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The solid phase syntheses of 5-isoleucine-angiotensin I and 5-isoleucine (10-14C(U),-leucine)-angiotensin I
Lüe Sciences Vol. 7, Part II, pp . 841-845, 1988, Printed in Great Britain.
Pergamon Press
TeE saLZD ~ sxNTHES~B ~ 5-ISOL~rcn~-ANOIOTENSIN I AND
l~
5-ISOLS~cn¢~ (lo-
o(U),-L~rcn~)-ANGIOTENSIN I*
N. S. Thampi, G . Schcellmann, M. W. Huret
and C. G . Hugging
Department of Biochemistry, Tulene University School of Medicine, New Orleans, Louisiana (Received 8 November 1987 ; in final form 1 Aprü 1988) During the course of our investigations of the angiotensin I converting enzyme it became obvious that relatively large amounts of angiotensia I would be required .
Since we could not procure substrate amounts from the usual sources
we resorted to the synthesis of the decapeptide making use of the Merrifield solid phase synthesis of polypeptides (1) .
We have followed the detailed in-
structions of Marshall and Merrifield (2) for this synthesis .
t-Hutylo~çy-
carbonyl amino acids, dicyclohexylcarbodümide and the Merrifield resin were purchased from chemical supply houses .
In addition the material has been pre-
pared by incorporating a radioactive label at LeulO so that the dipeptide Hig .Leu liberated by the angiotensin I converting enzyme would be radioactive and we could then use the radioactivity released as a measure of enzyme activity.
For the synthesis of radioactivity labeled angiotensin I we have used
t-BOC- 14 C(U)-L-Leucine which was synthesized according to the procedure of Schwyzer et el . (3) . ~rimental t-BOC-L-Leucinyl, copolystyrene-divinylbeazene A solution of 0.7 g (2 .8 mMoles) t-BOC-L- leucine hydrate and 0 .37 ml (2 .8 mMoles) of triethylemine in ~ ml of ethanol were allowed to react with 3 .8 g of the chloramethyl copolystyrene 2`~+ divinylbenzene (Merrifield resin) This investigation was supported in part by a Public Health Service Training Grant ~GM-0635-07 from the National Institutes of General Medical Sciences, sad in part by a Grant-in-Aid from American Heart Association ~65G116.
841
842
ANGIOTENSIN SYNTHESIS
at 75o for 24 hours . nol, water,
Vol. 7, No . 12
The esterified resin was filtered off, washed with etha-
and methanol and dried under vacuum .
The weight of the product
indicated an incorporation of 0 .24 g of the t-BOC amino acid (~+) . 5-Isoleucine-Angiotensin I The t-BOC-L-leucinyl resin (4 .04 g) was introduced into a Merrifield type of reaction flask in which the cycle for the incorporation of each amino acid consisted of the cleavage of the t-BOC group with hydrochloric acid in glacial acetic acid neutralization with triethylamine in dimethylformamide and coupling the free amino group with the appropriate t-BOC-amino acids, using N,N'dicyclohexylcarbodümide as the condensing agent .
The condensation steps in-
volved in the incorporation of t-BOC-N-imidazole-benzyl-L-histidine and tBOC-L-nitroarginine were carried out in specially purified dimethylformamide whereas the rest of the condensations were performed in methylene chloride (yield of the decapeptide resin 4.9 gm) . The protected decapeptide was cleaved from the polymer by bubbling a slow stream of dry HBr for 30 minutes at 23o through a suspension of polypeptide resin in trifluoroacetic acid .
The solution of the liberated protected deca
peptide in trifluoroacetic acid wen concentrated in a rotary evaporator e.nd the residue was lyophilized from glacial acetic acid solution (yield 1.0 g) . The remaining protecting groups from this product were removed by catalytic hydrogenation over 59~ palladium oxide on barium sulfate in a solution of methanol, acetic acid and water (10 :1 :1,
vw)
at 50 psi for 48 hours .
The
solution was filtered, evaporated and lyophilized from glacial acetic acid . white powder, not entirely soluble in water, was obtained . solved in glacial acetic acid, centrifuged, umes of dry ether. dried.
This was redis-
and precipitated with nine vol-
The residue was separated, washed with dry ether,
The precipitate was dissolved in water, centrifuged,
natant fluid lyophilized .
A
and
and the super-
This product was further purified by Bio-Gel-P-2
(100-200 mesh) filtration using 0 .1 M acetic acid as the eluting solvent .
