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Description  |
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This invention relates to the preparation of polypeptides by solid phase
synthesis wherein 3-nitro-4-amino methyl benzoyl amide resin and
3-nitro-4-bromomethyl benzoyl amide resin are prepared from
3-nitro-4-bromomethyl benzoic acid, and from which the protected peptide
acids or amides can be removed by photolysis in high yield, without
destroying acid base labile protected groups or aromatic amino acids.
The C-terminal amide group is present in several biologically active
peptides. Such peptides have been synthesized by solid phase methods (R.
B. Merrifield, J. Am. Chem. Soc. 85 2149 (1963), in which the C-terminal
amide of the protected peptide was removed from the resin by aminolysis or
by transesterification (H. C. Beyerman, H. Hindricks and E. W. B. deLeer,
J.C.S. Chem. Comm. 1668 (1968). However, these conditions necessitate the
use of side chain ester protecting groups which are resistant to
aminolysis or transesterification and therefore restrict the type of acid
labile protecting groups that can be used to synthesize the peptides.
Furthermore, peptides with hindered C-terminal residues such as valine in
secretin, can be difficult to remove from the resin. Thus new and improved
methods for preparation and removal of protected peptides from a solid
phase, as the C-terminal amide, is desirable.
The solid phase method of peptide synthesis introduced by Merrifield
(supra) is an effective method for the rapid synthesis of peptide.
However, the products prepared by this method are often difficult to
purify. Impurities, such as failure sequences caused by changes in the
physical-chemical properties of the polymer, accumulate during stepwise
synthesis and can be difficult to remove. It has been suggested that a
more homogeneous final product might be isolated by coupling pure
protected peptide fragments onto the solid support. Failure sequences
formed during synthesis, using fragment coupling, would differ
substantially from the desired product and would be more readily removed
by purification. If the fragment coupling is to become generally useful, a
convenient method for preparing protected peptide acids and amides, such
as Boc-peptide acids and amides, is needed. As described, these
derivatives have been prepared by solid phase synthesis but either
transesterification or hydrazinolysis reactions were required to remove
the Boc-protected peptides from the resin.
It is an object of this invention to provide a method whereby protected
peptide acids and amides can be removed by photolysis under conditions
which do not destroy aromatic residues, or cleave acid or base labile
protected groups, and it is a related object to produce and to provide
methods for producing new intermediates for use in the same as well as new
and improved polypeptides which result from same.
We have succeeded in the removal of protected amino acids and peptides by
photolysis from ortho-nitro chloromethyl polystyrene resins, as reported
in J. Chem. Soc. Chem. Comm. 610-11 (1973). Using this method, the
purified protected tripeptide Boc-Ser (Bzl)-Tyr (Bzl) -Gly
##STR1##
was obtained in 62% yield, based on starting Boc-Glycin resin. However, by
this method, the synthesis of longer peptides was not successful.
By continuation of the investigation, we have found that the deficiencies
can be overcome by the use of 3-nitro-4-bromomethyl benzoyl amide
polystyrene resin instead of the 3-nitro-4-chloromethylated derivative.
This is believed to result from the fact that the
3-nitro-4-bromo-methylated benzoyl amide polystyrene resin swells more in
non-polar solvents than the more polar 3-nitro-4-chloro-methylated
polystyrene resins and that the lower extent of swelling experienced by
the latter reduces penetration of the solvent and rates of reaction,
thereby to interfere with the preparation of longer chain protected
polypeptides.
The invention will now be described with reference to the synthesis of
3-nitro-4-bromomethyl benzoyl amide resin; the synthesis by the addition
of peptide onto the resin via Boc-amino acids or amides, for solid phase
peptide synthesis to form polypeptides which are capable of being
separated in a purified state by photolysis. The description will use, for
purposes of illustration, the synthesis of protected fragments of LH-RH,
but it will be understood that other polypeptides, acids and/or amides can
be produced by the method described including the coupling in various
combinations of Boc-amino acids or peptides including Boc-Pro, Boc-Arg
(Tos), Boc-Leu, Boc-Gly, Boc-Tyr (Pzl), Boc-Ser (Bzl), Boc-Ala, Boc-Val,
Boc-Ileu, Boc-Phe, Boc-Hypro, Boc-Thr (Bzl), Boc-Cys (Bzl), Boc-Met,
Boc-Asp (Bzl), Boc-Glu (Bzl), Boc-Lys (Bzl) and Boc-His (Bzl).
