Synthesis of Nα-tetrachlorophthaloyl (TCP)-protected amino acids under microwave irradiation (MWI)

Article abstract of DOI:10.1055/s-2001-15215

A range of N^α-tetrachlorophthaloyl protected amino acids have been synthesized by an easy and efficient condensation procedure of the corresponding amino acid and tetrachlorophthaloyl anhydride under irradiation in an unmodified commercial microwave oven.

Full text of DOI:10.1055/s-2001-15215

PAPER
1313
Synthesis of N -Tetrachlorophthaloyl (TCP)-Protected Amino Acids under
Microwave Irradiation (MWI)
Synthesis of
N
-
s
Tetrachlo
t
r
ophtha
h
loyl (TCP)-P
e
rotecte
d
A
r
mino Acids Cros, Marta Planas, Eduard Bardají*
Department of Chemistry, University of Girona, 17071 Girona, Spain
Fax +34(972)418150; E-mail: dqebr@xamba.udg.es
Received 10 January 2001; revised 5 April 2001
from tetrachlorophthalic anhydride and glycine using mi-
crowave irradiation in high yields.10
Abstract: A range of N -tetrachlorophthaloyl protected amino ac-
ids have been synthesized by an easy and efficient condensation
procedure of the corresponding amino acid and tetrachlorophthaloyl
anhydride under irradiation in an unmodified commercial micro-
wave oven.
We report in this paper an easy and efficient microwave-
induced synthesis of N-TCP protected amino acids suit-
able for SPPS by condensing the corresponding amino
acid with tetrachlorophthaloyl anhydride. This protocol
includes mixing of the amino acid with tetrachlorophtha-
loyl anhydride in DMF (Scheme 1). After 4–8 minutes of
irradiation (285 W) the reactions are complete. A range of
L- and D-amino acids, including tri-functional with side-
chain protection, have been protected and were obtained
with high yields (80–99%) after straightforward workup
(Table 1). Most N-TCP protected amino acids gave satis-
factory elemental analyses without further purification
(Table 2). In some syntheses the protected amino acid was
purified by simple organic extraction.
Key words: amino acids, microwave irradiation, protecting groups,
phthalimide, tetrachlorophthaloyl
The use of the phthaloyl moiety as primary amine protect-
ing group is extensively documented in the chemical liter-
ature.1 However, the use of this protective group has not
been practical in some sensitive substrates due to the quite
harsh conditions required for its removal. Addition of
electron-withdrawing groups to the phthalimide’s aromat-
ic ring greatly enhances deprotection under generally
milder conditions than those previously encountered with
the phthalimido group. In connection with their work in
amino sugars, Fraser-Reid's and Schmidt's groups have
described the use of the tetrachlorophthalimido function
as a masked amino group. Nevertheless, the common ap-
plication of the tetrachlorophthalimido protecting group
has been limited to sugar chemistry.2–4 To date, no efforts
have been made towards the synthesis of a wide range of
N-TCP protected amino acids suitable for SPPS (solid-
phase synthesis of peptides).5
Cl
MWI
(285 W, 4–8 min)
DMF
O
Cl
Cl
TCP-aa(P)-OH
H-aa(P)-OH
+
O
62–99%
1–16 (L-D)
O
Cl
aa = amino acid
P = side-chain protecting group
TCP = tetrachlorophthaloyl
Scheme 1
In general, N-substituted phthalimides are formed by con-
densation of phthalic anhydride and an amine by reflux2–4
(Dean–Stark water separator6) or by direct fusion proce-
dures.7 The phthaloyl group has also been introduced by
reacting the amine with 2-(ethoxycarbonyl)benzoic acid
activated by PyBOP (benzotriazol-1-yl-oxytripyrrolidino-
phosphonium hexafluoro phosphate), followed by the
thermally induced cyclization of the resulting phthalamic
ester.8
Whereas most of the amino acids were easily protected
starting with 1 mmol of both the amino acid and the anhy-
dride, same protections gave substantially better results
using 2.1 mmol of the amino acid and 2.0 mmol of the an-
hydride (entries 2, 10 and 14, Table 1). This approach can
be applied to the protection of a larger scale of amino acid
obtaining similar to better yields. Thus, we have synthe-
sized TCP-Leu-OH (4L), TCP-Ile-OH (3L) and TCP-
Met-OH (7L) starting from 4.2 mmol, 6.3 mmol, and 8.4
mmol of the corresponding amino acid, respectively (en-
tries 4, 6 and 12, Table 1). Moreover, we have shown that
using MWI methodology the addition of a tertiary amine
can be avoided, in contrast with previous reports that sug-
gested the need of such bases.10
On the other hand, the use of microwave irradiation in or-
ganic synthesis has been extensively reported.9 Micro-
wave assisted reactions can be carried out safely using
conventional glassware in domestic microwave ovens, re-
sulting on savings in reaction time and waste disposal.
Bose et al. described the synthesis of tetrachlorophthalim-
idoacetic acid as an intermediate to -amino- -lactams
Under microwave irradiation tert-butyl ethers, esters, car-
bamates and the trityl function of cysteine were stable.
Disappointingly, we observed some detritylation (5–
25%) during the synthesis of TCP-His(Trt)-OH (16L) and
TCP-D-His(Trt)-OH (16D); in these cases further purifi-
cation by flash chromatography was required. TCP-His-
OH formation was confirmed by treatment of 16L with
Synthesis 2001, No. 9, 29 06 2001. Article Identifier:
1437-210X,E;2001,0,09,1313,1320,ftx,en;E00201SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

1314
E. Cros et al.
PAPER
Table 1 Synthesis of N-TCP Protected Amino Acids 1–14 (L–D)
under MWI and Reflux (continued)
TFA-CH2Cl2 (1:1) and characterization of the product
thus obtained.
All compounds showed optical rotations in good agree-
ment with their stereochemical integrity, except for TCP-
Cys(Trt)-OH (15L-D) and TCP-His(Trt)-OH (16L-D)
where the large differences in [ ]D values indicate that
some racemization had occurred (Table 2).
