Design of a coumarin-based triketone as a fluorescent protecting group for primary amines

Article abstract of DOI:10.1021/jo801060p

(Chemical Equation Presented) A series of 3-acetyl-4-hydroxycoumarin and its derivatives were prepared and evaluated for their potential to function as a fluorescent primary amine protection group. When primary amines or amino acids react with the protecting group 3-acetyl-4-methoxy-7-N,N-dimethylaminocoumarin, the resulting compounds emit blue fluorescence with a quantum yield of 0.25-0.50 in methylene chloride. These protected compounds display satisfactory acid/base stability, and the protecting group can be removed with 5% hydrazine hydrate in DMF within 5 min at ambient temperature.

Full text of DOI:10.1021/jo801060p

Design of a Coumarin-Based Triketone as a
Fluorescent Protecting Group for Primary
Amines
Pei-Yu Kuo and Ding-Yah Yang*
FIGURE 1. Structure of the designed potential fluorescent protecting
group for primary amines.
Department of Chemistry, Tunghai UniVersity, 181,
Taichung-Kang Road Sec. 3, Taichung, Taiwan 407, ROC
ing of the amine protection/deprotection progress by fluores-
cence spectroscopy. Recently, we have reported some unique
properties of coumarin-based triketones.3 These molecules have
the potential to serve as a fluorescent amine protecting group,
since coumarins have been widely used as fluorescent dyes.4
In this paper, we report our efforts in design, synthesis, and
evaluation of coumarin-based triketones as selective fluorescent
protecting groups for primary amines.
yang@thu.edu.tw
ReceiVed May 18, 2008
Figure 1 shows our original design of the fluorescent
protection group. The introduction of a methyl group at
4-hydroxycoumarin aims to make the resulting coumarin moiety
sensitive to amine.5 The oxygen atom at the 3-carbonyl group
serves as a hydrogen bond acceptor to stabilize the protected
amine. The incorporation of a N,N-dimethylamino group at the
7-position of coumarin enhances its fluorescence emission after
protection. The relative stability of the protected amines in acidic
and basic conditions and the ease of deprotection by hydrazine
hydrate were also explored.
Scheme 1 depicts the synthesis of the potential coumarin-
based primary amine protection compounds 2 and 3. It began
with the acetylation of the readily available 7-N,N-dimethy-
lamino-4-hydroxycoumarin (1),3 followed by the cyanide-
catalyzed isomerization6 of the enol ester using triethylamine
as a base in methylene chloride to give the triketone 2.
Methylation of 2 with diazomethane generated from Diazald
in diethyl ether under room temperature afforded the target
compound 3 in a 72% yield. Condensation of 2 and 3 with
different amino acid hydrochlorides (Val-OH, Leu-OH, Ile-OH,
Phe-OH, Tyr-OH and Cys-OH) in the presence of triethylamine
as a base in methanol gave the amine-protected derivatives 4a-d
and 5a-g, respectively. Generally, the condensation reaction
finished in 24 h at room temperature with the yield ranging
A series of 3-acetyl-4-hydroxycoumarin and its derivatives
were prepared and evaluated for their potential to function
as a fluorescent primary amine protection group. When
primary amines or amino acids react with the protecting
group 3-acetyl-4-methoxy-7-N,N-dimethylaminocoumarin,
the resulting compounds emit blue fluorescence with a
quantum yield of 0.25-0.50 in methylene chloride. These
protected compounds display satisfactory acid/base stability,
and the protecting group can be removed with 5% hydrazine
hydrate in DMF within 5 min at ambient temperature.
Protecting groups are essential tools for the construction of
complex organic molecules. The usefulness of a protecting group
relies on its ease of synthesis and introduction, its stability to
different reaction conditions, and its efficient cleavage under
mild conditions. Among the various reported primary amine
protection groups, the 1-(4,4-dimethyl-2,6-dioxocyclohexylide-
ne)ethyl (Dde, a triketone derivative)1 has gained considerable
attention. It is relatively stable in trifluoroacetic acid (TFA) and
piperidine and can easily be removed from the amine with 2%
hydrazine in DMF.
(2) (a) Bloomberg, G. B.; Askin, G.; Gargaro, A. R.; Tanner, M. J.
Tetrahedron Lett. 1993, 34, 4709–4712. (b) Brugghe, H. F.; Timmermans,
H. A. M.; Van Unen, L. M. A.; Hove, G. J. T.; Van de Werken, G.; Poolman,
J. T.; Hoogerhout, P. Int. J. Peptide Protein Res 1994, 43, 166–172. (c) Truffert,
J.-C.; Lorthioir, O.; Asseline, U.; Thuong, N. T.; Brack, A. Tetrahedron Lett.
1994, 35, 2353–2356. (d) Eichler, J.; Lucka, A. W.; Houghten, R. A. Peptide
Res. 1994, 7, 300–307. (e) Dumy, P.; Eggleston, I. M.; Corvigni, S.; Sila, U.;
Sun, X.; Mutter, M. Tetrahedron Lett. 1995, 36, 1255–1258. (f) Ahlborg, N.
J. Immunol. Methods. 1995, 179, 269–275. (g) Kellam, B.; Chan, W. C.; Chhabra,
S. R.; Bycroft, B. W. Tetrahedron Lett. 1997, 38, 5391–5394. (h) Kellam, B.;
Bycroft, B. W.; Chhabra, S. R. Tetrahedron Lett. 1997, 38, 4849–4852. (i)
Chhabra, S. R.; Khan, A. N.; Bycroft, B. W. Tetrahedron Lett. 2000, 41, 1095–
1098. (j) Chhabra, S. R.; Khan, A. N.; Bycroft, B. W. Tetrahedron Lett. 2000,
41, 1099–1102.
Thus, Dde has become a useful tool for the construction of
branched, cyclic, side-chain modified peptides and polyamines
by Fmoc/t-Bu solid-phase synthesis.2 However, the Dde protec-
tion group itself lacks emission, which hampers in situ monitor-
(3) Chen, Y. S.; Kuo, P. Y.; Shie, T. L.; Yang, D. Y. Tetrahedron 2006, 62,
9410–9416.
* To whom correspondence should be addressed. Tel: 886-4-2359-7613. Fax:
886-4-2359-0426.
(4) Zahradnik, M. The Production and Application of Fluorescent Brightening
Agents: Wiley & Sons: New York, 1992.
(1) (a) Bycroft, B. W.; Chan, W. C.; Chhabra, S. R.; Teesdale-Spittle, P. H.;
Hardy, P. M. J. Chem. Soc., Chem. Commun. 1993, 776–777. (b) Bycroft, B. W.;
Chan, W. C.; Chhabra, S. R.; Hone, N. D. J. Chem. Soc., Chem. Commun. 1993,
778–779.
(5) (a) Krishnamurthy, N.; Ravindranath, B. Tetrahedron Lett. 1982, 23,
2233–2236. (b) Borisov, E. V.; Verenich, A. I.; Govorova, A. A.; Lyakhov,
A. S.; Khlebnikova, T. S. Russ. Chem. Bull. 2000, 49, 1068–1070.
(6) Montes, I. F.; Burger, U. Tetrahedron Lett. 1996, 37, 1007–1010.
10.1021/jo801060p CCC: $40.75
Published on Web 07/23/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 6455–6458 6455

