terminal Boc group was removed (Table 1, Steps 1-6), and 0.43 g of peptide-resin was removed for hydrogen fluoride cleavage. The secre
tin, , ,._-resin was cleaved for 70 minutes at 0° in the same manner 16-27
as described previously for secretin,__2y After gel filtration and lyophilization, 40 mg white powder was obtained. Amino acid analysis gave the following molar ratios: ammonia, 3.00 (3); arginine, 2.00
(2); serine, 0.73 (1); glutamic acid, 2.04 (2); glycine, 0.97 (1);
alanine, 0.97 (1); valine, 0.99 (1); leucine, 4.07 (4). Leucine amino peptidase cleavage gave the following molar ratios: arginine, 2.03 (2); glycine, 1.07 (1); alanine, 0.99 (1); valine, 1.17 (1);
leucine, 3.95 (4). Thin layer chromatography, using systems I,
III and IV gave single spots with R^=0.20, 0.59 and 0.16, respectively.
Synthesis of Arg-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly- Leu-Val-NH^. The title compound was prepared by continuation of the above synthesis and the coupling's went to completion as determined by the ninhydrin test. After the last coupling the N-terminal Boc group was removed (Table 1, Steps 1-6) , and 0.34 g secretin^_27-resin was removed for hydrogen fluoride cleavage. The peptide-resin was cleaved for 160 minutes at 0° in the same manner as described previously for secretin^ 27• After gel filtration and lyophilization, 31 mg white powder was obtained. Amino acid analysis gave the following molar ratios: ammonia, 2.86 (3); arginine, 2.84 (3); aspartic acid, 0.90
(1); serine, 0.75 (1); glutamic acid, 2.21 (2); glycine, 0.99 (1);
alanine, 0.93 (1); valine, 1.09 (1); leucine, 4.17 (4). Thin layer
chromatography, using systems I , III and IV gave single spots R^=
0.18, 0.67 and 0.05, respectively.
Purification of Secretin^ ^ Partition chromatography using solvent system B was used to purify secretin^^^gy The peptide (20 mg) was dissolved in 1 ml 0.2 77 HOAc and n, 2 ml upper phase was added.
The dissolved peptide was applied to a Sephadex G-25 .column, as pre
viously described for the purification of secretin,. . Analysis by the Folin-Lowry method gave two peaks, n, 0.45 and R^ < 0.19.
After gel filtration of each of the peaks, 2.2 mg and 7.0 mg of peptide material was obtained respectively. Amino acid analysis of the first peak gave the following molar ratios: ammonia, 3.04 (3); arginine, 3.09 (3); aspartic acid, 1.05 (1); serine, 0.98 (1); glutamic acid, 2.08 (2); glycine, 0.97 (1); alanine, 1.01 (1); valine, 1.09 (1);
leucine, 4.08 (4). Amino acid analysis of the latter peak gave the following molar ratios: ammonia, 3.49 (3); arginine, 2.16 (3);
aspartic acid, 0.60 (1); serine, 0.59 (1); glutamic acid, 2.02 (2);
glycine, 0.98 (1); alanine, 0.68 (1); valine, 1.02 (1); leucine, 3.98 (4).
Synthesis of Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Gln- Arg-Leu-Leu-Gln-Gly-Leu-Val-NH^. The title compound was prepared by
continuation of the above synthesis and the couplings went to comple
tion. After the last coupling the N-terminal Boc group was removed (Table 1, Steps 1-6) , and 0.32 g secreting^^y-1-65!11 was removed for hydrogen fluoride cleavage. The peptide-resin was cleaved for 30
minutes at 0° in the same manner as described previously for secre- tin,__27 • After gel filtration and lyophilization, 7 mg white powder was obtained. Amino acid analysis gave the following molar ratios:
ammonia, 3.05 (3); arginine, 4.14 (4); aspartic acid, 0.99 (1);
serine, 1.64 (2); glutamic acid, 3.19 (3); glycine, 0.98 (1); alanine, 1.00 (1); valine, 1.07 (1); leucine, 6.07 (6). Thin layer chromato
graphy, using systems I, II, III, IV and V, gave single spots R ^ O .24, 0.43, 0.76, 0.09 and 0.92, respectively.