The
fractions containing the biologically active material were pooled together and
Vol . 7, No . 12
ANGIOTENSIN SYNTHE3I3
849
lyophilized (yield 510 mg) " TABLE 1 Characterization of 5-Isoleucine (10-14C(U)- Leucine) Angioteaein I
Procedures Ae (282 mk) 0 .1 mg 0 .2 ma
Synthetic 14 5-Isoleucine-(10- C(U)Leucine) Angiotensin I
CIBA 6tandard 5-Valine Angiotenein I
0 .053 0 .109
0 .053
Ae (45o m~) with Pa~ly's Color Reagent
0 .29
0 .32
Paper Chromatography2 Glase Fiber Paper R f Whatman #1 Rf
0 .45 0 .13
0 .45 0 .13
Electrophoresis 3
4 cm (anode)
4 cm (anode)
0 .6 gm tension
0 .6 gm tension
0 .71 gm tension 28 mm Hg
0 .73 gm tension 27 mm gg
100
Inactivation
100
Inactivation
100 Inactivation 100, Inactivation
100` 100
Inactivation Inactivation
Parallel Assay Guinea Pig Ileum (4 x 10 - k8) Rabbit Aorta ( 20 Fib + 1 mg converting enzyme) Cat Blood Pressure (1 F+g) Enzyme Inactivation4 Leucine Aminopeptidase (pH 8 .3) Cerboxy peptidase B (~ 7 " 3) Nagarse (pH 6 .5)
Pauly's color was developed using a standard Pauly reagent (5) and the optical density measured in a Bechan DU Spectrophotometer . 2
The angiotensin was chromatographed on Whatmaa #1 filter paper (solvent system - n-butanol :acetic acid :water, 4 :1 :5, vw) end on silica gelimpregnated glass fiber paper, - I .T .L .C . type SG-Gelman Instrument Company (solvent system - n-butanol :ammonia :water, 120 :7 :7, vw) .
3 - Cellulose acetate (Sepraphore II) electrophoresis was performed with a Gelman Electrophoresis unit at 30 volte~cm (solvent system - 1 M acetic acid) . 4 Each enzyme was incubated with the two preparations of angiotensin I at 37o for 30 minutes and then tested for activity on ghe guinea pig ileum . One F18 oP leucine aminopeptidase, 0 .1 F18 carboxypeptidaee B, or 5 kg of Nagarse wereincubated with 10 F+g samples of angioteasin I .
ANGIOTEN3IId 3YNTHE3L4
844
Vol . 7, No . 12
5-Isoleucine (10- 1~C(U)-Leucine) Angiotensin 2 5-Isoleucine (10- 1~C(U)-Leucine) angiotensln I was synthesized according to the procedure described above for 5-isoleucine angiotensin 2, except that t-BOC- 1~C(U)-L- leucine (1 .2'( x 108 cts~min~mM) was used in place of t-BOC-Lleucine .
The specific activity of the material obtained by purification on
Bio-Gel-P-2 as described above was
9.6 x 107
cte~min~mM .
The percentage yield
was approximately the same ae that for the non-radioactive 5-isoleucine engiotensin 2 .
Its chemical and biological properties (see Table 1) were equiva-
lent to those obtained with a standard preparation of 5- valine -angiotensia I (CIBA) .
Table 1 shows data obtained concerning studies on the characteriza-
tion of the radioactive product in which it can be seen that 5-isoleucine (10- 1~C(U)- leucine)
angiotensin 2 was the same as that of e standard prepara-
tion of 5-valine angiotenain I (CI&4) .
In addition, we have found that leu-
tine aminopeptidase, carboxypeptidase, end Nagarse destroy the biological activity of our synthetic product at the same rate as they destroy the atandand preparation of angiotensin I .
Table 2 presents data showing the analysis
for the amino acid composition of the synthetic radioactive product and for comparison the analysis of a standard preparation of angiotenein I from CIBA . TABLE 2 Amino Acid Composition of 5-Isoleucine (10- 1~C(U)- Leucine) Angiotensin I and 5-Valine Angioteasin Amino Acid
Asp. Arg . Val . Tyr . Ile . His . Pro . Phe . Leu.
Synthetic 5-Isoleucine-(10- 1~C(U)Leucine) Angioteasin I
CIRA Standard 5-Valine Angiotensin 2
Theor.
Found
Theor .
Found
1 1 1 1 1 2 1 1 1
1.0
1 1 2 1
0 .7 0 .9 1 .8
0 .6
1 .0
0 .7 0 .9
2 .0 1 .1 1 .0 1 .0
-
2 1 1 1
0 .4
1 .9 0 .9 1 .0 1 .1
Vol. 7, No. 12
ANGIOTENSIN SYNTHESIS
645
References
412 (1962) .
