synthesis of protected peptide acids, amides and...
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SYNTHESIS OF PROTECTED PEPTIDE ACIDS, AMIDES AND ALKYL AMIDES USING
PHOTOLYTICALLY CLEAVABLE PS-BDODMA SUPPORTS
7.L Introduction
T he myriad of naturally occurring peptides and their biologically relevant
analogues, includes numerous examples where the C-terminal carboxyls are
modified as amides, aldehydes and esters. The peptide activity can be controlled
through changes at the C-terminal and therefore it is important to have efficient methods
for the solid phase synthesis of such derivatives. Chemical synthesis of C-terminal
modified peptides are important for the structure-activity relationship studies and
conformational ~tudies. ' .~ A large number of biologically active peptides contain an
amide or N-alkylamide group at the carboxy terminal. C-terminal modification of
peptides without affecting side-chain protecting groups and chiral centers has been a
great challenge in peptide chemistry. In classical Merrifield's solid phase peptide
synthesis, the C-terminal peptide amides are usually prepared by ammonolysis of the
polymer-peptide ester linkage.3 Another method for the synthesis of peptide amides is the
use of resins containing amino groups, which facilitates the coupling of the C-terminal
amino acid of the target peptide through an amide linkage and the final cleavage of the
synthesized peptide in the form of peptide amide by acid cleavage.G7 The major
limitation of these procedures is the lack of stability of side chain ester protecting groups.
Photolabile supports have been employed for 24 years in SPPS, since the
introduction of photosensitive 2-nitrobenzamido anchoring linkage between the polymer
support and the peptide.' This method permits the release of peptide amides under neutral
conditions at room temperature without affecting the side chain protecting groups and
N-protecting groups. Photolytic release of peptides offers a method of orthogonal
cleavage, which complements the traditional use of TFA or HF. The photolabile supports
have a light sensitive chromophore, which is stable to conditions of peptide ~ ~ n t h e s i s . ~ ~ ' ~
The photolabile linker can be easily introduced into the support and the C-terminal amino
acid can be easily incorporated to it. Photosensitive amino acids such as Trp and Tyr
should not remain intact at the wavelength of light used for photolysis and the by-product
formed should remain along with the polymer support. A large number of polymer
supports with various photolabile anchoring groups are used for the synthesis of peptides.
Nitration of Merrifield's resin results in very high loading of nitro group, which increases
the polarity of the resin and hence reduces the swelling property of the resin in organic
solvents with very low dielectric constants. A modified 4-bromomethyl 3-nitro and
4-aminomethyl 3-nitro tetraethyleneglycol diacrylate cross-linked polystyrene support
was used for the synthesis of protected peptide acids, amides and alkyl amides.''
PS-BDODMA resin anchored with photolabile 4-hromomethyl 3-nitro
benzamidomethyl and 4-aminomethyl 3-nitro benzamidomethyl anchoring groups were
successfully used for the solid phase peptide synthesis. This resin swells much effectively
than the Merrifield's resin in all solvents used for SPPS. This support contains only the
required number of nitro groups that are essential for the photochemical reaction. This
chapter describes the photolytic synthesis of fblly protected peptides, peptide amides and
peptide N-alkyl amides using PS-BDODMA resin.
7.2. Results and Discussion
7.2.a. Preparation of 4-chloromethyl3-nitro PS-BDODMA resin
The photolabile 4-chloromethyl 3-nitro PS-BDODMA resin was prepared by the
nitration of chloromethyl PS-BDODMA resin using fbming nitric acid at -10 "C. The
resin showed same swelling properties as that of chloromethyl PS-BDODMA resin
indicating no additional cross-linking during nitration. The resin shows characteristic IR
(Kl3r) absorption band at 690 cm-' and 1230 cm-' for chloromethyl group and 1350 cm-'
and 1545 cm-' for NO2 group (Fig.7-1).