SCHEME 1
SCHEME 2
of triketone 2 clearly indicates that the C-3 carbonyl moiety of
2 is coplanar and conjugated with the coumarin ring system by
an intramolecular hydrogen bond of the C-4 hydroxyl hydrogen
to the oxygen atom of C-3 carbonyl group. For compound 3,
conversely, the dipolar repulsions between the C-3 carbonyl
group and the other two oxygen atoms on the coumarin ring
system caused deformation of 3 from planarity and resulted in
its high susceptibility to a nucleophilic 1,4-addition and
subsequent elimination reaction.5 The preference of 3 to form
stable derivatives with primary amines is attributed to, as with
Dde, the strong intramolecular hydrogen bond in the resultant
protected derivatives.
from 74 to 90%. However, when an amino acid ester hydro-
chloride (for example, Phe-OMe) was used, the reaction went
much faster and the product (4e or 5g) precipitated out from
the solution in 4-6 h. Furthermore, this protecting reaction can
be carried out smoothly without the presence of any base if the
free amine is used instead of the corresponding hydrochloride
salt. For instance, the condensation of 2 and 3 with benzyl amine
in MeOH gave 6 and 7 within 10 min in 89 and 92% yield,
respectively (Scheme 2).
While no emission was observed for the synthesized series
4a-e in methylene chloride, compounds 5a-g exhibited blue
fluorescence under the same conditions. Table 2 lists the
fluorescence parameters of 3 and 5a-g in methylene chloride
with the quantum yields between 0.25 and 0.50. Derivatives
4a-e and 5a-g were then subjected to 50% TFA in CH2Cl2
and 20% piperidine in DMF at room temperature to investigate
their relative stability under both acidic and basic conditions.
The results are listed in Table 1. Most of the compounds showed
good acid stability, but some decomposed in 20% piperidine in
DMF within 1 min. Among the compounds tested, compounds
5a-g all displayed excellent acid/base stability. No sign of
decomposition was detected after exposure to trifluoroacetic acid
and piperidine for more than 37 h. The reasons that 5a-g are
more stable than the corresponding 4a-e in both acidic and
basic conditions are currently unclear.
The unsymmetrical nature of the triketone 2 predisposed the
resulting protected compounds to undergo a geometrical isomer-
1
ism. The Z:E ratios of 4a-e were determined from their H
NMR spectra and are shown in Table 1. It has been documented7
that NH protons in Z-isomers of 4a-e have higher δ values
than E-isomers, due to the stronger intramolecular hydrogen
bonding of the ester oxygen atom (Z-form) than the carbonyl
oxygen atom (E-form). The X-ray crystal structure of 5g8
unambiguously confirmed that the amine nitrogen attached to
the expected C-4 position of coumarin moiety. The detection
of a significantly downfield signal of 4-hydroxyl hydrogen
Since it is essential to demonstrate that the protecting group
can be removed smoothly from amino acids before applying in
synthetic procedures or solid state peptide synthesis, compounds
5a-e were treated with 5% hydrazine hydrate in DMF at
ambient temperature. Amino acids/esters were released quan-
titatively within 5 min with concomitant formation of hydrazine
derivative 8 (Scheme 3). Subsequent heating of 8 in methanol
formed the cyclized pyrazole derivative 9, whose structure was
confirmed by single-crystal X-ray diffraction analysis8 (see the
Supporting Information). Finally, the absence of detectable
racemization at the R-carbon of the protected amino esters
during the protection and deprotection steps was established by
1
absorption peak between 16.7-17.8 ppm on H NMR spectra
(7) Bandyopadhyay, C.; Sur, K. R.; Patra, P.; Sen, A. Tetrahedron 2000,
56, 3583–3587.
(8) Crystallographic data (excluding structure factors) for compounds 5g and
9 have been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication nos. CCDC-665112 and -665113, respectively. These
data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif,
by emailing data_request@ccdc.cam.ac.uk, or by contacting the Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
+44 1223 336033.
6456 J. Org. Chem. Vol. 73, No. 16, 2008