The sequence of reactions, to be described in the following examples, for
the synthesis of the 3-nitro-4-bromomethyl benzoyl amide resins may be
represented by the following:
EXAMPLE 1
Preparation of 3-nitro-4-bromomethyl benzoic acid 4
Briefly described, p-toluic acid 2 is refluxed with N-bromo-succinimide and
benzoyl peroxide in dry benzene to give .alpha.-bromo-p-toluic acid 3
which, upon reaction with nitric acid, is converted to
3-nitro-4-bromomethyl benzoic acid 4. This is believed to be a new
compound and represents a parent compound in the synthesis forming the
subject matter of this invention. At slightly higher temperatures, above
-10.degree. C, the corresponding dinitro derivative 4a predominates.
DETAILED PROCEDURE
Benzoyl peroxide (0.2 g) and N-bromosuccinimide (17.8 g, 100 mmol)
(recrystallized from hot H.sub.2 O) were added to a suspension of p-toluic
acid (13.6 g, 100 mmol) (recrystallized from CHCl.sub.3 /MeOH) in dry
benzene (100 ml). The mixture was heated at reflux for 24 hours. Removal
of the solvent in vacuo gave a white residue which was suspended in 100 ml
of boiling H.sub.2 O, collected by filtration and washed with boiling
H.sub.2 O (4 .times. 100 ml). The crude product was dried and
recrystallized from hot MeOH to give pure acid (17.5 g, 81.4%); m.p.
224.degree.-226.degree., i.r. (Nujol) 2800-2400, 1690 (COOH), 1560
cm.sup.-1 (Aromatic); nmr (CDCl.sub.3, DMSO-d6) .delta. 4.61 (s,2) 7.8
(8.4 J=8Hz), 10.4 (s, 1) UVmax (meOH) 232 m.mu. (t=1.32 .times. 10.sup.4),
285 m.mu. (t=1166). Anal. Calcd for C.sub.8 H.sub.7 BrO.sub.2 : C, 44.68;
H, 3.28; Br 37.15; found: C, 44.50; H, 3,18; Br, 37.02.
The bromoacid 3 (11.8 g) was added in portions over 0.5 hour to 100 ml of
90% HNO.sub.3 (white fumming) at -10.degree. C. The suspension was stirred
at -10.degree. for an additional 2 hours when the solution became clear
orange. This solution was poured onto crushed ice. The product was
collected by filtration, washed with ice cold H.sub.2 O (3 .times. 50 ml)
until the washings were neutral. Drying in vacuo followed by
crystallization twice from CH.sub.2 Cl.sub.2 /hexane gave pure nitro acid
4 (11.02 g, 85%): m.p. 125-126, i.r. (Nujol) 2800-2300, 1690, 1610 (COOH),
1600 (Aromatic), 1540, 1300 cm.sup.-1 (NO.sub.2); nmr (CDCl.sub.3,
DMSO-d6) 4.9 (S,2), 7.8 (d,1), 8.2 (dd,1), 8.6 (d,1) 10.8 (s,1); Rf(5)
0.55; Rf (1) 0.82; UVmax (CH.sub.3 OH) 227 m.mu. (t = 2.13 10.sup.4) 305
m.mu. (t 4.1 .times. 10.sup.3).
Anal. Calcd for C.sub.8 H.sub.6 NBrO.sub.4 : C, 36.95; H, 2.32; N, 5.38; Br
30.73; Found C, 37.16; H, 2.46; N, 5.47; Br 30.97.
