at r.t. the reaction mixture
was filtered. The filtrate was concentrated in vacuo and the residue
was diluted with a 5% solution of NaHCO3 (20 mL) and Et2O–
EtOAc (1:1, 20 mL), and the organic layer was extracted with a 5%
solution of NaHCO3 (2 × 20 mL). The aqueous layer was acidified
to pH 2–3 with a sat. solution of KHSO4 and extracted with EtOAc
(2 × 20 mL). The organic layer was washed with brine (2 × 20 mL),
dried over Na2SO4, and filtered. The solvent was evaporated under
reduced pressure and the residue was purified by column chroma-
tography (CHCl3–MeOH, 9:1).
Anal. Calcd for C37H35NO8S: C, 67.98; H, 5.40; N, 2.14. Found: C,
68.25; H, 5.57; N, 2.43.
4-(Fmoc-phenylalanyloxymethyl)phenoxyacetic Acid Phenacyl
Ester (4g)
1H NMR (200 MHz, CDCl3): d = 3.10 (d, J = 5.7 Hz, 2 H), 4.16–
4.25 (m, 1 H), 4.29–4.47 (m, 2 H), 4.64–4.74 (m, 1 H), 4.85 (s, 2 H),
5.05 (d, J = 11.8 Hz, 1 H), 5.14 (d, J = 11.9 Hz, 1 H), 5.27 (d, J =
8.3 Hz, 1 H), 5.47 (s, 2 H), 6.97 (d, J = 8.6 Hz, 2 H), 7.21–7.63 (m,
16 H), 7.77 (d, J = 7.2 Hz, 2 H), 7.90 (d, J = 8.6 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 191.44, 171.56, 168.62, 158.23,
155.75, 144.13, 143.97, 141.53, 135.80, 134.34, 134.10, 130.75,
129.60, 129.18, 128.81, 127.99, 127.95, 127.30, 125.36, 125.32,
120.21, 115.02, 67.18, 66.72, 65.28, 55.02, 47.38, 38.38.
1H NMR (200 MHz, CDCl3): d = 2.48–2.60 (m, 2 H), 4.18–4.29 (m,
3 H), 4.37–4.49 (m, 1 H), 4.61 (s, 2 H), 5.01 (s, 2 H), 6.83 (d, J =
8.6 Hz, 2 H), 6.94 (br, 2 H), 7.19–7.43 (m, 5 H), 7.68–7.73 (m, 3 H),
7.87 (d, J = 7.4 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 172.27, 171.45, 170.80, 158.26,
156.51, 144.44, 141.37, 130.17, 128.99, 128.32, 127.77, 125.89,
120.79, 114.95, 66.50, 65.14, 51.41, 47.27, 37.31.
Anal. Calcd for C41H35NO8: C, 73.53; H, 5.27; N, 2.09. Found: C,
73.25; H, 5.26; N, 2.37.
4-[Fmoc-(tert-butyl)serinyloxymethyl]phenoxyacetic Acid
Phenacyl Ester (4h)
MS (ESI): m/z = 517. 2 [M – H]–.
1H NMR (200 MHz, CDCl3): d = 1.11 (s, 9 H), 3.60 (dd, J = 8.9, 2.8
Hz, 1 H), 3.84 (dd, J = 8.9, 2.3 Hz, 1 H), 4.19–4.56 (m, 4 H), 4.78
(s, 2 H), 5.09 (d, J = 11.9 Hz, 1 H), 5.20 (d, J = 12.1 Hz, 1 H), 5.41
(s, 2 H), 5.79 (d, J = 8.9 Hz, 1 H), 6.96 (d, J = 8.5 Hz, 2 H), 7.26–
7.64 (m, 11 H), 7.75 (d, J = 7.4 Hz, 2 H), 7.86 (d, J = 7.2 Hz, 2 H).
4-[Fmoc-(NG-Pbf)-arginyloxymethyl]phenoxyacetic Acid (5b)
1H NMR (200 MHz, CDCl3): d = 1.37 (s, 6 H), 1.42–1.75 (m, 4 H),
2.01 (s, 3 H), 2.43 (s, 3 H), 2.50 (s, 3 H), 2.83 (s, 2 H), 4.02–4.17
(m, 1 H), 4.18–4.35 (m, 3 H), 4.48 (s, 2 H), 4.82 (br, 1 H), 5.85 (br,
1 H), 6.33 (br, 2 H), 6.76 (d, J = 6.0 Hz, 2 H), 7.14–7.36 (m, 6 H),
7.51 (d, J = 6.9 Hz, 2 H), 7.69 (d, J = 7.2 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 172.21, 170.10, 159.13, 158.10,
156.46, 156.39, 143.98, 143.80, 141.43, 138.72, 132.64, 130.79,
128.69, 127.94, 127.30, 125.32, 125.00, 120.18, 117.86, 114.79,
86.73, 67.31, 53.68, 47.16, 43.24, 28.73, 28.56, 19.47, 18.08, 12.69.
