Facile synthesis of N-(9-fluorenylmethyloxycarbonyl)-3-amino-3-(4,5- dimethoxy-2-nitrophenyl)propionic acid as a photocleavable linker for solid-phase peptide synthesis

Article abstract of DOI:10.1055/s-0032-1318314

A photocleavable linker, N-(9-fluorenylmethyloxycarbonyl)-3-amino-3-(4,5- dimethoxy-2-nitrophenyl)propionic acid was synthesized from veratraldehyde, with simple reaction and separation steps. This linker was stable under the normal solid-phase peptide sy

Full text of DOI:10.1055/s-0032-1318314

LETTER
▌733
letter
Facile Synthesis of N-(9-Fluorenylmethyloxycarbonyl)-3-amino-3-(4,5-dime-
thoxy-2-nitrophenyl)propionic Acid as a Photocleavable Linker for Solid-
Phase Peptide Synthesis
Photocleavable Linker for Solid-Phase Peptide Synthesis
Jaehi Kim,* San Kyeong, Dong-Sik Shin, Sewon Yeo, Joonhyuk Yim, Yoon-Sik Lee
School of Chemical and Biological Engineering, Seoul National University, Gwanak-Gu, Seoul 151-744, Korea
Fax +82(2)8769625; E-mail: yslee@snu.ac.kr
Received: 26.12.2012; Accepted after revision: 05.02.2013
methoxy-5-nitrophenoxy}butanoic acid (Fmoc-photola-
Abstract: A photocleavable linker, N-(9-fluorenylmethyloxycar-
bile linker) was synthesized from acetovanillone via con-
bonyl)-3-amino-3-(4,5-dimethoxy-2-nitrophenyl)propionic
was synthesized from veratraldehyde, with simple reaction and sep-
aration steps. This linker was stable under the normal solid-phase
acid
densation reaction with hydroxylamine (Figure 1, a),4a
and N-(9-fluorenylmethyloxycarbonyl)-3-amino-3-(2-ni-
peptide synthesis conditions and rearranged to a labile form when trophenyl)propionic acid (Fmoc-ANP linker) was synthe-
irradiated with UV light (λ = 365 nm). A model peptide sequence
(YGGFL) was synthesized and released from solid supports with
high efficiency by UV irradiation.
sized from 2-nitrobenzaldehyde via Perkin-type
condensation reaction (Figure 1, b).4b These linkers can
release synthesized peptides from solid phase, by radical
rearrangement of the o-nitrobenzyl group, when they are
exposed to UV light (λ = 365 nm). Since the peptides that
Key words: photochemistry, photocleavable linker, solid-phase
synthesis, amino acid derivative, peptides
are released from the polymer supports are of stable amide
form, the linkers are quite suitable for OBOC peptide li-
brary synthesis. However, these linkers have not been
Since its invention by R. B. Merrifield in 1963,1 the solid-
phase peptide synthesis (SPPS) method has been widely
used. By virtue of the SPPS method, the construction of
one-bead one-compound (OBOC) combinatorial peptide
libraries, for screening of various ligands or enzymatic
substrates, has been possible.2 For successful SPPS and
bioassay, a linker that connects peptides to polymer matri-
ces should be orthogonally stable to the reaction condi-
tions, for building up the peptide sequences on the
polymer supports. Various linkers that are labile to acid,
base, or chemicals have been developed to release pep-
tides from the polymer supports.3 However, a bioassay
system by the released peptides could be damaged under
these cleavage conditions. Since photolabile linkers are
stable with chemicals, and produce a minimum amount of
byproducts, they are more attractive than other linkers in
the SPPS and bioassay.
widely used, because their synthetic routes are trouble-
some. In the synthesis of the Fmoc-photolabile linker, the
hydrogenation of ketoxime to amine was tedious and
time-consuming.4a The synthetic route of the Fmoc-ANP
linker was quite simple, compared to the Fmoc-photola-
bile linker. However, the Perkin-type condensation reac-
tion of 2-nitrobenzaldehyde with malonic acid gave low
yield (27%), because of the electron-withdrawing effect
of the o-nitro group.4b Therefore, in this paper, we report
on the novel synthesis method of a photocleavable linker
that has high photocleavable efficiency and its application
in SPPS.