Fig.7-1: IR (KBr) spectrum of 4-chloromethyl 3-nitro PS-BDODhL4 resin
7.2.b. Preparation of 4-aminomethyl 3-nitro PS-BDODMA resin
4-aminomethyl 3-nitro P S - B D O D I ~ ~ resin was prepared from 4-chloromethyl
3-nitro PS-BDODMA resin by refluxing with potassium phthalimide in NMP followed
by hydrazinolysis. Amino capacity of the resin was estimated by picric acid titration method
and a quantitative reaction was observed. The resin showed IR (KBr) absorption band at
3440 c ~ ' @Hz) and 1340 cm.' and 1540 cm" corresponding to NO2 group (Fig. 7- 2).
Fig.7-2:IR (KBr) spectrum of 4-aminomethyl 3-nitro PS-BDODMA resin
7.2.c. Preparation of 4-bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin
The photolabile anchoring group 4-bromomethyl 3-nitro benzoic acid was prepared
by a two step reaction from p-toluic acid. p-Toluic acid was converted to 4-bromo-
methyl benzoic acid by the treatment with N-bromo succinimide (NBS). Nitration with
fuming nitric acid at -10 "C of 4-bromomethyl benzoic acid yielded 4-bromomethyl
3-nitro benzoic acid The pre-swollen aminomethyl resin in NMP was treated with HOBt
active ester of 4-bromomethyl 3-nitro benzoic acid yielded the photolabile
4-bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin (Scheme7-1). Estimation
of bromine capacity by Volhardt's method indicates the quantitative reaction. The resin
shows characteristic IR (KBr) bands at 1340 cm" and 1540 cm-' of the NO2 and
1650 cm'l (NHCO) (Fig.7-3) The resin shows the same extent of swelling as the
PS-BDODMA resin in all solvents used for SPPS.
I Stepwise incorporation of amino acids
Scheme. 7-1. Preparation and use of 3-nitro 4-bromomethyl benzamidomethyl PS-BDODMA resin.
Fig. 7-3.
10
n
u
I
Lad 4- am# Ism t w o IDDO YXI
, IR O<Br) spectrum of 3-nitro 4-bromomethyl benzamidomethyl PS-BDODMA resin.
7.2.d. Preparation of 4-aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin
4-Aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin can be prepared
from 4-bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin by refluxing with
potassium phthalimide in NMP followed by hydrazinolysis (Scheme. 7-2). Amino
capacity estimation by picric acid method indicates the quantitative reaction. The resin
showed characteristic IR (KBr) bands at 1342 cm-I and 1548 cm-I of the NOz, 1680 cm"
(NHCO) and 3450 cm-' (broad) of NH group (Fig. 7-4).
*0 )0
Fig. 7-4. IR (KBr) spectrum of 4-aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin
NO2 w &+&,@ O NMP, d I ~ O O C -
N2H4.H20
EtOH, 8 8 ~
0
Scheme. 7-2. Preparation of 3-nitro 4-aminomethyl benzamidomethyl PS-BDODMA resin
7.2.e Preparation of N-alkyl aminomethyl fni t ro benzamidomethyl PSBDODMA resin
4-Bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin was suspended in
DMF, dry methyl amine or ethyl amine gas was passed through the suspension at 0 'C
and the reaction mixture was shaken at room temperature for 24 h (Scheme. 7-3). The
N-alkyl resin was purified by washing and dried under vacuum. The resin shows IR
(KBr) absorption at 1342 cm-', 1540 cm-' (NOz), 3430 c ~ ' (broad) (NH) and 1640 cm-'
(NHCO). The amino capacity of the resin was estimated by picric acid method. The side
reactions like the formation of tertiary amine and quaternary ammonium salt of resin can
be eliminated by the use of large excess of amine.