Entry Product
MWIa
Yield
(%)b
Reflux
Yield
(%)c
26
27
28
29
30
TCP-D-Lys(Boc)-OH (14D)
95
86
95
70
69
TCP-Cys(Trt)-OH (15L)
TCP-D-Cys(Trt)-OH (15D)
TCP-His(Trt)-OH (16L)
TCP-D-His(Trt)-OH (16D)
Interestingly, all N-TCP protected amino acids showed
max UV absorption at 334–336 nm which allows selective
detection of N-TCP protected species and may be useful
for monitoring the reactivity of N-TCP protected com-
pounds. Furthermore, all are crystalline solids, so their
handling and purification are facilitated.
a Reactions carried out using 1 mmol of both the amino acid and tet-
rachlorophthalic anhydride.
Table 1 Synthesis of N-TCP Protected Amino Acids 1–14 (L–D)
under MWI and Reflux
b Isolated yield.
c Isolated yields determined via the synthesis of TCP-aa-OBu-t 17–
22L (Table 3). Hydrolysis of the t-Bu esters was quantitative.
d Results obtained from synthesis carried out using 2.1 mmol of the
amino acid and 2.0 of tetrachlorophthalic anhydride.
e Results obtained from synthesis carried out using 6.3 mmol of the
amino acid and 6.0 of tetrachlorophthalic anhydride.
f Results obtained from synthesis carried out using 4.2 mmol of the
amino acid and 4.0 of tetrachlorophthalic anhydride.
g Results obtained from synthesis carried out using 8.4 mmol of the
amino acid and 8.0 of tetrachlorophthalic anhydride.
Entry Product
MWIa
Yield
(%)b
Reflux
Yield
(%)c
1
2
TCP-Gly-OH (1)
85
98
90
TCP-Ala-OH (2L)
62 (87)d
80
3
TCP-D-Ala-OH (2D)
TCP-Ile-OH (3L)
4
82 (97)e
98
67
87
72
91
To demonstrate the simplicity and efficiency of the micro-
wave irradiation technique compared to the reflux meth-
odology we have also synthesized N-TCP protected
amino acids 1–6L using the latter protocol (Scheme 2).
This procedure consists of refluxing the amino acid tert-
butyl ester hydrochloride and tetrachlorophthalic anhy-
dride in the presence of triethylamine in CH2Cl2 for 22
hours, followed by treatment with acetic anhydride and
pyridine (1:1) in CH2Cl2 for 2.5 hours. Finally, hydrolysis
of the tert-butyl ester with TFA–CH2Cl2 (2:3) provided
the N-TCP protected amino acids. As shown in Table 1,
best yields and purities are generally achieved by the mi-
crowave-assisted reactions, which are also much faster,
with easier workup and consumption of less quantity of
solvent.
5
TCP-D-Ile-OH (3D)
TCP-Leu-OH (4L)
6
92 (89)f
97
7
TCP-D-Leu-OH (4D)
TCP-Val-OH (5L)
8
80
9
TCP-D-Val-OH (5D)
TCP-Phe-OH (6L)
88
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
90 (97)d
83
TCP-D-Phe-OH (6D)
TCP-Met-OH (7L)
90 (87)g
86
TCP-D-Met-OH (7D)
TCP- -Ala-OH (8)
64 (89)d
99
TCP-Tyr(Bu-t)-OH (9L)
TCP-D-Tyr(Bu-t)-OH (9D)
TCP-Ser(Bu-t)-OH (10L)
TCP-D-Ser(Bu-t)-OH (10D)
TCP-Thr(Bu-t)-OH (11L)
TCP-D-Thr(Bu-t)-OH (11D)
TCP-Asp(OBu-t)-OH (12L)
Cl
O
1) Et3N, CH2Cl2, reflux, 22 h
2) Ac2O, pyr, reflux, 2.5 h
Cl
88
H-aa(R)-OtBu·HCl
+
O
O
90
Cl
Cl
96
TFA-CH2Cl2 (2:3), r.t., 2 h
TCP-aa(R)-OtBu
TCP-aa(R)-OH
96
67–98%
17-22L
1-6L
96
Overall two steps
aa = amino acid
R = amino acid side-chain
TCP = tetrachlorophthaloyl
94
TCP-D-Asp(OBu-t)-OH (12D) 12D 98
Scheme 2
TCP-Glu(OBu-t)-OH (13L)
TCP-D-Glu(OBu-t)-OH (13D)
TCP-Lys(Boc)-OH (14L)
99
89
88
We checked the stability of the TCP function towards
standard SPPS treatments. Different portions of a TCP-
Gly-Val-PAL-PEG-PS resin11 were treated at room tem-
Synthesis 2001, No. 9, 1313–1320 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of N -Tetrachlorophthaloyl (TCP)-Protected Amino Acids
1315
noted. Microwave irradiations were conducted in a commercial
Moulinex Optimo microwave oven. Peptide-resins (5–10 mg) were
hydrolized in 12 N HCl–propionic acid (1:1, v/v) for 2 h at 160 °C.