Starting from veratraldehyde, our synthetic pathway for a
photocleavable linker consists of serial six-step reactions
(Scheme 1). Compared to 2-nitrobenzaldehyde, the alde-
hyde group of veratraldehyde is less hindered, and two
methoxy groups are expected to boost the Perkin-type
condensation reaction by their electron-donation effect.5
Taking these into consideration, 3-amino-3-(3,4-dime-
thoxyphenyl)propionic acid was synthesized under mild
conditions, in higher yield than from 3-amino-3-(2-nitro-
phenyl)propionic acid. Prior to performing nitration, the
carboxylic acid and the amino groups were protected with
conventional methods (95% and 85%, respectively). After
nitration, we found that the nitro group was introduced
only on the 2-position, in the same way as veratraldehyde
was nitrated. The nitration was selectively performed be-
cause of the electron-donating effect of m-methoxy
groups and steric hindrance. After deprotection of the tri-
fluoroacetyl group and hydrolysis of methyl ester by sa-
ponification, 9-fluorenylmethyloxycarbonyl (Fmoc)
group was introduced to the free amino group under the
Schotten–Baumann conditions. After following these
O
(b)
O
(a)
O
NH
O
NH
OH
O2N
O2N
O
OMe
O
O
OH
Figure 1 Photoreactive linkers
Holmes et al. and Geysen et al. reported the linkers that
can be rearranged to the labile form by UV irradiation.4 4-
{4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2-
SYNLETT 2013, 24, 0733–0736
Advanced online publication: 04.03.2013
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
DOI: 10.1055/s-0032-1318314; Art ID: ST-2012-U1101-L
© Georg Thieme Verlag Stuttgart · New York
steps,
N-(9-fluorenylmethyloxycarbonyl)-3-amino-3-

734
J. Kim et al.
LETTER
O
O
O
O
O
H3N
OMe
Cl
H2N
OH
O
H
HO
OH
Cl
MeOH
F3C
O
CF3
O
O
pyridine
NH4OAc
EtOH
OMe
OMe
OMe
OMe
OMe
OMe
1
2 (60%)
3 (95%)
O
O
O
F3C
NH
OMe
F3C
O2N
NH
OMe
O
O2N
NH
OH
HNO3
i) NaOH
O
O
O
O
ii)
O
OMe
OMe
OMe
O
O
N
OMe
4 (85%)
OMe
5 (85%)
OMe
6 (81%)
O
THF, pH 9
Scheme 1 Synthetic pathway of the Fmoc-PCA linker
(4,5-dimethoxy-2-nitrophenyl) propionic acid (Fmoc- As a performance test of the PCA linker in the solid-phase
PCA linker, named after photocleavable amino acid) was peptide synthesis, Leu-enkephaline (YGGFL) was syn-
synthesized in overall 33% yield. It is notable that the thesized on the HiCore resin with Fmoc/t-Bu chemistry
overall yield through our synthetic strategy is higher than (see experimental procedures for details). After building
that from the literature,4 and no chromatographic separa- up the peptide on the resin, Leu-enkephaline was released
tion is required during all the reaction steps.
as an amide form from the resin by UV irradiation. The
purity of the released peptide was confirmed by HPLC
(Figure 2) and MALDI-TOF MS analyses {[M + H]+:
555.62, [M + Na]+: 577.56, Figure 3}. Overall, this result
proves that the PCA linker is stable under acidic and basic
conditions in Fmoc/t-Bu chemistry, including piperidine
or trifluoroacetic acid treatment.
As a preliminary test, the photocleavage yield of the PCA
linker was estimated by measuring the amount of released
product, Fmoc-Phe-NH2. For this, PCA linker and Fmoc-
Phe-OH were serially introduced to the core-shell-type
HiCore resin, which was proved to be ideal for solid-
phase photochemical reaction.6 Then, Fmoc-Phe-NH2
was released by UV irradiation (λ = 365 nm) on the resin. In conclusion, we successfully synthesized the Fmoc-
The photocleavage yield of Fmoc-Phe-NH2 increased PCA linker from veratraldehyde in six steps, without any
over time, and reached ca. 50% in 20 minutes. This result column chromatography. The Fmoc-PCA linker was sta-