EXAMPLE 2
Preparation of 3-nitro-4-bromomethyl benzoyl polystyrene resin 7
Briefly described, the amino methyl polystyrene resin 6 is produced by
reaction of the chloro methylated polystyrene resin with anhydrous ammonia
in methylene chloride. Other solvents can be used but when reacted in the
presence of methylene chloride, the degree of cross linking of the resin
is relatively unchanged while, at higher temperatures and in the presence
of different solvents, such as methanol, methanoldioxane or dimethyl
formamide, while the ammonation takes place, the degree of resin cross
linking is increased. Treatment of the amino resins with the nitro acids 4
and dicyclohexylcarbodiimide in dimethylformamide is followed by
acetylation of the residual amino group by reaction with acetic anhydride
and diisopropyl ethylamine.
DETAILED PROCEDURE
Aminomethylated polystyrene resin 6 (1.0 g 0.4 mmol NH.sub.2 /g) was added
to a solution of the nitro-acid 4 (0.52 g, 2 mmol) and
dicyclohexylcarbodiimide (DCC) (0.42 g, 2 mmol) in dimethylformamide (DMF)
(10 ml). The suspension was stirred at room temperature for 18 hours and
filtered. The resin was washed with MeOH, CH.sub.2 Cl.sub.2, MeOH (3
.times. 20 ml for 1 minute each), dried in vacuo and placed again in a
solution of 0.260 g of nitro acid 4 and 0.206 g of DCC in 10 ml DMF. After
the same workup (vide supra), the resin was suspended in CH.sub.2 Cl.sub.2
(25 ml) and acetic anhydride (0.61 g, 6 mmol) and diisopropylethylamine
(0.774 g, 6 mmol) were added. The suspension was stirred at room
temperature for 1 hour, washed with CH.sub.2 Cl.sub.2, MeOH (3 .times. 20
ml for 1 minute) and dried in vacuo to give the desired resin 7 (1.08 g)
ir (KBr) 1600 (NH.sub.2), 1560, 1350 (NO.sub.2) cm.sup.-1. The resin
contained 0.3 mmol/g of bromine and no free amine (Dorman).sup.31 ; 100 mg
of resin swelled to 1.0 mg in dry CH Cl.sub.3.
Anal. Calcd for 0.3 mmol Br.sup.- /g, 0.6 mmol N/g Br, 2.4; N 0.84; Found:
Br 2.08; N 0.70.
The light yellow product contained 0.3 mmol bromine/g resin and no
detectable free amino groups. The high bromine content and correct
nitrogen analysis indicated that little if any alkylation of resin amino
groups by bromomethyl groups had occurred. The nitro resin 7 swells in
chloroform and all other solvents used in solid phase synthesis to the
same extent as does the chloromethylated polystyrene starting material.
Alternatively, the bromine group on the 3-nitro-4-bromomethyl benzoic acid
4 is replaced by an amino group which is then protected by a Boc group for
resin coupling in accordance with the following equation:
##STR3##
Compound 5, which is also believed to be new, reacts in the same way with
the methylamino resin (6) to produce the Boc protected derivative in
accordance with the following equation:
##STR4##
EXAMPLE 3
Preparation of 3-nitro-4-aminomethyl benzoic acid 4b
Dry liquid ammonia (15 ml) was added to 2.0 g (7.7 mmol) of
3-nitro-4-bromomethyl benzoic acid 4 in a pressure resistant bottle. The
solution was stirred at 0.degree.-5.degree. for 24 hours. The bottle was
then cooled to -78.degree. C, opened and the ammonia evaporated. The
residue was suspended in methanol, collected by filtration, washed with
methanol and dried to give acid 4b: m.p. 235.degree.-237.degree. C, yield
1.089 g (72%).