1.
Merrifield, R. B., Federation Proc . 21,
2.
Marshall, G . R. and Merrifield, R . B., Biochem.
3.
Schwyzer, R ., Sieber, P ., and Kappeler, H ., Helv Chim . Acta ,
4.
Khosla, M. C ., Smeby, R . R.,
5.
Greenstein, J . P., and Winitz, M., Chemistr, Vol . 3, John Wiley and Sons, Inc ., New York
4, 2394 (1965) .
(1959) .
and Bumpus, F . M ., Biochem .
42, 2622
6, 754 (1967) .
of the Amino Acids, p . 181,
19 2 .
Pergamon Press
TeE saLZD ~ sxNTHES~B ~ 5-ISOL~rcn~-ANOIOTENSIN I AND
l~
5-ISOLS~cn¢~ (lo-
o(U),-L~rcn~)-ANGIOTENSIN I*
N. S. Thampi, G . Schcellmann, M. W. Huret
and C. G . Hugging
Department of Biochemistry, Tulene University School of Medicine, New Orleans, Louisiana (Received 8 November 1987 ; in final form 1 Aprü 1988) During the course of our investigations of the angiotensin I converting enzyme it became obvious that relatively large amounts of angiotensia I would be required .
Since we could not procure substrate amounts from the usual sources
we resorted to the synthesis of the decapeptide making use of the Merrifield solid phase synthesis of polypeptides (1) .
We have followed the detailed in-
structions of Marshall and Merrifield (2) for this synthesis .
t-Hutylo~çy-
carbonyl amino acids, dicyclohexylcarbodümide and the Merrifield resin were purchased from chemical supply houses .
In addition the material has been pre-
pared by incorporating a radioactive label at LeulO so that the dipeptide Hig .Leu liberated by the angiotensin I converting enzyme would be radioactive and we could then use the radioactivity released as a measure of enzyme activity.
For the synthesis of radioactivity labeled angiotensin I we have used
t-BOC- 14 C(U)-L-Leucine which was synthesized according to the procedure of Schwyzer et el . (3) . ~rimental t-BOC-L-Leucinyl, copolystyrene-divinylbeazene A solution of 0.7 g (2 .8 mMoles) t-BOC-L- leucine hydrate and 0 .37 ml (2 .8 mMoles) of triethylemine in ~ ml of ethanol were allowed to react with 3 .8 g of the chloramethyl copolystyrene 2`~+ divinylbenzene (Merrifield resin) This investigation was supported in part by a Public Health Service Training Grant ~GM-0635-07 from the National Institutes of General Medical Sciences, sad in part by a Grant-in-Aid from American Heart Association ~65G116.
841
842
ANGIOTENSIN SYNTHESIS
at 75o for 24 hours . nol, water,
Vol. 7, No . 12
The esterified resin was filtered off, washed with etha-
and methanol and dried under vacuum .
The weight of the product
indicated an incorporation of 0 .24 g of the t-BOC amino acid (~+) . 5-Isoleucine-Angiotensin I The t-BOC-L-leucinyl resin (4 .04 g) was introduced into a Merrifield type of reaction flask in which the cycle for the incorporation of each amino acid consisted of the cleavage of the t-BOC group with hydrochloric acid in glacial acetic acid neutralization with triethylamine in dimethylformamide and coupling the free amino group with the appropriate t-BOC-amino acids, using N,N'dicyclohexylcarbodümide as the condensing agent .
The condensation steps in-
volved in the incorporation of t-BOC-N-imidazole-benzyl-L-histidine and tBOC-L-nitroarginine were carried out in specially purified dimethylformamide whereas the rest of the condensations were performed in methylene chloride (yield of the decapeptide resin 4.9 gm) . The protected decapeptide was cleaved from the polymer by bubbling a slow stream of dry HBr for 30 minutes at 23o through a suspension of polypeptide resin in trifluoroacetic acid .
The solution of the liberated protected deca
peptide in trifluoroacetic acid wen concentrated in a rotary evaporator e.nd the residue was lyophilized from glacial acetic acid solution (yield 1.0 g) . The remaining protecting groups from this product were removed by catalytic hydrogenation over 59~ palladium oxide on barium sulfate in a solution of methanol, acetic acid and water (10 :1 :1,
vw)
at 50 psi for 48 hours .