is comparable to those with a commercially available ble under typical solid-phase peptide synthesis conditions,
Fmoc-photolabile linker.6,7 Similar or better efficiency of and ca. 50% of the synthesized peptide was released by 20
photocleavage reaction confirms that the PCA linker is minutes of UV irradiation. Based on these results, we en-
applicable to on-bead assays.2b,c
vision that the Fmoc-PCA linker can be used in solid-
phase peptide synthesis for on-bead assay. Moreover, our
Figure 2 (a) HPLC analysis data of purified H-YGGFL-NH2 and (b) photocleaved H-YGGFL-NH2 by using the Fmoc-PCA linker
Synlett 2013, 24, 733–736
© Georg Thieme Verlag Stuttgart · New York

LETTER
Photocleavable Linker for Solid-Phase Peptide Synthesis
735
Figure 3 MALDI-TOF MS analysis of H-YGGFL-NH2 synthesized from HiCore resin by using Fmoc-PCA linker
two layers were partitioned. The aqueous layer was thrown, and the
remaining organic layer was washed with aq 0.5 N NaHCO3 solu-
tion, collected, and dried by using MgSO4. The dried organic layer
was filtered and evaporated under reduced pressure. As a result,
methyl 3-(3,4-dimethoxyphenyl)-3-(2,2,2-trifluoroacetamido)pro-
panoate (335.28 g/mol) was obtained as yellow crystals; yield:
1.921 g (85%); mp 85–86 °C. 1H NMR (400 MHz, DMSO-d6): δ =
7.01–6.86 (m, 3 H), 5.24 (qr, J = 7.7 Hz, 1 H), 3.76 (s, 3 H), 3.74 (s,
3 H), 3.60 (s, 3 H), 2.99 (dd, J = 9.3, 9.6 Hz, 1 H), 2.87 (dd, J = 6.1,
5.4 Hz, 1 H). 13C NMR (100 MHz, DMSO-d6): δ = 170.45, 148.84,
148.41, 132.91, 118.61, 111.73, 110.50, 55.51, 51.50, 50.06 ppm.
ESI-HRMS (+): m/z calcd: 335.28; found: 336.1055.
synthetic strategy of the PCA linker is more convenient
and efficient than the previously reported Fmoc-photola-
bile linkers. Therefore, we expect that the PCA linker can
be widely used in SPPS for on-bead assay.
3-Amino-3-(3,4-dimethoxyphenyl)propionic Acid (2)
Well-crushed veratraldehyde (2.5 g, 15.05 mmol), NH4OAc (4.64
g, 60.18 mmol), and malonic acid (6.26 g, 60.18 mmol) were dis-
solved in EtOH (150 mL). The reaction solution was refluxed dur-
ing 18 h. After reaction, the white solids could be separated by
filtration. This white compound was gently washed with cold EtOH
about three or more times till the filtrate become colorless, and dried
in vacuo. 3-Amino-3-(3,4-dimethoxyphenyl)propionic acid (225.24
g/mol) was obtained as a white powder, after drying; yield: 2.046 g
Synthesis Methyl 3-(4,5-Dimethoxy-2-nitrophenyl)-3-(2,2,2-tri-
fluoroacetamido)propanoate (5)
Nitration of methyl 3-(3,4-dimethoxyphenyl)-3-(2,2,2-trifluoro-
acetamido)propanoate was performed as follows. About 20 mL of
70% HNO3 was slowly added to methyl 3-(3,4-dimethoxyphenyl)-
3-(2,2,2-trifluoroacetamido)propanoate (1.921 g, 5.73 mmol) in an
ice bath. The solution was stirred in a dark place for 2 h, and the re-
sulting orange-colored reaction solution was quenched by pouring
cold H2O (ca. 500 mL, about ten or more times the HNO3 volume)
into the reaction solution. A light yellow solid was obtained from
the solution by chilling at 4 °C overnight and filtration. The collect-
ed solid was washed with H2O three times. Methyl 3-(4,5-dime-
thoxy-2-nitrophenyl)-3-(2,2,2-trifluoroacetamido)propanoate
(380.27 g/mol) was obtained as ivory powder after drying in vacuo;
yield: 1.844 g (85%); mp 167–169 °C. 1H NMR (400 MHz, DMSO-
d6): δ = 7.57 (s, 1 H), 7.28 (s, 1 H), 5.24 (qr, J = 7.7 Hz, 1 H), 3.76
(s, 3 H), 3.74 (s, 3 H), 3.60 (s, 3 H), 2.99 (dd, J = 9.3, 9.6 Hz, 1 H),
2.87 (dd, J = 6.1, 5.4 Hz, 1 H). 13C NMR (100 MHz, DMSO-d6): δ
= 169.99, 153.28, 147.89, 140.15, 129.90, 109.98, 107.63, 56.27,
56.09, 39.93, 39.51 ppm. ESI-HRMS (+): m/z calcd: 380.27; found:
381.0859.