EXAMPLE 4
Preparation of 3-nitro-4-t-butoxycarbonyl (Boc) aminomethyl benzoic acid 5a
Triethylamine (0.3 ml, 2 mmol) and tert-butyl oxycarbonyl azide (0.15 ml, 1
mmol), were added to a solution of acid 4b (0.196 g, 1 mmol) in
dimethylsulfoxide (6 ml). The reaction mixture was stirred at 25.degree. C
for 18 hours. The solution was diluted with water (10 ml) and washed with
ether (10 ml). The aqueous layer was cooled to 0.degree. C and solid
citric acid was added until the pH was between 2 and 3. The solution was
washed three times with ethyl acetate, the ethyl acetate layers were dried
and evaporated to dryness in vacuo to give acid 5a. Acid 5a was dissolved
in methylene chloride (5 ml) to which was added an etheral solution of
dicyclohexylamine (1 ml/3 ml ether). The salt 5b was crystallized from
ether to give pure 5b: 0.4 g, 85%; m.p. 205.degree.-207.degree. C.
Anal. Calculated for C.sub.25 H.sub.39 N.sub.3 O.sub.6 ; C 62.87, H 8.23, N
8.80. Found: C, 62.61, H 8.46 and N 8.55.
EXAMPLE 5
Preparation of 3-nitro-4-t-butoxycarbonyl (Boc) aminomethyl benzoyl amide
resin 7a
To a solution of acid 5a (0.889 g, 3 mmol) in 10 ml DMF, was added 0.609 g
(3 mmol) DCC in 10 ml DMF. The amino resin 5 g, 0.3 mmol NH.sub.2 /g) was
added to the solution and the suspension stirred for 18 hours. The resin
was filtered, washed three times with 20 ml portions each of DMF,
methanol, methylene chloride, methanol before drying in vacuo to yield
resin 7a: 0.3 mmol amine group/g resin after the t-Boc group is removed.
It will be apparent from the above that the term "Boc", as used herein,
refers to t-butoxycarbonyl protective group. Other protective groups, well
known to the art, may be used instead of the t-butoxycarbonyl (BOC) groups
for protection of the peptide acids and amides.
Similarly, starting with the resin represented by the compound 7a,
polypeptides, such as the C-terminal peptide amide LH-RH can be produced
by solid phase synthesis, followed by removal of the purified protected
polypeptide by photolysis, in accordance with the following equations:
##STR5##
The attachment of one or more Boc amino acids to the resins 7 or 7a by
solid phase synthesis and release of the fragment from the resin by
photolysis are represented by the following equations:
##STR6##
The compound 9a is believed to exist as an intermediate that is formed in
response to irradiation of the Boc protected amino (peptide) resin.
The attachment of the Boc-amino acid to the nitro resin 7 is effected by
heating under reflux with triethylamine (TEA) or preferably with
diisopropylethylamine, in ethyl acetate. Less quaternization of the resin
is experienced with the use of diisopropylethylamine. No racemization has
been detected either during attachment or during photo-chemical removal of
the amino acid derivative by photolysis. Boc-amino acids are attached to
resin 7a using a suitable coupling reagent such as DCC.
The Boc-amino acids and peptides are released from the resin 7 or 7a by
photolysis in methanol or other short chained C.sub.1 to C.sub.5 alcohol,
under anaerobic conditions, preferably in the absence of oxygen which has
the tendency to reduce yield as well as purity of the product.
In accordance with the practice of this invention, any number or any
combination of amino acids or amides (peptides) can be attached to the
resins 7 or 7a by solid phase synthesis, such as described for the
attachment of a single Boc protected amino acid or amide to the resin, and
then the combination of multiple amino acids or amides, in block
arrangement, or in any arrangement or number desired, can be removed from
the resin by the described method of photolysis to produce the formed
polypeptide. With the Boc group or other protective group present,
multiple polypeptides, such as in combinations of 10 + 10 + 10 + 10 can be
joined with subsequent separation of the combination from the resin for
photolysis to produce purified tailor-made enzymes of the type which have
been the subject of extensive research by the most highly skilled in the
art.
The example: Boc peptide chains of 10 amino acids can be split off by
photolysis from the resin, in accordance with the practice of this
invention, for subsequent addition by the described solid phase synthesis
into a compound 7a or 8 with 10 or more protected amino acids to produce a
purified Boc protected polypeptide chain of some 20 peptides. Upon
separation by photolysis, this chain can be added for combination with
another resin chain whereby 10, 20, 30, 40, 50 and more amino acids or
amides of various types and in various combinations can be produced in a
purified form upon separation by photolysis from the nitro resin.