Amino acid analyses were performed on a Beckman 6300 Analyzer
with a sulfated polystyrene cation-exchange column (0.4 cm 21
cm).
perature with DMF (24 h), piperidine–DMF (1:4, 30 min)
and (i-Pr)2NEt–DMF (1:19, 30 min) followed by coupling
with Boc-Phe-OH. Then, the resin was subjected to cleav-
age with TFA–H2O (19:1, 2 h) and the crude product mix-
ture was analyzed by HPLC and mass spectrometry. In all
conditions tested, TCP-Gly-Val-NH2 was obtained exclu-
sively. Thus, the TCP protecting group is stable to stan-
dard Fmoc and Boc removal conditions, as well as, to
TFA cleavage treatments. These observations agree with
the results previously reported in the literature that TCP-
sugar derivatives are stable to mildy basic and to very
acidic conditions.2c,d
Microwave-Induced Synthesis of N-TCP Protected Amino
Acids; General Procedure
A mixture of tetrachlorophthalic anhydride (286 mg, 1 mmol) and
the appropriate amino acid (conveniently protected if necessary) (1
mmol) was thoroughly grounded and transferred to a 250 mL bea-
ker. DMF (1.7 mL) was added, the beaker was covered with a watch
glass, placed in the microwave oven, and subjected to irradiation at
285 W at 20 s intervals for 4 to 8 min.12 Reaction progress was fol-
lowed by TLC. Then, the beaker was removed from the oven and
allowed to cool to r.t. before the addition of cold H2O (30 mL). The
resultant precipitate was filtered, washed with cold H2O and dried
in vacuo over P2O5 to afford the N-TCP protected amino acids 1–
16(L-D) with 91% average yield (Tables 1 and 2). When purifica-
tion was necessary the precipitate was taken up in EtOAc (20 mL)
and washed with aq sat. NaCl solution (20 mL). The organic layer
was dried (MgSO4) and concentrated under reduced pressure.
Preliminary studies on solid-phase TCP removal were
carried out on a TCP-Gly-Val-PAL-PEG-PS resin11 and
we observed that after treatment with hydrazine–DMF
(3:17, 1 h, 40°C) or with ethylenediamine–DMF (1:199,
30 min, 50°C) the TCP group was removed quantitative-
ly.
In summary, we have described the synthesis of a range of
N -tetrachlorophthaloyl protected amino acids suitable for
solid phase synthesis of peptides in high yields and puri-
ties by a convenient procedure using domestic microwave
ovens.
TCP-His(Trt)-OH (16L)
The general procedure described above was used with the following
reagents and amounts: H-His(Trt)-OH (397 mg, 1 mmol, 1 equiv),
tetrachlorophthalic anhydride (0.286 g, 1 mmol, 1 equiv) in DMF
(1.7 mL). Purification of the crude product by column chromatog-
raphy on silica gel (CHCl3–MeOH, 2:1) provided 0.466 mg of 16L
(70%) as a white solid.
Commercially available reagents were used throughout without pri-
or purification, except solvents, which were distilled. Mps were de-
termined with a Mel-Temp device. Spectrophotometric analyses
were performed on a Shimadzu 160 spectrophotometer. Optical ro-
tations were measured on a Perkin Elmer 243B polarimeter at
20 °C. Aluminum-backed plates coated with silica gel (Scharlau or
Macherey-Nagel UV254) were used for analytical TLC; spots were
visualized by UV. Analytical reversed phase HPLC analyses were
carried out with a Thermo Separation Products P2000 binary pump,
a Perkin Elmer LC290 UV/VIS detector, and a Spherisorb ODS-2
column (0.46 25 cm; 5 m particle size). Low-resolution fast atom
bombardment mass spectroscopy (FABMS) was carried out in a
3-nitrobenzyl alcohol (3-NBA) matrix on a VG Quattro spectrome-
ter at Servicios Xerais de Apoio á Investigación, Universidade da
Coruña, Spain. Elemental analyses were determined with a Thermo
Instruments elemental analyzer EA 1110 CHNS/O (Universitat de
Girona) or with a Carlo Erba Instruments elemental analyzer EA
1108 CHNS/O (Universidade da Coruña). 1H NMR spectra were re-
corded at 200 MHz on a Bruker OPX200 Avance instrument.
Chemical shifts were expressed in ppm relative to TMS as internal
reference, J values are given in Hz. 13C NMR spectra were recorded
on a Bruker OPX200 Avance instrument operating at 50.33 MHz.
Amino acid abbreviations denote L-configuration unless otherwise
TCP-D-His(Trt)-OH (16D)
The general procedure described above was used with the following
reagents and amounts: H-D-His(Trt)-OH (397 mg, 1 mmol, 1
equiv), tetrachlorophthalic anhydride (0.286 g, 1 mmol, 1 equiv) in
DMF (1.7 mL). Purification of the crude product by column chro-
matography on silica gel (CHCl3–MeOH, 2:1) provided 0.459 mg
of 16D (69%) as an off-white solid.
TCP-His-OH:
TCP-His(Trt)-OH (16L) (78 mg) was dissolved in TFA–CH2Cl2
(1:1, 10 mL). After 15 min stirring at r.t. the clear solution was con-
centrated to dryness under reduced pressure followed by addition of
Et2O. The resulting solid was collected by filtration and washed
with Et2O to yield pure TCP-His-OH.
1H NMR (200 MHz, DMSO-d6): = 3.23–3.46 (m, 2 H, -CH2),
4.85–4.87 (m, 1 H, -CH), 6.82 (s, 1 H, CHimid), 7.61 (s, 1 H,
CHimid).
Table 2 Characterization of Compounds 1–16(L-D)
1H NMR
, J (Hz)
13C NMR,
FAB-MS
m/z (%)
c
Prod-
ucta
Rfb
tR
Mp (°C)
[ ]D
(c)d
1
0.55
18.1
288–292
-
4.42 (s, 2 H, -CH2)e
40.01 ( -CH2), 127.98,
[M + H]+ 341.98 (17),
(dec.)
128.56, 138.85 (Carom), 162.86 344.0 (16), 346.0 (10),
(CON), 168.36 (CO2H)e
[M – CO2H]+ 295.9 (57),
297.9 (70), 299.9 (42)
2L
0.68
19.4
280–286
(dec.)