Stepwise incorporation of amino acids
(a & b)
(a) R=CH, (b) R=CzH5
Scheme. 7-3. Preparation 'and use of 3-nitro 4-N-alkylaminomethyl benzamidomethyl PS-BDODMA resin
7.2.f. Synthesis of protected peptide acids, amides and alkyl amides
The synthetic utility of the resins 4-chloromethyl 3-nitro PS-BDODMA,
4-aminomethyl 3-nitro PS-BDODMA, 4-bromomethyl 3-nitro benzamidomethyl
PS-BDODMA, 4-aminomethyl 3-nitro benzamidomethyl PS-BDODMA, N-methyl
aminomethyl 3-nitro benzamidomethyl PS-BDODMA and N-ethyl aminomethyl 3-nitro
benzamidomethyl PS-BDODMA are illustrated with the synthesis of some representative
peptide acids, amides and N-alkyl amides. The cesium salt method was used for the
attachment of the C-terminal amino acid to the 4-chloromethyl 3-nitro resin. The
peptides were assembled on the resin using the pre-formed HOBt active ester of Boc or
Fmoc-amino acids. For resins 4-aminomethyl 3-nitro PS-BDODMA, 4-aminomethyl
3-nitro benzamidomethyl PS-BDODMA, N-methyl aminomethyl 3-nitro
benzamidomethyl PS-BDODMA and N-ethyl aminomethyl 3-nitro benzamidomethyl
PS-BDODMA, the C-terminal amino acid was attached using its pre-formed HOBt
active ester. After the incorporation of amino acids in the target sequence, the peptides
were cleaved from the resin by photolysis carried out in TFElDCM solution. The
peptides were characterized by HPLC and amino acid analysis. The following peptide
acids, amides and N-alkyl amides were synthesized by photolysis.
1. Boc-NH-Gly-Leu-Ala-Leu-Ala-Gly
2. Boc-NH-Leu-Ala-Gly-Leu-Ala-Gly
3. Boc-NH-Gly-Ile-Cys(Acm)-Pro
4. Fmoc-NH-Ile-Leu-Ala-Gly
5. ~ m o c - ~ ~ - ~ e u - ~ s ~ ( ~ ~ u ~ - ~ e u - G l ~ - A l a - G l ~
6. Ile-Ala-Val-Gly-NH2
7. Boc-NH-Pro-Val-NHZ
8. Boc-NH-Gly-Phe-Pro-NH2
9. Boc-NH-Leu-Ala-Gly-Val-NH2
10. Boc-NH-Ala-Gly-Leu-Ile-Gly-N&
1 1. Fmoc-NH-Ala-Gly-Leu-Ile-Gly-NH2
12. Boc-NH-Leu-Ala-Val-NHMe
13. Boc-Val-Leu-Ala-Val-NHMe
14. Boc-NH-Leu-Ala-Val-NHEt
15. Boc-NH-Val-Leu-Ala-Val-NHEt
7.2.g. Mechanism of photolytic cleavage
The mechanism of photolytic cleavage of nitro benzyl and related system is well
documented The reaction involves a light induced internal oxidation-reduction reaction
of aromatic nitro compounds containing a carbon-hydrogen bond ortho to the nitro group.
The reaction follows the reduction of nitro group to the nitroso group when oxygen is
inserted to the carbon-hydrogen bond at the 2-position resulting in the oxidation of <Hz-
group to -CHO group.'2 (Scheme. 7- 4)
Scheme. 7- 4. Mechanism of photolytic cleavage of peptide from the resin
eoo eoo rooo MassICharge
Fig. 7.5. MALDI TOF MS of (a) Boc-NH-Gly-Leu-Ala-Leu-Ala-Gly (b) ~ m o c - N H - ~ e u - ~ s ~ ( ~ ~ u ' ) - ~ e u - ~ l ~ - ~ l a - ~ l ~
All the observations illustrate the applicability of the modified PS-BDODMA
resin as a photo-removable polymeric support for solid phase synthesis of fully protected
peptide acids, peptide amides and peptide alkyl amides. This method has a unique
advantage of avoiding the formation of diketopiperazine and the unwanted side reactions
in trans-esterification procedure, thus increasing the overall yield of the peptide. The
peptides obtained are in fully protected form, which can be applicable for segment
condensation. Photolytic cleavage can be conveniently employed for peptides containing
sterically hindered C-terminal amino acid like Val, Ile etc.