TABLE 1. Relative Stability of 4a-e, 5a-g, 6, and 7 in Acidic and Basic Conditions
time (h)a
20% piperidine/ DMF
entry
compd
R1
R2
Z:E
50%TFA/CH2Cl2
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
5f
CH(CH3)2
CH2CH(CH3)2
CH(CH3)CH2CH3
CH2Ph
H
H
H
H
Me
H
H
H
H
1:37
1:23
1:4
1:16
1:9
17
19
20
26
14
>37
>37
>37
>37
>37
>37
35
1 min
1 min
1 min
1 min
1 min
>37
>37
>37
>37
>37
>37
30
CH2Ph
CH(CH3)2
CH2CH(CH3)2
CH(CH3)CH2CH3
CH2Ph
CH2Ph-p-OH
CH2SH
9
10
11
12
13
14
H
H
Me
5g
6
7
CH2Ph
1:36
14
30
3 min
>37
a Time of new spots detected by TLC analysis.
TABLE 2. Fluorescence Parameters for 3 and 5a-g in CH2Cl2
EtOAc/hexanes) to give a yellow solid in a 72% yield: mp 134-135
°C; 1H NMR (CDCl3, 300 MHz) δ 7.66 (d, J ) 9.0 Hz, 1H), 6.60
(dd, J ) 9.0, 2.4 Hz, 1H), 6.42 (d, J ) 2.4 Hz, 1H), 3.95 (s, 3H),
3.07 (s, 6H), 2.67 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 199.5,
166.8, 161.9, 154.6, 153.5, 125.1, 108.8, 105.1, 104.5, 96.4, 61.6,
39.6, 31.9; IR ν (KBr) 1711, 1578, 1348, 821, 764 cm-1; HRMS
(EI) m/z calcd for C14H15NO4 261.1001, found 261.1007 (M+).
General Procedure for Preparation of Compounds 4a-d. To a
solution of 2 (100 mg, 0.40 mmol) in methanol (5 mL) were added
amino acid (0.40 mmol) and triethylamine (41 mg, 0.40 mmol) at
room temperature for 1 day. After completion of the reaction, the
solvent was concentrated in vacuo. This mixture was poured into
water, 2 N NaOH was added to the aqueous solution until pH ∼12,
and the aqueous solution was washed with methylene chloride. A
HCl solution (6 N) was added to the aqueous solution until pH
∼2, and the aqueous solution was extracted with methylene
chloride. The organic layer was dried over MgSO4 and concentrated
in vacuo to give the pure product.
compd λex (nm) λem (nm) Stokes shift (cm-1
)
quantum yield (%)
3
373
371
372
371
374
368
372
373
419
446
446
445
450
415
424
449
2943
4533
4460
4482
4516
3078
3297
4538
0.18
0.40
0.27
0.31
0.26
0.50
0.25
0.43
5a
5b
5c
5d
5e
5f
5g
SCHEME 3
2- (1-[7-N,N-Dimethylamino-2,4-dioxochroman-(3E)-ylidene-
]ethylamino)-3-methyl butyric acid (4a): yellow solid; yield 76%;
mp 157-158 °C; [R]28D ) 32.26 (c 0.006, DMSO). Only the data
of the major isomer (E form) is given: 1H NMR (CDCl3, 300 MHz)
δ 14.63 (s, 1H), 7.92 (d, J ) 9.0 Hz, 1H), 7.06 (bs, 1H), 6.57 (dd,
J ) 9.0, 2.4 Hz, 1H), 6.30 (d, J ) 2.4 Hz, 1H), 4.40 (dd, J ) 8.1,
4.8 Hz, 1H), 3.04 (s, 6H), 2.67 (s, 3H), 2.50-2.40 (m, 1H), 1.16
(d, J ) 6.9 Hz, 3H), 1.11 (d, J ) 7.2 Hz, 3H); 13C NMR (DMSO-
d6, 75 MHz) δ 176.1, 171.4, 155.2, 154.6, 127.1, 126.5, 110.0,
108.8, 96.4, 95.6, 61.8, 40.0, 31.1, 29.6, 19.0, 18.8, 17.7; IR ν (KBr)