The following equations will further illustrate the solid phase synthesis
and separation by photolysis for buildup of the polypeptide chain in the
manner described.
##STR7##
15a' is produced by the procedure illustrated by equation 15c except that
7a is used instead of 7.
The high yields of purified polypeptide isolated by photolysis indicates
that the hindered amino acid derivative can be removed efficiently by
photolysis under mild conditions. In addition to the Boc protective
groups, the O-benzyl group on Ser, Tyr, Asp and Glu, and the Tos group on
Arg and His are not removed by photolysis but can be removed from the
separate hindered polypeptides by reaction with sodium in liquid ammonia,
or other methods.
The preparation of polypeptides, in accordance with the procedures of this
invention, by solid phase synthesis, and the separation by photolysis will
now be illustrated by the following representative examples.
EXAMPLE 6
Solid phase synthesis
In general, the solid phase synthesis was carried out on a Beckman model
990 Peptide synthesizer using the following procedure: (1) deprotection
was achieved by two successive washes (5 min and 30 min) of 25% TEA in
CH.sub.2 Cl.sub.2 which contained 1 mg/ml of indole; (2) two equivalents
of each Boc-amino acid per equivalent Boc-glycine resin was used; (3) a
second coupling of each amino acid in 50% DMF/CH.sub.2 Cl.sub.2 was
performed; (4) the resin was washed with ethanol and dried with nitrogen
at the end of each synthesis.
EXAMPLE 7
Removal of protected peptides from the nitro resins
A suspension of resin in anhydrous MeOH or EtOH was placed in a flask
surrounded by a jacket containing a 40% CuSO.sub.4 solution. Dissolved air
was removed from the suspension by passing prepurified, O.sub.2 free,
nitrogen for two hours through the solution which was under a slight
vacuum. The suspension was then irradiated at 3500 A for 18-24 hours. Upon
completion of photolysis the suspension was filtered and the resin washed
three times for two minutes with 20 ml portions of each of the following
solvents: EtOH, CH.sub.2 Cl.sub.2, 50% CH.sub.2 Cl.sub.2 -EtOH, EtOH. The
filtrate and washings were evaporated in vacuo. The crude product was
purified by chromatography over a Sephadex LH-20 column (100 g 2.5 .times.
80 cm., flow rate 30 ml/hr, fraction volume of 5 ml each). The elution was
monitored by a dual beam UV detector from Instrumentation Specialties Co.
The products were checked for homogeneity in the following thin layer
chromatograph (tlc) solvent systems: 1 (acidic), n-butanol: acetic acid:
water: ethylacetate (1:1:1:1); 2 (basic), n-butanol: NH.sub.4 OH (7.3); 3,
n-butanol: acetic acid: water (4:1:5 upper layer); 4, chloroform: methanol
(7.5:25); 5, chloroform: methanol (1:1).
Specific Examples
EXAMPLE 8
Preparation of Glycine Resin 8
The 3-nitro-4-bromomethyl resin 7 (4.0 g 0.3 mmol Br.sup.- /g) was added
slowly to a solution of Boc-glycine (0.70 g, 4 mmol) in 20 ml of EtOAc.
Diisopropyl ethyl amine (0.52 g, 4 mmol) was added and the suspension was
gently heated at reflux for 48 hours. The resin was collected by
filtration, washed with EtOAc, MeOH, CH.sub.2 Cl.sub.2, MeOH (3 .times. 25
ml for 2 minutes), and dried in vacuo to give the desired product 8 (4.2
g). The resin contained 0.3 mmol/g of Boc-Glycine and no detectable
bromine (Dorman method).