7.3. Experimental
7.3.a. Preparation of 4-Chloromethyl f nitro PS-BDODMA resin
Chloromethyl PS-BDODMA resin (1 g, 0.68 mmol) was added to fuming nitric
acid (100 mL) in portion wise at -10 OC over 30 min. The suspension was kept for 2 h at
-10 "C with occasional swirling. The reaction mixture was then poured in to a beaker
containing ice-cold water. The resin was collected by filtration and washed with ice-cold
water until the washings were neutral. The resin was washed thoroughly with DCM (6 x
30 mL), methanol (6 x 30 mL) and ether (6 x 30 mL) and dried under vacuum.
7.3.b. Preparation of 4-Aminomethyl 3-nitro PS-BDODMA resin
4-Chloromethyl 3-nitro PS-BDODMA resin (500 mg, 0.66 mmol) was suspended
in NMP (20 mL), potassium phthalimide (1.2 g, 6.6 mmol) was added and the reaction
mixture was kept at 1 10- 120 "C with occasional shaking for 12 h. The resin was filtered,
washed with NMP (6 x 25 d ) , dioxane (6 x 25 mL), EtOH (6 x 25 d ) , MeOH (6 x
25 mL) and dried under vacuum. The dried resin was then suspended in EtOH (100 mL)
and refluxed with hydrazine hydrate (0.33 mL, 6.6 mmol) for 8 h, the resin was collected
by filtration, washed with EtOH (6 x 25 mL) and MeOH (6 x 25 mL). The resin was
then dried in vacuum. Amino capacity of the resin=0.65 mmol/g.
7.3.c. Preparation of 4-Bromomethyl benzoic acid
p-Toluic acid (13.6 g, 100 mmol) was suspended in dry benzene (100 mL) and a
mixture of benzoyl peroxide (200 mg) and N-bromosuccinimide (17.8 g, 100 mmol)
were added and refluxed for 24 h. The solvent was removed under vacuum and the
residue was suspended in boiling water (100 mL) for 10 min. The precipitate was filtered
and washed with boiling water (6 x 25 mL). The crude product was recrystallized from
hot MeOWDCM mixture.
mp=227.5 "C
IR W r ) : 2800-2400 cm'l, 1682 cm-' (COOH), 1556 cm-', 690 cm-'(aromatic)
7.3.d. Preparation of 4-Bromomethyl3-nitro benzoic acid
Add portion wise 4-bromomethyl benzoic acid to hming nitric acid (100 mL) at
-10 OC over 30 min. The suspension was stirred for 2 h at -10 OC. The reaction mixture
was then poured in to a beaker containing ice-cold water. The precipitate formed was
collected by filtration and washed with ice-cold water until the washings were neutral.
The precipitate was dried and recrystallised from petroleum ether. mp=125-126 "C.
IR (KBr): 2800-2300 cm-', 1688 cm-' (COOH), 1592, 700 cm-' (aromatic), 1542 cm-',
13 10 cm" (nitro).
7.3.e. Preparation of 4-Bromomethyl3-nitro benzamidomethyl PS-BDODMA resin
To the pre-swollen aminomethyl PS-BDODMA resin (1 g, 0.67 mmol) in DCM, a
mixture of 4-bromomethyl3-nitro benzoic acid ( mg, 2 mmol), HOBt (270 mg, 2 mmol),
HBTU ( 760 mg, 2 mmol) and DIEA (350 pL, 2 mmol) were added and kept at room
temperature. Afier 1 h, the resin was filtered, washed with DCM (6 x 10 mL) and a
second coupling was performed. The resin was collected by filtration, washed with DCM
(5 x 20 mL), DMF (5 x 20 rnL) and MeOH (5 x 20 mL). Bromine content of the
resin = 0.64 mmollg.