–4.10
(1.162)
1.70 (d, 3 H, J = 7.4,
-
15.08 ( -CH3), 49.09 ( -CH), [M + H]+ 355.9 (46),
357.9 (55), 359.9 (28),
(Carom), 163.58 (CON), 171.39 [M – CO2H]+ 309.8 (89),
(CO2H)f
311.9 (100), 313.9 (53)
CH3), 5.00 (q, 1 H, J = 7.4, 129.11, 129.91, 140.07
-CH)f
Synthesis 2001, No. 9, 1313–1320 ISSN 0039-7881 © Thieme Stuttgart · New York

1316
E. Cros et al.
PAPER
Table 2 Characterization of Compounds 1–16(L-D) (continued)
c
Prod-
ucta
Rfb
tR
Mp (°C)
[ ]D
1H NMR
, J (Hz)
13C NMR,
FAB-MS
m/z (%)
(c)d
2D
3L
0.67
19.7
283–286
(dec)
+4.72
(1.121)
Identical to 2L
Identical to 2L
[M + H]+ 355.9 (36),
357.9 (46), 359.9 (23),
[M – CO2H]+ 309.9 (46),
311.9 (57), 313.9 (30)
0.73
21.6
174–176
–24.80
(0.138)
1.02 (t, 3 H, J = 7.4, -CH3), 11.70 ( -CH3), 17.48 ( -CH3), [M + H]+ 397.9 (85),
1.14–1.38 (m, 1 H, -CH2), 26.79 ( -CH2), 35.72 ( -CH), 399.9 (100), 401.9 (47),
1.27 (d, 3 H, J = 5.0, -
58.45 ( -CH), 129.26, 130.49, [M – CO2H]+ 351.9 (78),
CH3), 1.68–1.82 (m, 1 H, - 140.62 (Carom), 164.29 (CON), 353.9 (95), 355.9 (47)
CH2), 2.57–2.71 (m, 1 H, - 169.94 (CO2H)f
CH), 4.83 (d, 1 H, J = 8.0,
-CH)f
3D
4L
0.69
0.70
22.0
21.8
173–175
226–228
+24.20
(0.124)
Identical to 3L
Identical to 3L
[M + H]+ 398.0 (56),
400.0 (66), 401.9 (32),
[M – CO2H]+ 352.0 (82),
354.0 (100), 356.0 (49)
–6.75
0.97 (d, 3 H, J = 6.4, -
21.23 ( -CH3), 23.43 ( -CH3), [M + H]+ 397.9 (17),
(0.126)
CH3), 1.01 (d, 3 H, J = 6.4, 25.55 ( -CH), 37.80 ( -CH2), 400.0 (19), 402.0 (9),
-CH3), 1.51–1.60 (m, 1 H, 51.84 ( -CH), 128.94, 130.05, [M – O2H]+ 352.0 (26),
-CH), 2.02 (ddd, 1 H,
140.21 (Carom), 163.83 (CON), 354.0 (31), 356.0 (17)
J = 4.5, 10.0, 14.2, -CH2), 170.52 (CO2H)f
2.34 (ddd, 1 H, J = 4.2,
11.3, 14.2, -CH2), 5.01
(dd, 1 H, J = 4.5, 11.3,
-
CH)f
4D
5L
0.66
0.70
21.9
20.8
229–231
210–212
+4.66
(0.118)
Identical to 4L
Identical to 4L
[M + H]+ 397.9 (56),
399.9 (67), 401.9 (32),
[M – CO2H]+ 352.0 (60),
354.0 (72), 355.9 (37)
–36.29
0.98 (d, 3 H, J = 6.7, -
19.68 ( -CH), 21.19 ( -CH3), [M + H]+ 383.9 (58),
(1.017)
CH3), 1.22 (d, 3 H, J = 6.7, 28.65 ( -CH), 58.68 ( -CH), 386.0 (71), 387.9 (35),
-CH3), 2.75 (dq, 1 H, 128.87, 130.12, 140.24
[M – CO2H]+ 338.0 (81),
J = 6.7, 8.0, -CH), 4.65 (d, (Carom), 163.91 (CON), 169.50 340.0 (100), 341.9 (50)
1 H, J = 8.0, -CH)f
(CO2H)f
5D
6L
0.69
0.67
21.0
21.6
212–214
250–254
+37.25
(1.023)
Identical to 5L
Identical to 5L
[M + H]+ 384.0 (44),
385.9 (51), 387.9 (25),
[M – CO2H]+ 338.0 (81),
340.0 (100), 342.0 (48)
–150.40
3.62 (dd, 1 H, J = 11.0,
34.95 ( -CH2), 54.71 ( -CH), [M + H]+ 431.9 (12),
(0.124)
14.3, -CH2), 3.78 (dd, 1 H, 127.61, 128.49 (Carom),
J = 5.1, 14.3, -CH2), 5.40 129.37, 129.68 (CHarom),
433.9 (15), 436.1 (10),
[M – CO2H]+ 385.9 (9),
387.9 (11), 390.0 (7)
(dd, 1 H, J = 5.1, 11.0,
-
130.12, 138.05, 140.48
(Carom), 163.52 (CON), 169.70
(CO2H)f
CH), 7.29–7.40 (m, 5 H,
f
CHarom
)
6L
7L
0.67
0.65
21.8
20.3
253–256
171–173
+144.30
(0.096)
Identical to 6L
Identical to 6L
[M + H]+ 431.9 (14),
433.9 (18), 435.9 (9), [M
– CO2H]+ 385.9 (14),
387.9 (18), 389.9 (10)
–24.77
(0.107)
2.03 (s, 3 H, SCH3), 2.27– 14.47 (SCH3), 27.56 ( -CH2), [M + H]+ 415.9 (62),
2.46 (m, 2 H, -CH2), 2.48– 29.86 ( -CH2), 51.31 ( -CH), 417.9 (79), 419.9 (40)
2.90 (m, 2 H, -CH2), 5.18 127.93, 128.53, 138.75
(dd, 1 H, J = 5.6, 8.8,
-
(Carom), 162.99 (CON), 169.90
CH)e
(CO2H)e
7D
0.71
20.9
172–174
+22.85
(0.116)
Identical to 7L
Identical to 7L