13C NMR (75 MHz, CDCl3): d = 191.47, 170.83, 168.62, 158.08,
156.39, 144.26, 144.03, 141.49, 134.33, 134.04, 130.37, 129.16,
129.05, 127.99, 127.35, 125.49, 125.43, 120.24, 114.96, 73.67,
67.40, 66.99, 66.74, 65.22, 62.33, 54.98, 47.35, 27.52.
Anal. Calcd for C39H39NO9: C, 70.36; H, 5.90; N, 2.10. Found: C,
70.27; H, 6.12; N, 2.35.
MS (ESI): m/z = 811.5 [M – H]–.
4-(Fmoc-tryptophyloxymethyl])phenoxyacetic Acid Phenacyl
Ester (4k)
4-[Fmoc-(g-tert-butyl)glutamyloxymethyl]phenoxyacetic Acid
(5c)
1H NMR (200 MHz, CDCl3): d = 3.19–3.29 (m, 2 H), 4.13–4.26 (m,
1 H), 4.30–4.45 (m, 2 H), 4.72–4.81 (m, 1 H), 4.85–4.97 (m, 3 H),
5.20 (d, J = 11.6 Hz, 1 H), 5.37 (d, J = 8.5 Hz, 1 H), 5.51 (s, 2 H),
6.94 (d, J = 8.6 Hz, 2 H), 7.02–7.65 (m, 16 H), 7.76 (d, J = 7.1 Hz,
2 H), 7.91 (d, J = 8.6 Hz, 2 H), 8.31 (br, 1 H).
1H NMR (200 MHz, CDCl3): d = 1.38 (s, 9 H), 1.67–2.10 (m, 2 H),
2.12–2.38 (m, 2 H), 4.03–4.19 (m, 1 H), 4.23–4.54 (m, 5 H), 4.97
(s, 2 H), 5.70 (d, J = 7.7 Hz, 1 H), 6.76 (d, J = 7.0 Hz, 2 H), 7.12–
7.38 (m, 6 H), 7.53 (d, J = 7.0 Hz, 2 H), 7.70 (d, J = 7.3 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 172.39, 172.19, 157.83, 156.34,
144.11, 143.84, 143.45, 141.42, 130.25, 127.90, 127.58, 127.48,
127.27, 127.11, 125.31, 120.17, 81.11, 67.31, 67.08, 53.77, 47.25,
31.62, 30.75, 28.25, 27.50, 25.27, 25.04.
13C NMR (75 MHz, CDCl3): d = 191.52, 180.12, 171.91, 169.30,
157.98, 155.97, 144.19, 144.01, 141.49, 136.21, 134.55, 133.92,
131.42, 129.28, 128.03, 127.89, 127.28, 125.44, 123.59, 121.97,
120.16, 119.69, 118.69, 114.63, 111.45, 109.01, 67.28, 66.90,
64.76, 55.06, 47.37, 28.29.
MS (ESI): m/z = 588.3 [M – H]–.
Anal. Calcd for C43H36N2O8: C, 72.87; H, 5.12; N, 3.95. Found: C,
73.05; H, 5.06; N, 3.78.
4-(Fmoc-glycyloxymethyl)phenoxyacetic Acid (5d)
1H NMR (200 MHz, DMSO-d6): d = 3.75 (d, J = 6.1 Hz, 2 H), 4.19–
4.29 (m, 3 H), 4.60 (br, 1 H), 4.62 (s, 2 H), 5.01 (s, 2 H), 6.84 (d,
J = 7.1 Hz, 2 H), 7.23–7.77 (m, 8 H), 7.85 (d, J = 7.3 Hz, 2 H).
4-[Fmoc-(tert-butyl)tyrosyloxymethyl]phenoxyacetic Acid
Phenacyl Ester (4l)
1H NMR (200 MHz, CDCl3): d = 1.31 (s, 9 H), 2.95–3.15 (m, 2 H),
4.09–4.20 (m, 1 H), 4.28–4.45 (m, 2 H), 4.62–4.72 (m, 1 H), 4.81
(s, 2 H) 5.04 (d, J = 11.8 Hz, 1 H), 5.12 (d, J = 12.0 Hz, 1 H), 5.42
(s, 2 H), 5.48 (d, J = 8.3 Hz, 1 H), 6.83–7.00 (m, 5 H), 7.25–7.61
(m, 12 H), 7.75 (d, J = 7.2 Hz, 2 H), 7.86 (d, J = 8.6 Hz, 2 H).