IR (KBr): 1650 cm-' (NHCO), 1340 cm-', 1540 cm-' (NOz)
7.3.f. Preparation of 4-Aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin
4-Bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin (500 mg, 0.32 mmol)
was suspended in NMP (20 mL), potassium phthalimide (550 m g, 3.2 rnmol) was added and
the reaction mixture was kept at 110-120 OC with occasional shaking for 12 h. The resin was
filtered, washed with NMP (6 x 25 mL), dioxane (6 x 25 mL), EtOH (6 x 25 mL), MeOH (6 x
25 mL) and dried under vacuum. The dried resin was then suspended in EtOH (100 rnL) and
refluxed with hydrazine hydrate (0.15 mL, 3.2 mmol) for 8 4 the resin was then colkcted by
filtration, washed with EtOH (6 x 25 mL) and MeOH (6 x 25 mL). The resin was then dried in
vacuum. Amino capacity of the resin= 0.63 mmollg.
7.3.g. Preparation of 4-Methyl aminomethyl 3-nitro benzamidomethyl PS- BDODMA resin
4-Bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin (100 mg,
0.064 mmol) was suspended in DCM (20 mL) in a stoppered bottle and was kept at
0-5 'C. Dry methyl amine gas was bubbled through the reaction mixture for 12 h. The
reaction bottle was stoppered well and shaken for 12 more hours at room temperature.
The resin was filtered, washed with DCM (3 x 2 mL x 3 min), THF (3 x 20 mL x
3 min), water (3 x 20 mL x 3 min), MeOH (3 x 20 rnL x 3 min) and dried in vacuum.
Amino capacity of the resin = 0.6 mmollg.
IR (KBr): 1650 cm-' (NHCO), 1340 cm-l, 1530 cK1 (NO& 3400 cm-I (NH)
7.3.h. Preparation 4-Ethyl aminomethyl 3-nitro benzamidomethyl PSBDODMA resin
4-Bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin (100 mg,
0.064 mmol) was treated with dry ethyl mine. The above protocol was used for the
synthesis. Amino capacity of the resin= 0.6 mmol/g.
IR (KBr): 1652 cm" (NHCO), 1340 cm", 1530 cm-' (NOz), 3440 cm-' (NH).
7.3.i. General synthetic protocol for peptides using solid supports.
1. Synthesis of peptides using Boc-amino acids
Peptide synthesis was carried out manually in a silanised glass reaction vessel
with a glass filter at one end and a calcium chloride guard tube at the other end. The
C-terminal amino acid was attached to the resin by cesium salt method. The first
Boc- amino acid attached resin was taken in the reaction vessel and swelled in DCM.
Boc-protection was removed by using 30% TFA/DCM and neutralization was achieved
by 5% DIEA/DCM. The resin was washed thoroughly with DCM and D m . Boc-amino
acid (3 equiv corresponding to the halogen capacity of the resin) along with HOBt
(3 equiv), HBTU (3 equiv) and DIEA (3 equiv) were added and kept for 1 h at room
temperature. The coupling procedure was proceeds till the target peptide sequence was
completed. Each coupling was performed twice for 100% reaction. The coupling
reactions were monitored by Kaiser's test.
2. Synthesis of peptides using Fmoc-amino acids
The Fmoc-protection from the C-terminal amino acid attached resin was removed
by 20% piperidinelDMF. The resin was washed thoroughly with DMF and the
subsequent Fmoc-amino acids (3 equiv) were coupled by using HOBt (3 equiv) and
HBTU (3 equiv) in presence of DIEA (3 equiv). Each coupling steps were monitored by
ninhydrin test.
7.3.1. General procedure for photolysis
The peptidyl resin was suspended in a mixture of 30% TFE in DCM (100 mL)
and placed in an immersion type photochemical reactor. The suspension was degassed
for 1 h with dry nitrogen and irradiated with Philips HPK 125 W medium pressure
mercury lamp at 340-350 nm for 24 h. A solution of CuS04 was circulated throughthe
outer jacket of the photochemical reactor to filter off light waves below 320 nm. After
photolysis, the resin was filtered, washed with ethanol (3 x 25 mL) and DCM (3 x
25 mL). Combined filtrate and washings were evaporated on a rotary evaporator under
reduced pressure The residue was collected and purified by chromatography on a
sephadex G-10 column using suitable solvents.