EXAMPLE 9
Preparation of tetrapeptide 13
The peptide resin 13 was synthesized as described in Example 6 by adding
Boc glycine resin 8 (2.0 g, 0.23 mmol glycine/g), to successive solutions
of Boc-Pro, Boc-Arg (Tos) and Boc-Leu. Amino acid analysis gave
Leu.sub.1.0 Arg.sub.0.92 Pro.sub.0.91 Gly.sub.1.01. A suspension of 1.0 g
of 13a in absolute EtOH was photolyzed and purified as described in the
preceding Example 7. The purified tetrapeptide 13 was obtained in 56%
(0.098 g): m.p. 122.degree.-124.degree.; tlc Rf.sub.1 0.93, Rf.sub.2 0.29,
Rf.sub.3 0.46, Rf.sub.4 0.24; UV max (MeOH) 255 m.mu. (t = 1200); nmr was
consistent with structure; amino acid analysis: Leu.sub.1.04 Arg.sub.1.0
Pro.sub.1.0 Gly.sub.1.03 [.alpha.].sup.27 D - 14 (C 1, CH.sub.3 CO.sub.2
H)
Anal. Calcd. for C .sub.31 H.sub.49 N.sub.7 SO.sub.9 ; C, 53.51; H, 7.10;
N, 14.09; S, 4.61 Found: C, 53.58; H, 7.20; N, 14.28; S, 4.63.
EXAMPLE 10
Preparation of heptapeptide 14
The synthesis of the peptide resin 14a was done following the method of
Example 6 using Boc resin 13a (2.0 g, 0.3 mmol glycine/g). Amino acid
analysis gave Gly.sub.2.0, Ser.sub.1.3, Pro.sub.1.2, Leu.sub.1.1,
Tyr.sub.1.4, Arg.sub.0.8. A suspension of 0.5 g of 14a in absolute MeOh
was photolyzed and purified as described in Example 7. The purified
heptapeptide 14 was obtained in 50% yield (0.057 g): m.p.
135.degree.-138.degree.; tlc Rf.sub.1 0.86; Rf.sub.2 0.44; Rf.sub.3 0.87;
Rf.sub.4 0.91; UV max (MeOH) 264 m.mu. (t = 3300); the nmr was consistent
with the structure. The amino acid composition was Gly.sub.2.0
Ser.sub.1.06 Pro.sub.0.88 Leu.sub.1.12 Tyr.sub.0.87
Arg.sub.0.73,[.alpha.].sup.27 D - 12 (c 1, CH.sub.3 CO.sub.2 H).
Anal. Calcd for C.sub.59 H.sub.78 N.sub.10 SO.sub.14 ; C, 59.88; H, 6.64;
N, 11.84; S, 2.71. Found: C, 59.96; H, 6.64; N, 11.69; S, 2.89.
EXAMPLE 11
Preparation of decapeptide 15
The synthesis of the peptide resin 15 was done according to the method
described in Example 6 by building on Boc glycine resin (2.0 g, 0.3 mmol
Gly/g) or by using resin 14a and adding Boc-Trp, Boc-His (Bzl) and
Pyro-Glu. Amino acid analysis gave Gly.sub.2.0 Ser.sub.0.96 Pro.sub.1.05
Glu.sub.1.05 Leu.sub.1.06 Tyr.sub.1.13 Arg.sub.1.10 BzlHis.sub.0.86. A
suspension of 1.1 g of 13 in absolute ethanol was photolyzed and purified
as described in Example 7. The purified protected decapeptide 15 was
obtained in 64% yield (0.257 g): m.p. 155.degree.-159.degree.; tlc
Rf.sub.1 0.80, Rf.sub.2 0.11, Rf.sub.3 0.75, Rf.sub.4 0.04; UV max (MeOH)
262 m.mu. (t = 6000); the nmr was consistent with the structure;
[.alpha.].sup.27 D - 22 (c 1 (CH.sub.3 CO.sub.2 H); the amino acid
composition was Gly.sub.1.90 Ser.sub.0.92 Pro.sub.1.0 Leu.sub.1.0
Tyr.sub.0.92 Arg.sub.1.02 Glu.sub.1.02.