1754, 1669, 1602, 1476, 1193, 777, 698 cm-1; HRMS (EI) m/z
calcd for C18H22N2O5 346.1529, found 346.1525 (M+).
General Procedure for Preparation of Compounds 5a-f. To a
solution of 3 (100 mg, 0.38 mmol) in methanol (5 mL) were added
amino acid (0.38 mmol) and triethylamine (39 mg, 0.38 mmol) at
room temperature for 1 day. This mixture was poured into water,
2 N NaOH was added to the aqueous solution until pH ∼12, and
the aqueous solution was washed with with methylene chloride.
HCl (6 N) was added o the aqueous solution until pH ∼2, and the
aqueous solution was extracted with methylene chloride. The
organic layer was dried over MgSO4 and concentrated in vacuo to
give the pure product.
specific rotation comparisons of the amino esters released from
hydrazine deprotection with the authentic compounds.
In summary, we have developed a fluorescent coumarin-based
primary amine protection group from readily available 7-N,N-
dimethylamino-4-hydroxycoumarin. The protected amines emit
strong blue fluorescence in methylene chloride and are resistant
to a range of acidic and basic conditions. The amines can be
released quantitatively with 5% hydrazine hydrate in DMF
within 5 min.
2-(3-Acetyl-7-N,N-dimethylamino-2-oxo-2H-chromen-4-ylamino)-
3-methylbutyric acid (5a): yellow solid; yield 79%; mp 178-180
1
°C; [R]29 ) -76.92 (c 0.004, DMSO); H NMR (CDCl3, 300
D
Experimental Section
MHz) δ 12.83 (d, J ) 8.7 Hz, 1H), 7.58 (d, J ) 9.6 Hz, 1H),
6.50 (dd, J ) 9.6, 2.4 Hz, 1H), 6.32 (d, J ) 2.4 Hz, 1H), 5.22
(bs, 1H), 4.59 (dd, J ) 8.1, 5.4 Hz, 1H), 3.03 (s, 6H), 2.67 (s,
3H), 2.46-2.38 (m, 1H), 1.14 (d, J ) 6.9 Hz, 1H), 1.04 (d, J )
6.9 Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ 201.4, 173.6, 162.7,
161.8, 156.2, 153.8, 128.0, 108.4, 101.4, 97.9, 95.8, 65.7, 39.8,
Preparation of 3-Acetyl-7-N,N-dimethylamino-4-methoxychromen-
2-one (3). To a stirred mixture of 2 (1 g, 4.04 mmol) in EtOAc (10
mL) was added an ethereal solution of diazomethane at 0 °C for
0.5 h. After completion of the reaction, the solution was evaporated
and the residue was purified by column chromatography (1:6
J. Org. Chem. Vol. 73, No. 16, 2008 6457

33.0, 32.9, 19.1, 17.8; IR ν (KBr) 1672, 1595, 1471, 1391, 823
cm-1; HRMS (EI) m/z calcd for C18H22N2O5 346.1529, found
346.1530 (M+).
Supporting Information Available: Synthesis of compounds
3, 4a-e, 5a-g, 6, 7, and 9, experimental details, and additional
spectra. X-ray structure details for compounds 5g and 9 (CIF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. The financial assistance provided by
National Science Council of Republic of China is thankfully
acknowledged.
JO801060P
6458 J. Org. Chem. Vol. 73, No. 16, 2008