7.3.k Synthesis of Boc-NH-Gly-Leu-Ala-Leu-Ala-Gly, Boc-NH-Leu-Ala-Gly-Leu- Ala-Gly, Boc-NH-Gly-Ile-Cys(Acm)-Pro.
C-terminal Boc-amino acids of these peptides were attached to 4-bromomethyl
3-nitro benzamidomethyl PS-BDODMA resin (50 mg 0.64 mmol Brlg) by cesium salt
method and the extent of attachment was measured by picric acid method. After
Boc-deprotection with 30% TFA in DCM and neutralization with 5% DIEA in DCM the
remaining amino acids (2.5 mmol excess compared to the amino capacity) in the target
sequences were coupled successively as their HOBt active ester. The resin was washed
thoroughly with MeOH/DCM (6 x 10 mL), NMP (6 x 10 mL) and DCM (6 x 10 mL).
The peptidyl resin was suspended in TFEIDCM (30% vlv) and photolysed
according to the general procedure described above. The crude peptides were dissolved
in acetic acid-water mixture and eluted through a sephadex G-10 column. The peptidyl
fractions were collected and lyophilized. The purity of the peptides were checked by
HPLC and characterized by amino acid analysis.
Boc-NH-Gly-Leu-Ala-Leu-Ala-Gly (Yield= 13.8 mg, 72 %).
Amino acid analysis: Gly, 2.1 (2); Leu, 1.92 (2); Ala, 2.03 (2).
MALDI TOF MS: m/z 600.8 [(M+H)+, 100%], C27hgN609, requires M' 599.7.
Boc-MI-Leu-Ala-Gly-Leu-Ala-Gly (Yield= 14.4 mg, 75 %).
Amino acid analysis: Gly, 2.0 (2); Leu, 1.98 (2); Ala, 1.95 (2).
Boc-NH-Gly-Ile-Cys(Acm)-Pro (Yield= 11.7 mg, 68 %).
Amino acid analysis: Gly, 1.0 (I); Ile, 0.87 (1); Pro, 0.87 (1); Cys, 0.78 (1).
73.1 Synthesis of Fmoc-NH-&Leu-Ah-, F m o c - N B - L e u - ~ s ~ ( ~ ~ u ~ ~ e n - ~ ~ ~ - ~ l a ~ ~
C-terminal amino acid Boc-Gly (35 mg, 0.2 mrnol) was attached to the resin (100 mg
0.64 mmol Brlg) by cesium salt method and the extent of incorporation was estimated by
picric acid method. The resin was washed thoroughly with DCM and Boc-protection was
removed by 30% TFNDCM (10 d ) , the resin was washed thoroughly with DCM (6 x
10 d ) , NMP (6 x 10 d ) and DMF (6 x 10 d ) . Fmoc-Ala was attached to the resin
by HOBVHBTU method. After removing Fmoc protection with 20% piperidine /Dm, it
was washed with DMF and the remaining amino acids (Fmoc-protected) were assembled
till the target sequences were formed.
The resins were suspended in TFE/DCM (30% vtv) mixture and photolysed
according to the general procedure. The crude peptides were dissolved in 5% acetic
acidtwater and purified by passing through a sephadex G-10 column. The purity of the
peptides was monitored by HPLC.
Fmoc-NH-Ile-Leu-Ala-Gly (Yield= 13 mg, 68%)
Amino acid analysis: Ile, 0.93 (1); Leu, 1.03 (1); Ala, 1.1 (1); Gly, 1.04 (1).
~ m o c - ~ e u - ~ s ~ ( ~ ~ u ' ) - ~ e u - ~ l ~ - A l a - G l ~ (Yield= 17 mg, 65%)
Amino acid analysis: Leu, 2.1 (2); Asp, 0.91 (1); Gly, 2.12 (2); Ala, 0.98 (1).