1
(60%); mp 213–216 °C. H NMR (400 MHz, D2O/K2CO3): δ =
7.04–6.99 (m, 3 H), 4.55 (t, J = 7.1 Hz, 1 H), 3.83 (s, 3 H), 3.81 (s,
3 H), 2.86 (dd, J = 7.9, 9.1 Hz, 1 H), 2.75 (dd, J = 6.8, 9.9 Hz, 1 H).
13C NMR (100 MHz, D2O/NaOH): δ = 183.00, 150.78, 149.84,
140.76, 121.70, 114.64, 112.88, 58.58, 58.52, 55.47, 49.80 ppm.
ESI-HRMS (–): m/z calcd: 225.24; found: 224.0928.
1-(3,4-Dimethoxyphenyl)-3-methoxy-3-oxopropan-1-aminium
Chloride (3)
3-Amino-3-(3,4-dimethoxyphenyl)propionic acid (1.6 g, 7.10
mmol) was suspended in anhyd MeOH (150 mL), and acetyl chlo-
ride (1.5 mL, 21.37 mmol) was added to the suspension. The reac-
tion mixture was stirred and refluxed for
3 h. 1-(3,4-
Dimethoxyphenyl)-3-methoxy-3-oxopropan-1-aminium chloride
(275.73 g/mol) was obtained as white salt form, after evaporation of
MeOH and drying in vacuo; yield: 1.860 g (95%); mp 183–185 °C.
1H NMR (400 MHz, CD3OD): δ = 7.52–6.86 (m, 3 H), 4.66 (t, J =
7.1 Hz, 1 H), 3.84 (s, 3 H), 3.80 (s, 3 H), 3.65 (s, 3 H), 3.17 (dd, J =
7.9, 9.1 Hz, 1 H), 3.01 (dd, J = 6.8, 9.9 Hz, 1 H). 13C NMR (100
MHz, CD3OD): δ = 171.86, 151.38, 150.97, 129.88, 121.37, 113.17,
112.22, 56.87, 56.64, 53.12, 52.88, 39.42 ppm. ESI-HRMS (+): m/z
calcd: 275.73; found: 240.1229.
Synthesis of Fmoc-3-amino-3-(4,5-dimethoxy-2-nitrophe-
nyl)propionic Acid (6)
Methyl
3-(4,5-dimethoxy-2-nitrophenyl)-3-(2,2,2-trifluoroacet-
amido)propanoate (1.844 g, 4.85 mmol) was dissolved into 0.1 M
NaOH solution (150 mL). The solution was stirred and refluxed for
5 h and cooled to r.t. After that, the pH of the solution was adjusted
to about 7 by adding 6 M HCl, until the color of solution changed to
yellow. The resulting solution was stirred in an ice, followed by the
addition of DIPEA (1.2 mL). After that, a solution of 1.083 g of
Fmoc-OSu in THF (50 mL) was added slowly to the reaction mix-
ture and stirred for 1 h in an ice bath, and overnight at r.t. After THF
was evaporated from the reaction mixture by reduced pressure, the
remaining aqueous solution was slowly acidified to pH 2–3 with 6
Synthesis of Methyl 3-(3,4-Dimethoxyphenyl)-3-(2,2,2-trifluo-
roacetamido)propanoate (4)
Methyl 1-(3,4-dimethoxyphenyl)-3-methoxy-3-oxopropan-1-amin-
ium chloride (1.860 g, 6.75 mmol) was dispersed into pyridine (80
mL). The reaction solution was cooled to 0 °C with an ice bath, and
TFAA (1.2 mL, 8.50 mmol) was added to the vigorously stirred so-
lution. After 1 h, the reaction solution was poured into a separation
funnel with EtOAc. This solution was acidified with 6 M HCl until
the pH value of the solution reached about 2–3, and the resulting
© Georg Thieme Verlag Stuttgart · New York
Synlett 2013, 24, 733–736

736
J. Kim et al.
LETTER
M HCl, and the precipitated product was separated by centrifuga-
tion. Fmoc-3-amino-3-(4,5-dimethoxy-2-nitrophenyl)propionic
h. The resins were washed with TFA, CH2Cl2, and MeOH, about
three times. About 10 mg of the resin was sampled in 2 mL micro-
tube, and MeOH (1 mL) was added. UV light (λ = 365 nm, 50 mW)
was irradiated to the microtube for 20 min. The peptide MeOH so-
lution was sampled and analyzed by HPLC and MALDI-TOF.