[M + H]+ 415.9 (74),
417.9 (96), 419.9 (49)
Synthesis 2001, No. 9, 1313–1320 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of N -Tetrachlorophthaloyl (TCP)-Protected Amino Acids
1317
Table 2 Characterization of Compounds 1–16(L-D) (continued)
c
Prod-
ucta
Rfb
tR
Mp (°C)
230–240
[ ]D
1H NMR
, J (Hz)
13C NMR,
FAB-MS
m/z (%)
(c)d
8
0.73
18.4
-
2.70 (t, 2 H, J = 10.1,
-
32.03 ( -CH2), 34.36 ( -CH2), [M + H]+ 355.8 (17),
CH2), 3.90 (t, 2 H, J = 10.1, 128.15,128.40, 138.31(Carom), 357.9 (18), 359.9 (9)
-CH2)e
163.23 (CON), 172.03
(CO2H)e
9L
0.72
22.9
69–72
–147.38
1.27 (s, 9 H, 3 CH3), 3.50 28.70 (3 CH3), 33.57
[M + H]+ 504.0 (2), 506.0
(0.122)
(dd, 1 H, J = 6.1, 14.4,
-CH2), 3.60 (dd, 1 H,
J = 10.7, 14.4, -CH2), 5.19 127.02 (Carom), 129.19
(dd, 1 H, J = 6.1, 10.7, (CHarom), 129.76, 131.03,
-CH), 6.85 (d, 2 H, J = 8.5, 140.28 (Carom), 154.22
( -CH2), 53.81 ( -CH), 78.53 (2), 508.1 (1), [M – t-Bu
(CBu-t), 124.48 (CHarom),
+ H]+ 447.0 (3), 449.0
(5), 451.0 (3)
CHarom), 7.06 (d, 2 H,
(Carom-OBu-t), 162.60 (CON),
g
J = 8.5, CHarom
)
172.04 (CO2H)g
9D
0.71
0.73
22.9
21.8
66–69
+150.97
(0.164)
Identical to 9L
Identical to 9L
[M + H]+ 504.0 (1), 506.0
(1), 508.0 (0.6), [M – t-
Bu + H]+ 447.9 (3), 449.9
(4), 451.9 (2)
10L
173–175
–27.24
(0.112)
1.21 (s, 9 H, 3 CH3), 4.00 27.31 (3 CH3), 52.64 ( -
[M + H]+ 428.0 (10),
430.0 (12), 432.0 (6),
[M – t-Bu + H]+ 372.0
(dd, 1 H, J = 9.2, 9.4,
-CH2), 4.17 (dd, 1 H,
J = 6.7, 9.4, -CH2), 5.09
(dd, 1 H, J = 6.7 Hz, 9.2,
-CH)g
CH), 58.31 ( -CH2), 75.09
(CBu-t), 127.34, 129.98,
140.43 (Carom), 162.72 (CON), (79), 373.9 (100), 375.9
169.87 (CO2H)g
(49)
10D
11L
0.70
0.74
21.7
22.3
176–178
92–94
+20.20
(0.098)
Identical to 10L
Identical to 10L
[M + H]+ 428.0 (11),
430.0 (13), 432.0 (7),
[M – t Bu + H]+ 371.9
(79), 373.9 (100), 375.9
(49)
–72.27
1.21 (s, 9 H, 3 CH3), 1.45 21.46 ( -CH3), 28.26 (3
[M + H]+ 442.0 (6), 444.0
(0.627)
(d, 3 H, J = 6.2, -CH3),
CH3), 56.71 ( -CH), 66.15 ( - (8), 446.0 (4), [M – t-Bu
4.22–4.35 (m, 1 H, -CH), CH), 76.18 (CBu-t), 127.24, + H]+ 385.9 (77), 387.9
4.87 (d, 1 H, J = 7.0, -CH)g 129.96, 140.43 (Carom), 162.57 (100), 389.9 (49)
(CON), 168.75 (CO2H)g
11D
12L
0.70
0.74
22.8
21.6
88–91
+77.53
(0.688)
Identical to 11L
Identical to 11L
[M + H]+ 442.0 (5), 444.0
(6), 446.0 (3), [M – t-Bu
+ H]+ 385.9 (78), 387.9
(100), 389.9 (50)
256–258
–15.84
(0.627)
1.43 (s, 9 H, 3 CH3), 3.11 27.90 (3 CH3), 34.66
(dd, 1 H, J = 9.3, 16.7, ( -CH2), 48.79 ( -CH), 82.03 458.0 (11), 460.0 (6),
[M + H]+ 455.9 (10),
-
CH2), 3.29 (dd, 1 H, J = 5.6, (CBu-t), 127.30, 130.04,
16.7, -CH2), 5.39 (dd, 1 H, 140.48 (Carom), 162.51
[M – t-Bu + H]+ 399.9
(77), 401.9 (100), 403.9
(49)
J = 5.6, 9.3, -CH)g
Identical to 12L
(CON), 168.92 (CO2H),
171.85 (CO2Bu-t)g
12D
13L
0.75
0.76
21.3
22.2
252–255
122–126
+19.71
(0.614)
Identical to 12L
[M + H]+ 455.9 (11),
457.9 (13), 460.0 (7),
[M – t Bu + H]+ 399.9
(78), 401.9 (100), 403.9
(49)
–22.90
1.44 (s, 9 H, 3 CH3),
23.72 ( -CH2), 27.97
[M + H]+ 470.0 (9), 472.0
(0.677)
2.38–2.30 (m, 2 H, -CH2), (3 CH3), 32.00 ( -CH2),
(10), 474.0 (5), [M – t- Bu
2.66–2.45 (m, 2 H, -CH2), 51.85 ( -CH), 81.22 (CBu-t), + H]+ 413.9 (77), 415.9
5.00 (dd, 1 H, J = 4.8, 9.4, 127.33, 130.00, 140.43
(100), 417.9 (50)
-CH)g
(Carom), 162.77 (CON), 171.40
(CO2H), 172.83 (CO2Bu-t)g
Synthesis 2001, No. 9, 1313–1320 ISSN 0039-7881 © Thieme Stuttgart · New York