MALDI TOF MS: d z 822.9 [(M+H)+, 100°h], C ~ ~ H S ~ N ~ O I ~ , requires ~ + 8 2 1 . 2 .
7.3.m. Synthesis of Ile-Ala-Val-Gly-NHt
Fmoc-Gly (45 mg, 0.15 mmol) was attached to the 4-aminomethyl 3-nitro
benzamidomethyl PS-BDODMA resin (75 mg, 0.05 mmol) by HOBt active ester
method Afier Fmoc-deprotection, the remaining amino acids in the target sequences
were incorporated by Fmoc-solid phase synthetic strategy. Fmoc-protection from the
peptidyl resin was removed by 20% piperidine1DMF (10 d ) , washed with DMF (6 x
10 d ) , DCM (6 x 10 mL) and dried. The peptidyl resin was suspended in TFE/DCM
mixture and photolysed. The crude peptide was passed through a sephadex G-10 column
and the purity was checked by HPLC.(Yield= 11 mg, 69%)
Amino acid analysis: Ile, 0.93 (1); Ala, 1.05 (1); Val, 0.92 (1); Gly, 1.1 (1).
7.3.11. Synthesis of Boc-NH-Pro-Val-NHz, Boc-NH-Gly-Phe-Pro-NHz, Boc-Leu-Ala- Gly-Val-NHz, Boc-Ala-Gly-Leu-Ile-Gly-NH2
The C-terminal amino acid (0.15 mmol) was attached to the 4-aminomethyl
3-nitro benzamidomethyl PS-BDODMA resin (75 mg, 0.05 mmol) by using HOBt
(20 mg, 0.15 mmol), HBTU (57 mg, 0.15 mmol) in presence of DIEA (26 pL). After the
Boc-deprotection with 30% TFAfDCM (10 mL), the remaining amino acids in the target
sequences were coupled by Boc-solid phase synthetic protocol. The peptidyl resin was
suspended in TFElDCM mixture and photolysed. The crude peptide obtained was
dissolved in 5% acetic acidlwater and passed through a sephadex G-10 column. The
peptidyl fractions were collected and lyophilized. The purity of the peptides was
monitored by HPLC.
Boc-NH-Pro-Val-NH2 (Yield= 10.4 mg, 74%)
Amino acid analysis: Pro, 0.91 (1); Val, 1.0 (1).
Boc-NH-Gly-Phe-Pro-NHz (Yield= 15 mg, 80%)
Amino acid analysis: Gly, 0.96 (1); Phe, 1.0 (1); Pro, 0.98 (1).
Boc-Leu-Ala-Gly-Val-Nfi (Yield= 14.8 mg, 71%)
Amino acid analysis. Leu, 1.0 (1); Ala, 0.95 (1); Gly, 1.02 (1); Val, 0.97 (1).
Boc-Ala-Gly-Leu-Ile-Gly-N& (Yield= 17.5 mg, 73%)
Amino acid analysis: Ala, 1.12 (1); Gly, 2.02 (2); Leu, 0.91 (1); Ile, 1.04 (1).
7.3.0. Synthesis of Fmoc-NH-Ala-Gly-Leu-Ile-Gly-NH2
The C-terminal Fmoc-Gly (45 mg, 0.15 mmol) was attached to the 4-aminomethyl
3-nitro benzamidomethyl PS-BDODMA resin (75 mg, 0.05 mmol) by using HOBt (20 mg,
0.15 mmol), HBTU (57 mg, 0.15 mmol) in presence of DIEA (26 pL). The extent of
attachment was determined by measuring the OD of the adduct formed by treating a definite
amount of amino acid attached resin with 20% piperidine in DMF (10 mL). The remaining
amino acids were coupled by Fmoc-synthetic strategy. Atter the synthesis the peptidyl resin
was suspended in TFEiDCM and photolysed. The crude product was eluted through a
sephadex G-10 column. The peptidyl fractions were collected and lyophilized. The purity of
the peptide was checked by HPLC.(Yield= 22.5 mg, 75%)
Amino acid analysis: Ala, 1.06 (1); Gly, 1.92 (2); Leu, 1 .O1 (1); Ile, 1.1 (1).