Anal. Calcd for C.sub.83 H.sub.98 N.sub.16 SO.sub.16 CH.sub.2 Cl.sub.2 : C,
59.56; H, 5.95; N, 13.23; S, 1.89. Found: C, 59.80; H, 5.44; N, 13.20; S,
1.80
EXAMPLE 12
Preparation of tripeptide 17
The peptide resin 17a was synthesized according to the procedure of Example
6 using Boc glycine resin 8 (2.0 g, 0.3 mmol Gly/g) and adding Boc
Tyr(Bzl) and Boc Ser (Bzl). Amino acid analysis gave Ser.sub.1.0
Tyr.sub.0.74 Gly.sub.1.26. A suspension of 0.5 g of 17a was photolyzed and
purified as described in Example 7. The purified tripeptide 17 was
obtained in 50% yield (0.045 g) and was found to be identical.sup.1 to a
sample prepared by solution procedure: m.p. 136.degree.-137.degree.; tlc
Rf.sub.1 0.71, Rf.sub.2 0.85, Rf.sub.3 0.56, Rf.sub.4 0.45; UV max (MeOH)
258 m.mu. (t = 1800); nmr spectrum was consistent with the structure;
amino acid composition: Gly.sub.1.16 Tyr.sub.0.89 Ser.sub.1.0, [.alpha.]
.sup.27 D - 8 (c 1, CH.sub.3 CO.sub.2 H);
Anal. Calcd for C.sub.33 H.sub.39 N.sub.3 O.sub.8 : C, 65.44; H, 6.49; N,
6.94. Found: C, 65.21; H, 6.39; N, 7.08.
It will be apparent from the foregoing that protective peptide acids
suitable for fragment coupling in solution or on a solid support can be
synthesized in good yield, using resin 7 or 7a. The Boc, Bzl, Tosyl
protecting groups are stable to photolysis conditions.
The important configuration for solid phase synthesis and for clean
separation by photolysis is represented by the ortho nitro benzyl group.
The presence of the nitro group in the ortho position is effective to
prevent loss during treatment with TFA and contributes materially to the
separation of a pure derivative in high yield, without the introduction of
contaminants which otherwise require removal and are difficult to remove.
The carboxyl group (--COOH) on the C.sub.1 position of the compounds
represented by equations 2, 3, 4, 4b, 5 can be replaced by an RCOOH in
which R is methyl, ethyl, propyl or other C.sub.1 -C.sub.8 alkyl or
substituted alkyl group although it is preferred to make use of a carboxyl
group attached directly to the C.sub.1 carbon atom.
While the invention has been described with reference to the use of
polystyrene resins, other solid supports can be used such as polyamide
resins, glass beads and the like to which the ortho nitro benzyl group can
be attached.
It will be apparent from the foregoing examples that the protected peptide
segments can be added onto the support in the form of amino amides as in
##STR8##
with the splitting off occurring along the broken line during photolysis,
or in the form of amino acids as in
##STR9##
with splitting off occurring along the broken line in response to
photolysis.
The ortho-nitro benzoic acid group appears to have a unique function in the
described solid phase synthesis in that in addition to its ability to act
as a coupling agent for joining the amino acids and/or peptides onto the
resin or other support, it allows clean separation of the coupled segment
or segments of amino acids and peptides in response to photolysis or
activation by light.
This unique function of the ortho-nitro benzoic acid, as a coupling agent
and as a release agent responsive to light, finds important utility in a
number of other applications. One that appears very interesting and unique
is in the field of agriculture wherein certain herbicides, fungicides,
fertilizers, cytokinin compounds and the like having free hydroxyl, amino
or carboxyl groups can be coupled via the ortho-nitro benzoic acid group
onto a suitable solid support for subsequent application, pre-emergent or
post-emergent, to crops, plants, grasses, trees, and the like. Slow or
calculated release of the coupled component, be it a herbicide, fungicide,
fertilizer, cytokinin or the like, will take place cleanly from the
support in response to activation by daylight. Thus such materials can be
applied in a combined or coupled form and released for availability in
uncombined form over an extended period of time whereby fuller and more
effective and efficient utilization can be made of such materials.
It will be apparent that a large number of other organic materials can be
coupled via the ortho-nitro benzoic acid group onto a suitable support for
subsequent clean and controlled release by photolysis, as in response to
light.
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