1318
E. Cros et al.
PAPER
Table 2 Characterization of Compounds 1–16(L-D) (continued)
c
Prod-
ucta
Rfb
tR
Mp (°C)
118–120
[ ]D
1H NMR
, J (Hz)
13C NMR,
FAB-MS
m/z (%)
(c)d
13D
0.74
22.3
+20.37
(0.707)
Identical to 13L
Identical to 13L
[M + H]+ 470.0 (10),
472.0 (12), 474.0 (6),
[M – t- Bu + H]+ 413.9
(78), 415.9 (100), 417.9
(50)
14L
0.79
21.2
125–128
–15.80
(1.045)
1.38 (s, 9 H, 3 CH3),
1.38–1.56 (m, 4 H, , -
CH2), 2.64 (dt, 2 H, J = 7.5, 30.07 ( -CH2), 40.46
24.20 ( -CH2), 28.55
(3 CH3), 28.77 ( -CH2),
[M + H]+ 513.0 (2), 515.0
(2), 517.0 (1),
[M – C5H8O2 + H]+ 413.0
8.0, -CH2), 3.08 (t, 2 H,
J = 6.8, -CH2), 4.95
(t, 1 H, J = 7.5, -CH)g
( -CH2), 53.53 ( -CH), 78.27 (80), 415.0 (100), 417.0
(CBut), 128.99, 130.06,
140.19 (Carom), 156.60
(CO-Boc), 163.80 (CON),
170.25 (CO2H)g
(49)
14D
15L
0.72
0.72
21.3
121–124
+13.49
(1.052)
Identical to 14L
Identical to 14L
[M + H]+ 513.0 (1), 515.0
(1), 517.0 (0.7),
[M – C5H8O2 + H]+ 413.0
(79), 415.0 (100), 417.0
(49)
26.1h
82–84
–29.75
(0.079)
3.04 (dd, 1 H, J = 4.4, 13.8, 30.22 ( -CH2), 52.27 ( -CH), [M + H]+ 632.0 (0.04),
-CH2), 3.43 (dd, 1 H,
67.65 (CTrt), 126.90 (CHarom), [Trt]+ 243.2 (100),
J = 11.5, 13.8, -CH2), 4.35 127.28 (Carom), 128.23, 129.41 [Trt + H]+ 244.2 (23)
(dd, 1 H, J = 4.4, 11.5, (CHarom), 129.84, 140.29,
(dec.)e
-
CH), 7.20–7.44 (m, 15 H, 144.09 (Carom), 162.45 (CON),
g
CHarom
)
170.91 (CO2H)g
15D
16L
0.70
0.72
25.0h
24.6h
83–85
(dec.)
+11.84
(0.076)
Identical to 15L
Identical to 15L
[M + H]+ 632.0 (0.07),
[Trt]+ 243.2 (100),
[Trt + H]+ 244.2 (72)
142–158
–70.20
3.46 (dd, 1 H, J = 9.60,
26.43 ( -CH2), 53.70 ( -CH), [M + H]+ 664.0 (0.99),
(dec.)
(0.095)
15.3, -CH2), 3.82 (dd, 1 H, 77.20 (CTrt), 119.42 (CHimid), 666.0 (1.24), 668.0
J = 6.0, 15.3, -CH2), 5.16 128.18 (Carom), 128.18, 129.48 (0.65), [M – C19H14 + H]+
(dd, 1 H, J = 6.0, 9.6,
-
(CHarom), 135.10 (Carom),
421.9 (4), 423.9 (5),
CH), 6.59 (s, 1 H, CHimid), 137.28 (CHimid), 139.79,
425.9 (2), [M – Trt-CO2H
7.00–7.39 (m, 15 H,
141.18, 146.85 (Carom), 162.85 + H]+ 376.0 (2), 378.0
CHarom), 7.68 (s, 1 H,
(CON), 170.21 (CO2H)g
(3), 380.0 (1), [Trt]+
243.2 (100), [Trt + H]+
244.2 (78)
g
CHimid
)
16D
0.66
24.2h
138–151
(dec.)
+59.01
(0.142)
Identical to 16L
Identical to 16L
[M + H]+ 664.0 (0.45),
666.0 (0.55), 668.0
(0.28), [M – C19H14 + H]+
421.9 (3), 423.9 (4),
425.9 (2), [M – Trt –
CO2H + H]+ 376.0 (2),
378.0 (3), 380.0 (2),
[Trt]+ 243.2 (100),
[Trt + H]+ 244.2 (77)
a Satisfactory microanalyses obtained: C ±0.40, H ±0.31, N ±0.40, S ±0.26 ( Exceptions: 9L, C –1.24, N +0.92; 9D, C –1.33, N +1.51; 11L,
H +0.48; 12D, C –0.52, N +1.36; 13D, N +0.65; 14L, N +0.54; 14D, C –1.06, N +1.06; 15L, C –2.56, N +0.68, S –0.7; 15D, H +0.49, N +1.27;
16L, C +0.45, H –0.52; 16D, C –0.75, N +0.43). Compounds 10L, 11L, and 13D contained occluded water to an extent of 0.5 H2O (10L, 11L),
1 H2O (13D, 16D) and 2 H2O (16L).
b EtOAc–MeOH–AcOH (2:3:0.3).
c Analytical HPLC was developed at 1.0 mL/min with 0.1% aq TFA–MeCN (2 min at 1:0, 1:0 to 0:1 over 23 min, then 5 min at 0:1); detection
at 220 nm.
d EtOH.
e DMSO-d6.
f Acetone-d6.
g CDCl3.
h Analytical HPLC was carried out at 1.0 mL/min with H2O–MeCN (2 min at 2.3:1, 2.3:1 to 1:9 over 23 min, 1:9 to 0:1 over 10 min, then 10
min at 0:1); detection at 220 nm.