7.3.p. Preparation of Boc-NH-Val-N(CH3)-Resin
4-Methyl aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin (100 mg,
0.06 mmol) was suspended in NMP (10 mL). The HOBt active ester of Boc-Val (61 mg,
0.18 mmol) was added to the reaction mixture and kept for 1 h with occasional shaking. The
resin was collected by filtration and washed with NMP (6 x 10 mL), DCM (6 x 10 mL),
MeOH (6 x 10 mL) and ether (6 x 10 mL). Amino capacity= 0.58 mmol/g.
7.3.q. preparation of Boc-NH-Val-N(CzH5)-resin
4-Ethyl aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin (100 mg,
0.06 mrnol) was suspended in NMP (10 mL). The HOBt active ester of Boc-Val(61 rng,
0.18 mmol) was added to the reaction mixture and kept for 1 h with occasional shaking.
The resin was collected by filtration and washed with NMP (6 x 10 mL), DCM (6 x 10 mL),
MeOH (6 x 10 mL) and ether (6 x 10 mL). Amino capacity= 0.57 mmoYg
7.3.r. Synthesis of Boc-NH-Leu-Ala-Val-NHMe, Boc-NH-Val-Leu-Ala-Val-NHMe, Boc-NH-Val-Leu-Ala-Val-NHEt, Boc-NH-Leu-Ala-Val-NHEt
Boc-Val was attached to the resins N-methyl aminomethyl 3-nitro
benzamidomethyl PS-BDODMA (50 mg, 0.03 rnmol) and N-ethyl aminomethyl 3-nitro
benzamidomethyl PS-BDODMA (50 mg, 0.03 mmol) by using HOBt (13.5 mg,
0.09 mmol), HBTU (37.9 mg, 0.09 mmol) and DIEA (14 pL). The successive amino
acids (3 equiv) were coupled and finally the peptides were detached kom the resins by
photolysis. The purity of the peptides was checked by HPLC.
Boc-NH-Leu- Ala-Val-NHMe
Amino acid analysis: Ala, 1.0 (1); Leu, 0.9 (1); Val, 1.12 (1).
Boc-NH-Val-Leu-Ala-Val-NHEt
Amino acid analysis. Ali, 1.0 (1); Leu, 1.21 (1); Val, 1.98 (2).
References
1. Stimson, E. R.; Meinwald, Y. G.; Montelione, G. T.; Scheraga, H. A. Inf. J Peptide Protein Res. 1986, 27, 569.
2. Mammi, S.; Goodman, M. Int. J. Peptide Protein Res. 1986,28, 29.
3. Manning, M. J. Am. Chem. Soc. 1968,90, 1348.
4. Matsueda, G. R.; Stewart, J. M. Peptides 1970, 2, 45.
5. Pietta, P. A,; cavello, P. F.; Takahashi, K.; Marshall, G. R. J. Org. Chem. 1974,39,44.
6 . Orlowski, R. C.; Walter, R.; Winkler, D. J. Org. Chem. 1976,41,3701.
7. Penke, B.; Rivier, J. J. Org. Chem. 1987, 52, 1197.
8. Rich, D. H.; Gunvara, S. K. J. Am. Chem. Soc. 1975,97,1575.
9. Pillai, V. N. R. Synthesis 1980, 1
10. Pillai, V. N. R. "Organic Photochemishy" Padwa, A Eds., Vo1.9 ,Marcel Dekker, New York, 1987, pp.225-312.
11. Kumar, K. S.; Pillai, V. N. R. Tetrahedron, 1999, 55,10437.
12. Patchornik, A,; Amit, B.; Woodward, R. B. J Am. Chem. Soc. 1970,92, 6333.
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