13C NMR (75 MHz, DMSO-d6): d = 170.76, 158.33, 157.16,
144.44, 141.38, 130.55, 128.92, 128.29, 127.73, 125.83, 120.77,
114.98, 66.42, 66.34, 65.09, 47.23, 42.86.
MS (ESI): m/z = 460.2 [M – H]–.
13C NMR (75 MHz, CDCl3): d = 191.48, 171.70, 168.64, 158.19,
155.83, 154.64, 144.14, 143.99, 141.51, 134.34, 134.03, 130.71,
130.08, 129.16, 128.63, 128.00, 127.34, 125.38, 124.40, 120.25,
115.00, 67.14, 66.77, 65.20, 55.18, 47.36, 37.71, 29.08.
4-[Fmoc-(e-Boc)-lysyloxymethyl]phenoxyacetic Acid (5e)
1H NMR (200 MHz, CDCl3): d = 1.39 (s, 9 H), 0.81–1.86 (m, 6 H),
2.84–3.11 (m, 2 H), 4.06–4.18 (m, 1 H), 4.32–4.76 (m, 5 H), 4.95
(d, J = 11.6 Hz, 1 H), 5.07 (d, J = 11.7 Hz, 1 H), 5.67 (d, J = 7.0
Synthesis 2006, No. 16, 2789–2793 © Thieme Stuttgart · New York

PAPER
Synthesis of 4-(Fmoc-aminoacyloxymethyl)phenoxyacetic Acids
2793
Hz, 1 H), 6.80 (d, J = 7.3 Hz, 2 H), 7.15–7.39 (m, 6 H), 7.55 (d, J =
7.3 Hz, 2 H), 7.72 (d, J = 7.4 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 172.60, 158.21, 156.52, 156.33,
144.11, 143.91, 141.47, 130.43, 127.93, 127.28, 125.33, 120.20,
115.27, 115.22, 79.69, 67.22, 66.92, 53.99, 47.29, 40.32, 32.17,
29.69, 28.61, 22.44.
MS (ESI): m/z = 622.4 [M – H]–.
4-[Fmoc-(g-tert-butyl)glutamyloxymethyl]phenoxyacetate Res-
in
Aminomethyl PS-DVB resin (100 mg, 1 mmol NH2/g) was placed
into a syringe for solid-phase peptide synthesis and left to swell for
30 min. Derivative 5c (240 mg, 0.4 mmol), HOBt (64 mg, 0.4
mmol), and PyBOP (0.4 mmol) were dissolved into DMF (1 mL)
and after addition of DIPEA (104 mg, 0.8 mmol) the solution was
transferred into the reaction syringe. The resin was gently agitated
for 4 h. Then the reaction mixture was washed with DMF (6 × 2
mL), CH2Cl2 (6 × 2 mL), Et2O (2 × 2 mL), and dried; yield: 152 mg
(97%); the Kaiser ninhydrine test was negative; Fmoc estimation
0.66 mmol/g.
MS (ESI): m/z = 631.4 [M – H]–.
4-(Fmoc-methionyloxymethyl)phenoxyacetic Acid (5f)
1H NMR (200 MHz, CDCl3): d = 2.00 (s, 3 H), 1.85–2.23 (m, 2 H),
2.33–2.52 (m, 2 H), 4.07–4.43 (m, 4 H), 4.51 (s, 2 H), 5.06 (s, 2 H),
5.53 (br, 1 H), 6.83 (d, J = 7.6 Hz, 2 H), 7.16–7.46 (m, 6 H), 7.56
(d, J = 7.5 Hz, 2 H), 7.74 (d, J = 7.3 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 174.18, 172.23, 157.90, 156.24,
144.03, 143.80, 141.45, 130.39, 128.73, 127.95, 127.28, 125.27,
120.21,115.17, 67.24, 53.37, 47.26, 31.90, 29.99, 15.62.
Acknowledgment
MS (ESI): m/z = 534.3 [M – H]–.
This work was supported by funds from ‘Special Account for Re-
search Grants’ of the National and Capodistrian University of
Athens.
4-(Fmoc-phenylalanyloxymethyl)phenoxyacetic Acid (5g)
1H NMR (200 MHz, CDCl3): d = 2.96–3.10 (m, 2 H), 4.18–4.55 (m,
6 H), 4.97 (s, 2 H), 5.41 (d, J = 7.8 Hz, 1 H), 6.80 (d, J = 7.5 Hz, 2
H), 6.96–7.39 (m, 11 H), 7.50 (d, J = 6.3 Hz, 2 H), 7.72 (d, J = 7.3
Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 171.73, 163.73, 158.13, 155.91,
144.05, 143.90, 141.48, 135.87, 130.60, 129.57, 128.79, 128.54,
127.95, 127.30, 125.37, 125.32, 120.21, 115.18, 67.20, 67.11,
55.05, 47.27, 47.13, 38.26, 37.12, 32.00.