Synthesis 2001, No. 9, 1313–1320 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of N -Tetrachlorophthaloyl (TCP)-Protected Amino Acids
1319
N-TCP Protected Amino Acids by Reflux; General Procedure
Tetrachlorophthalic anhydride (1 equiv) was added to a solution of
the amino acid tert-butyl ester hydrochloride (1 equiv) and Et3N (2
equiv) in CH2Cl2 (100 mL). The resultant solution was heated to re-
flux and stirred for 22 h. After cooling to r.t., the mixture was con-
centrated under reduced pressure. To the off white residue a
solution of Ac2O–pyridine (1:1, 40 mL) and CH2Cl2 (40 mL) was
added, and the mixture was refluxed for 2.5 h. Then, it was allowed
to cool to r.t. and concentrated in vacuo. 0.1 M HCl (50 mL) was
added to the residue and the suspension was left 15 min at 4 °C. The
resultant precipitate was collected by filtration and washed with
cold 0.05 M HCl and water. The solid was dissolved in EtOH and
the solution concentrated to dryness; this process was repeated
twice. The N-TCP amino acid tert-butyl esters 17–22L (Table 3)
obtained were dried in vacuo over P2O5.
piperidine–DMF (3:7, 2 + 10 min), and finally washed with DMF
(3 1 min) and CH2Cl2 (3 1 min). Fmoc-Val-OH (24 mg, 0.072
mmol, 3 equiv) was coupled to the resin with DIPCI (11 l, 0.072
mmol, 3 equiv) and HOAt (10 mg, 0.072 mmol, 3 equiv) in DMF
(0.8 mL) for 3 h, followed by washing with DMF (5 2 min) and
CH2Cl2 (5 2 min). After Fmoc removal and the washings de-
scribed above, TCP-Gly-OH (25 mg, 0.072 mmol, 3 equiv) was in-
corporated onto the resin with DIPCI (11 l, 0.072 mmol, 3 equiv)
and HOAt (10 mg, 0.072 mmol, 3 equiv) in DMF at 25 °C for 5.5 h
(Kaiser ninhydrin test13 negative after this time). The resin was
washed with DMF (3 1 min) and CH2Cl2 (3 1 min) and dried in
vacuo. An aliquot of this TCP-Gly-Val-PAL-PEG-PS resin was hy-
drolyzed for amino acid analysis: Val 1.0, Gly 0.89.
TCP-Gly-Val-NH2 was characterized prior to the stability studies.
A portion of the TCP-Gly-Val-PAL-PEG-PS resin (10 mg) was
subjected to cleavage with TFA–H2O (19:1) for 2 h, the filtrates
were drawn off from the vessel with positive nitrogen pressure, and
the cleaved resin was washed (2 0.5 mL) with further TFA–H2O
(19:1). The combined filtrates were evaporated to dryness, the resi-
due dissolved in MeCN and analyzed by analytical HPLC (gradient
in Table 1) and mass spectrometry: HPLC: tR 18.9 min.
Compounds 17–22L were dissolved in TFA–CH2Cl2 (2:3, 30 mL).
After stirring for 2 h at r.t. the clear solution was concentrated to
dryness under reduced pressure. The resultant solid was dissolved
in CH2Cl2, following by evaporation of the solvent; this process was
repeated twice, yielding the corresponding N-TCP protected amino
acids 1–6L (Tables 1, 2) which were dried in vacuo over P2O5.
FAB-MS: m/z = [M + H]+ 441.8, [M + H – CONH2]+ 396.9, [M.]–
440.8.
Studies of the Stability of the TCP Function Towards Standard
SPPS Treatments
Fmoc-PAL-PEG-PS resin (150 mg, 0.024 mmol, 0.16 mmol/g) was
washed with DMF (3 2 min) and CH2Cl2 (3 2 min), treated with
Portions of this resin (10 mg per experiment) were subjected to var-
ious TCP stability experiments, as follows: TCP-Gly-Val-PAL-
Table 3 Characterization of Compounds 17–22L
Product
Rfa
1H NMR
13C NMR
, J (Hz)
TCP-Gly-OBu-t
(17)
0.79
1.51 (s, 9 H, 3 CH3), 4.37 (s, 2 H, -CH2)b 27.98 (3 CH3), 40.20 ( -CH2), 83.35
(CBu-t), 127.65, 129.92, 140.32 (Carom),
162.86 (CON), 165.49 (CO2Bu-t)b
TCP-Ala-OBu-t
(18L)
0.85
0.72
1.49 (s, 9H, 3 CH3), 1.70 (d, 3 H, J = 7.4, - 14.99 ( -CH3), 27.83 (3 CH3), 49.10 ( -
CH3), 4.91 (q, 1 H, J = 7.4, -CH)c
CH), 82.85 (CBu-t), 127.46, 129.76, 140.17
(Carom), 162.74 (CON), 167.79 (CO2Bu-t)d
TCP-Ile-OBu-t
(19L)
0.91 (t, 3 H, J = 7.2, -CH3), 1.00–1.18 (m, 1 11.12 ( -CH3), 16.93 ( -CH3), 26.26 ( -
H, -CH2), 1.14 (d, 3 H, J = 6.8, -CH3), 1.47 CH2), 27.93 (3 CH3), 37.78 ( -CH), 58.84
(s, 9 H, 3 CH3), 1.47–1.61 (m, 1 H, -CH2), ( -CH), 82.75 (CBu-t), 127.32, 129.83,
2.45–2.62 (m, 1 H, -CH), 4.63 (d, 1 H,
140.28 (Carom), 163.20 (CON), 167.12
J = 7.8, -CH)b
(CO2Bu-t)b
TCP-Leu-OBu-t
(20L)
0.80
0.97 (d, 3 H, J = 6.6, -CH3), 0.98 (d, 3 H,
21.01( -CH3), 23.11 ( -CH3), 25.27 ( -CH),
J = 6.4, -CH3), 1.47 (s, 9 H, 3 CH3), 1.43– 27.89 (3 CH3), 37.11 ( -CH2), 52.41 ( -
1.54 (m, 1 H, -CH), 1.95 (ddd, 1 H, J = 4.6, CH), 82.91 (CBu-t), 127.42, 129.81, 140.25
10.0, 14.3, -CH2), 2.31 (ddd, 1 H, J = 4.4, (Carom), 163.14 (CON), 167.99 (CO2Bu-t)b
11.4, 14.3, -CH2), 4.88 (dd, 1 H, J = 4.6,
11.4, -CH)b
TCP-Val-OBu-t
(21L)
0.82
0.84
0.95 (d, 3 H, J = 6.8, -CH3), 1.17 (d, 3 H,
J = 6.8, -CH3), 1.47 (s, 9 H, 3 CH3), 2.76 CH3), 28.56 ( -CH), 59.43 ( -CH), 82.76
(dq, 1 H, J = 6.8, 8.0, -CH), 4.54 (d, 1 H,
J = 8.0, -CH)b
19.70 ( -CH3), 21.01 ( -CH3), 27.91 (3
(CBu-t), 127.30, 129.84, 140.29 (Carom),
163.17 (CON), 166.98 (CO2Bu-t)b
TCP-Phe-OBu-t
(22L)
1.52 (s, 3 H, 3 CH3), 3.51 (dd, 1 H, J = 10.6, 27.91 (3 CH3), 34.42 ( -CH2), 54.83 ( -
14.3, -CH2), 3.75 (dd, 1 H, J = 6.6, 14.3, - CH), 83.33 (CBu-t), 126.91, 127.16, 128.62,
CH2), 5.14 (dd, 1 H, J = 6.6, 10.6, -CH),
128.65, 129.70, 136.58, 140.19 (Carom),
b
7.17–7.30 (m, 5 Harom
)
162.74 (CON), 166.98 (CO2Bu-t)b
a EtOAc–MeOH–AcOH (2:3:0.3).