Synthesis of Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser- Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH^■ The title compound was prepared by continuation of the above synthesis and the couplings went to completion. After the last coupling the N-terminal Boc group was removed (Table 1, Steps 1-6) , and 0.24 g secretin,, ^g-tesin was removed for hydrogen fluoride cleavage. The peptide-resin was cleaved for 30 minutes at 0° in the same manner as described previously for secretin^_27 (first synthesis). After gel filtration and lyophiliza
tion, 6 mg white powder was obtained. Amino acid analysis gave the following molar ratios: Ammonia, 3.26 (3); arginine, 3.90 (4);
aspartic acid, 0.98 (1); threonine, 1.48 (2); serine, 2.40 (3); glu
tamic acid, 3.16 (3); glycine, 1.00 (1); alanine, 1.01 (1); valine, 1.05 (1); leucine, 6.04 (6); phenylalanine, 0.84 (1). Thin layer chromatography gave single spots in systems I, II, III, IV and V, with R^=0.24, 0.72, 0.81, 0.09 and 0.96, respectively.
Synthesis of Secretin^. ^ Using 50% TFA. The title compound was prepared by continuation of the synthesis from the secreting
stage. Instead of using 25% TFA and 2 minute:20 minute deblocking times, 50% TFA and 2 minute:30 minute:30 minute conditions were employed. The synthesis used 0.6 g secreting_27'"resi-n and all other conditions were the same. After the last coupling, the N-terminal Boc group was removed, and 0.16 g secretint-_27-resin was removed for hydrogen fluoride cleavage. The peptide-resin was cleaved for 90 min
utes at 0° in the same manner as described previously for secretin^ ^ (first synthesis). After gel filtration and lyophilization, 18 mg white powder was obtained. Amino acid analysis gave the following molar ratios: ammonia, 3.18 (3); arginine, 4.03 (4); aspartic acid,
1.01 (1); threonine, 1.47 (2); serine, 2.71 (3); glutamic acid, 2.88 (3); glycine, 1.00 (1); alanine, 1.02 (1); valine, 1.17 (1); leucine, 6.00 (6); phenylalanine, 0.88 (1).
Activity Studies of Secretin Intermediates. The assay was the same as that previously described for secretin,. ^ and was done by Dr.
Gardner; the log-dose-response curves are given in Figure 2. The first peak in the partition chromatography spectrum of secretin^ ^ was fully active, and the latter peak was inactive. Crude secre- tin^_27 was almost as active as purified secretin^ Only secre-tin^_27, from the 30 minute hydrogen fluoride cleavage, was tested.
oI
ux u
fHrt
u(U
■MO a)
V)i G
• H
•MO
Gu (D
C/5
1.00
0.80 Secretin
14-27
0.60
0.40--Secret in, 9-27 Secretin
0 . 2 0
--Secretin 5-27
6 5
Concentration (AT)
OJ
C O
Figure 2. Activity of Secretin Fragments
Effect of Trifluoroacetic Acid Concentration and Phenol on the Incorporation of the Last Three Amino Acids into Secretin
The first synthesis of secretin^^y showed failure sequences and the latter synthesis of secretin^ while possessing activity- identical to purified secretin^_2y> still showed, on amino acid anal
ysis, a slightly less than theoretical amount of phenylalanine. In
complete coupling of amino acids, and/or incomplete deprotection of alpha amino groups, could be responsible for failure sequences appear
ing in the later stages of the synthesis. Failure sequences should be more pronounced at the end of the synthesis, and the only histidine
residue in secretin is the N^-terminal amino acid. Thus the amount of histidine present in the amino acid analysis spectrum should indicate coupling and/or deblocking efficiency. Phenol has been reported by Yamashiro, Blake and Li (1976) to make free amino groups more access
ible during coupling steps. The couplings (Table 1) for each amino acid went for 20 minutes, after which time phenol (to make a 10% solu
tion) and diisopropylethylamine (six drops) were added; each coupling went for a total of 30 minutes. In the couplings without phenol,