References
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(2) (a) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide
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Int. J. Pept. Protein Res. 1981, 18, 459.
MS (ESI): m/z = 550.3 [M – H]–.
(5) Atherton, E.; Logan, C. J.; Shepard, R. C. J. Chem. Soc.,
4-[Fmoc-(tert-butyl)serinyloxymethyl]phenoxyacetic Acid (5h)
1H NMR (200 MHz, CDCl3): d = 1.10 (s, 9 H), 3.59 (dd, J = 9.0, 3.0
Hz, 1 H), 3.83 (dd, J = 9.1, 2.7 Hz, 1 H), 4.19–4.29 (m, 1 H), 4.34–
4.43 (m, 2 H), 4.50–4.55 (m, 1 H), 4.60 (s, 2 H), 5.08 (d, J = 12.0
Hz, 1 H), 5.18 (d, J = 12.1 Hz, 1 H), 5.82 (d, J = 9.0 Hz, 1 H), 6.87
(d, J = 8.6 Hz, 2 H), 7.21–7.49 (m, 6 H), 7.61 (d, J = 7.3 Hz, 2 H),
7.76 (d, J = 7.2 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 172.88, 170.94, 157.87, 156.58,
144.18, 143.94, 141.49, 130.42, 129.06, 127.96, 127.62, 127.34,
127.16, 125.45, 125.39, 125.14, 120.22, 114.84, 73.80, 67.55,
67.04, 65.06, 62.29, 54.94, 47.29, 27.48, 25.18, 25.04.
Perkin Trans. 1 1981, 538.
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Williams, B. J. J. Chem. Soc., Chem. Commun. 1981, 336.
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Perkin Trans. 1 1981, 538.
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Tetrahedron Lett. 1989, 30, 4341.
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(11) (a) Greene, T. W.; Wuts, P. G. M. Protective Groups in
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1999, 393–394. (b) Greene, T. W.; Wuts, P. G. M.
Protective Groups in Organic Synthesis, 3rd ed.; J. Wiley &
Sons: New York, 1999, 728.
MS (ESI): m/z = 546.2 [M – H]–.
4-(Fmoc-tryptophyloxymethyl]) (5k)
1H NMR (200 MHz, CDCl3): d = 2.94–3.27 (m, 2 H), 4.01–4.18 (m,
1 H), 4.195–4.558 (m, 3 H), 4.601–5.037 (m, 3 H), 5.53 (d, J = 7.7
Hz, 1 H), 6.45 (s, 1 H), 6.66 (d, J = 6.3 Hz, 2 H), 6.81–7.49 (m, 12
H), 7.71 (d, J = 7.4 Hz, 2 H), 8.44 (s, 1 H).
(12) Kokinaki, S.; Leondiadis, L.; Ferderigos, N. Org. Lett. 2005,
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3741.
13C NMR (75 MHz, CDCl3): d = 172.18, 157.78, 156.28, 144.07,
143.86, 141.45, 136.33, 130.71, 130.60, 127.95, 127.81, 127.31,
125.39, 123.52, 122.21, 120.20, 119.80, 118.66, 114.95, 111.66,
109.24, 67.39, 66.87, 55.11, 47.23.
MS (ESI): m/z = 589.5 [M – H]–.
4-[Fmoc-(O-tert-butyl)-tyrosyloxymethyl] (5l)
1H NMR (200 MHz, CDCl3): d = 1.27 (s, 9 H), 2.83–3.14 (m, 2 H),
3.98–4.15 (m, 1 H), 4.21–4.47 (m, 4 H), 4.53–4.69 (m, 1 H), 4.95
(s, 2 H), 5.48 (d, J = 6.7 Hz, 1 H), 6.70–6.90 (m, 5 H), 7.10–7.39
(m, 8 H), 7.51 (d, J = 6.5 Hz, 2 H), 7.72 (d, J = 7.2 Hz, 2 H).
13C NMR (75 MHz, CDCl3): d = 171.75, 158.01, 155.92, 154.53,
144.07, 143.92, 141.48, 130.71, 130.43, 130.00, 127.93, 127.62,
127.47, 127.37, 127.28, 127.15, 127.12, 125.33, 124.35, 120.19,
115.36, 78.74, 67.23, 67.03, 55.09, 47.30, 37.68, 29.02, 25.27,
24.98.
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Synthesis 2006, No. 16, 2789–2793 © Thieme Stuttgart · New York