b CDCl3.
c DMSO-d6.
d Acetone-d6.
Synthesis 2001, No. 9, 1313–1320 ISSN 0039-7881 © Thieme Stuttgart · New York

1320
E. Cros et al.
PAPER
(3) Castro-Palomino, J. C.; Schmidt, R. R. Tetrahedron Lett.
PEG-PS resin was swollen in DMF (2 5 min), treated with DMF
(24 h), piperidine–DMF (1:4, 30 min) or (i-Pr)2NEt–DMF (1:19, 30
min), and washed with DMF (5 2 min). Then, Boc-Phe-OH (2 mg,
0.008 mmol, 5 equiv) was coupled to each of the resins with DIPCI
(1.2 l, 0.008 mmol, 5 equiv) and HOAt (1 mg, 0.008 mmol, 5
equiv) in DMF (0.2 mL) for 4.5 h. The resins were washed with
DMF (3 1 min) and CH2Cl2 (3 1 min) and dried in vacuo. Final
release of the peptide from the resins was carried out with TFA–
H2O (19:1) for 2 h as described above. The resulting residue was
dissolved in MeCN and analyzed by analytical HPLC (gradient in
Table 2). TCP-Gly-Val-NH2 (tR 18.9 min) was detected exclusively
for all the conditions tested.
1995, 36, 5343.
(4) Stangier, P.; Hindsgaul, O. Synlett 1996, 179.
(5) El-Naggar, A. M.; Zaher, M. R.; El-Basiouny, M.; Khalaf,
N. S. Il Farmaco-Ed. Sc. 1984, 39, 154; They reported the
synthesis in solution of some N-tetrachlorophthaloyl
protected amino acids and dipeptide derivatives to test their
biological activity against a number of microorganisms. The
reported synthesis did not include any deprotection step of
the TCP protecting group.
(6) (a) Bose, A. K.; Greer, F.; Price, C. C. J. Org. Chem. 1958,
23, 1335. (b) Bose, A. K. Org. Synth., Coll. Vol. V 1973,
973.
(7) Scheehan, J. C.; Chapman, D. W.; Roth, R. W. J. Am. Chem.
Soc. 1952, 74, 3822.
Acknowledgment
(8) Aguilar, N.; Moyano, A.; Pericàs, M. A.; Riera, A. Synthesis
1998, 313.
We thank CIRIT (QFN-4620) and University of Girona (UdG98/
404) for the financial support. We are also grateful to Prof. G. Bar-
any for the amino acid analyses and to Medichem S.L. for the opti-
cal rotation determinations.
(9) (a) Bram, G.; Loupy, A.; Majdoub, M.; Gutiérrez, E.; Ruiz-
Hitzky, E. Tetrahedron 1990, 46, 5167. (b) Bose, A. K.;
Manhas, M. S.; Banik, B. K.; Robb, E. W. Res. Chem.
Intermed. 1994, 20, 1. (c) Caddick, S. Tetrahedron 1995,
51, 10403.
(10) Bose, A. K.; Jayaraman, M.; Okawa, A.; Bari, S. S.; Robb,
E. W.; Manhas, M. S. Tetrahedron Lett. 1996, 37, 6989.
(11) PAL, peptide amide linker: 5-(4-aminomethyl-3,5-
dimethoxyphenoxy)valeric acid; PEG-PS, polyethylene
glycol-polystyrene (graft support).
References and Notes
(1) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 3rd Ed.; Wiley: New York, 1999.
(2) (a) Debenham, J. S.; Madsen, R.; Roberts, C.; Fraser-Reid,
B. J. Am. Chem. Soc. 1995, 117, 3302. (b) Debenham, J. S.;
Fraser-Reid, B. J. Org. Chem. 1996, 61, 432.
(12) Continuous heating resulted in burning of the reaction
mixture.
(13) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. Anal.
Biochem. 1970, 34, 595.
(c) Debenham, J. S.; Debenham, S. D.; Fraser-Reid, B.
Bioorg. Med. Chem. 1996, 4, 1909. (d) Debenham, J. S.;
Rodebaugh, R.; Fraser-Reid, B. Liebigs Ann./Recueil 1997,
791.
Synthesis 2001, No. 9, 1313–1320 ISSN 0039-7881 © Thieme Stuttgart · New York