acid (Fmoc-PCA linker) was obtained as a brown powder after
freeze drying; yield: 1.942 g (81%); mp 210–215 °C. 1H NMR (400
MHz, DMSO-d6): δ = 12.38 (s, 1 H), 8.12 (d, J = 8.0 Hz, 1 H), 7.88–
7.24 (m, 10 H), 5.57 (qr, J = 7.4 Hz, 1 H), 4.27 (d, J = 5.8 Hz, 2 H),
4.18 (t, J = 6.7 Hz, 1 H), 3.87 (s, 3 H), 3.85 (s, 3 H), 2.70 (d, J = 6.6
Hz, 2 H). 13C NMR (100 MHz, DMSO-d6): δ = 171.37, 155.28,
153.17, 147.38, 143.93, 143.54, 140.73, 140.69, 139.91, 133.08,
127.61, 126.98, 126.90, 125.04, 120.14, 120.10, 109.90, 107.31,
65.41, 56.20, 56.04, 47.56, 46.62 ppm. ESI-HRMS (+): m/z calcd:
492.48; found: 493.1613.
Acknowledgment
This research was supported by the Pioneer Research Center Pro-
gram, through the National Research Foundation of Korea funded
by the Ministry of Education, Science and Technology (Grant Num-
ber 2012-0000460).
Preparation of Fmoc-Phe-NH2 for the Calculation of the Photo-
cleavage Yield
References
HiCore resin (0.3 mmol/g, 100 mg) was swollen in DMF. Fmoc-
PCA linker was introduced on the resin with the BOP coupling
method for 2 h, and the Fmoc group was removed by using 20%
(v/v) piperidine in DMF for 1 h. Fmoc-Phe-OH was coupled onto
the resin with BOP coupling in the same manner. After perfect dry-
ing, about 10 mg of resin was transferred into 2 mL microtubes, and
DMF (1 mL) was added. The resin was irradiated by UV light (λ =
365 nm, 50 mW) for a certain period of time (1–20 min). Samples
were analyzed by HPLC, and the amount of Fmoc-Phe-NH2 was
calculated with standard curve.
(1) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
(2) (a) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.;
Kazmierski, W. M.; Knapp, R. J. Nature (London) 1991,
354, 82. (b) Kim, Y. G.; Shin, D. S.; Kim, E. M.; Park, H. Y.;
Lee, C. S.; Kim, J. H.; Lee, B. S.; Lee, Y. S.; Kim, B. G.
Angew. Chem. Int. Ed. 2007, 119, 5408. (c) Shin, D. S.; Kim,
Y. G.; Kim, E. M.; Kim, M.; Park, H. Y.; Kim, J. H.; Lee, B.
S.; Kim, B. G.; Lee, Y. S. J. Comb. Chem. 2008, 10, 20.
(3) (a) Rink, H. Tetrahedron Lett. 1987, 28, 3787. (b) Atherton,
E.; Logan, C. J.; Sheppard, R. C. J. Chem. Soc., Perkin
Trans. 1 1981, 538. (c) Kenner, G. W.; McDermott, J. R.;
Sheppard, R. C. J. Chem. Soc., Chem. Commun. 1971, 636.
(4) (a) Holmes, C. P.; Jones, D. G. J. Org. Chem. 1995, 60,
2318. (b) Brown, B. B.; Wagner, D. S.; Geysen, H. M. Mol.
Diversity 1995, 1, 4.
Preparation and Analysis of H-YGGFL-NH2
HiCore resin (0.3 mmol/g, 100 mg) was swollen in DMF. Fmoc-
PCA linker was introduced on the resin with the BOP coupling
method for 2 h, and the Fmoc group was removed by using 20%
(v/v) piperidine in DMF for 1 h. The reaction mixture of the result-
ing resin and 4 equiv of Fmoc-amino acids, BOP, HOBt, and 8
equiv of DIPEA in DMF was shaken for 2 h. When each coupling
reaction was ended, the resins were washed with DMF, CH2Cl2, and
MeOH about three times. The completion of coupling was con-
firmed by Kasier’s ninhydrin test. After whole peptide sequences
were coupled on the resin, the side-chain protecting groups were re-
moved by using the cleavage cocktail (TFA–3,6-dioxa-1,8-octane-
dithiol–H2O–triisopropylsilane = 94:2.5:2.5:1, volume ratio) for 1
(5) Tan, C.; Weaver, D. Tetrahedron 2002, 58, 7449.
(6) Kim, H.; Cho, J. K.; Chung, W. J.; Lee, Y. S. Org. Lett.
2004, 6, 3273.
(7) Cho, J. K.; Park, B. D.; Lee, Y. S. Tetrahedron Lett. 2000,
41, 7481.
Synlett 2013, 24, 733–736
© Georg Thieme Verlag Stuttgart · New York

Copyright of Synlett is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or
emailed to multiple sites or posted to a listserv without the copyright holder's express written permission.
However, users may print, download, or email articles for individual use.