The
solution was filtered, evaporated and lyophilized from glacial acetic acid . white powder, not entirely soluble in water, was obtained . solved in glacial acetic acid, centrifuged, umes of dry ether. dried.
This was redis-
and precipitated with nine vol-
The residue was separated, washed with dry ether,
The precipitate was dissolved in water, centrifuged,
natant fluid lyophilized .
A
and
and the super-
This product was further purified by Bio-Gel-P-2
(100-200 mesh) filtration using 0 .1 M acetic acid as the eluting solvent .
The
fractions containing the biologically active material were pooled together and
Vol . 7, No . 12
ANGIOTENSIN SYNTHE3I3
849
lyophilized (yield 510 mg) " TABLE 1 Characterization of 5-Isoleucine (10-14C(U)- Leucine) Angioteaein I
Procedures Ae (282 mk) 0 .1 mg 0 .2 ma
Synthetic 14 5-Isoleucine-(10- C(U)Leucine) Angiotensin I
CIBA 6tandard 5-Valine Angiotenein I
0 .053 0 .109
0 .053
Ae (45o m~) with Pa~ly's Color Reagent
0 .29
0 .32
Paper Chromatography2 Glase Fiber Paper R f Whatman #1 Rf
0 .45 0 .13
0 .45 0 .13
Electrophoresis 3
4 cm (anode)
4 cm (anode)
0 .6 gm tension
0 .6 gm tension
0 .71 gm tension 28 mm Hg
0 .73 gm tension 27 mm gg
100
Inactivation
100
Inactivation
100 Inactivation 100, Inactivation
100` 100
Inactivation Inactivation
Parallel Assay Guinea Pig Ileum (4 x 10 - k8) Rabbit Aorta ( 20 Fib + 1 mg converting enzyme) Cat Blood Pressure (1 F+g) Enzyme Inactivation4 Leucine Aminopeptidase (pH 8 .3) Cerboxy peptidase B (~ 7 " 3) Nagarse (pH 6 .5)
Pauly's color was developed using a standard Pauly reagent (5) and the optical density measured in a Bechan DU Spectrophotometer . 2
The angiotensin was chromatographed on Whatmaa #1 filter paper (solvent system - n-butanol :acetic acid :water, 4 :1 :5, vw) end on silica gelimpregnated glass fiber paper, - I .T .L .C . type SG-Gelman Instrument Company (solvent system - n-butanol :ammonia :water, 120 :7 :7, vw) .
3 - Cellulose acetate (Sepraphore II) electrophoresis was performed with a Gelman Electrophoresis unit at 30 volte~cm (solvent system - 1 M acetic acid) . 4 Each enzyme was incubated with the two preparations of angiotensin I at 37o for 30 minutes and then tested for activity on ghe guinea pig ileum . One F18 oP leucine aminopeptidase, 0 .1 F18 carboxypeptidaee B, or 5 kg of Nagarse wereincubated with 10 F+g samples of angioteasin I .
ANGIOTEN3IId 3YNTHE3L4
844
Vol . 7, No . 12
5-Isoleucine (10- 1~C(U)-Leucine) Angiotensin 2 5-Isoleucine (10- 1~C(U)-Leucine) angiotensln I was synthesized according to the procedure described above for 5-isoleucine angiotensin 2, except that t-BOC- 1~C(U)-L- leucine (1 .2'( x 108 cts~min~mM) was used in place of t-BOC-Lleucine .
The specific activity of the material obtained by purification on
Bio-Gel-P-2 as described above was
9.6 x 107
cte~min~mM .
The percentage yield
was approximately the same ae that for the non-radioactive 5-isoleucine engiotensin 2 .
Its chemical and biological properties (see Table 1) were equiva-
lent to those obtained with a standard preparation of 5- valine -angiotensia I (CIBA) .
Table 1 shows data obtained concerning studies on the characteriza-
tion of the radioactive product in which it can be seen that 5-isoleucine (10- 1~C(U)- leucine)
angiotensin 2 was the same as that of e standard prepara-
tion of 5-valine angiotenain I (CI&4) .
In addition, we have found that leu-
tine aminopeptidase, carboxypeptidase, end Nagarse destroy the biological activity of our synthetic product at the same rate as they destroy the atandand preparation of angiotensin I .
Table 2 presents data showing the analysis
for the amino acid composition of the synthetic radioactive product and for comparison the analysis of a standard preparation of angiotenein I from CIBA . TABLE 2 Amino Acid Composition of 5-Isoleucine (10- 1~C(U)- Leucine) Angiotensin I and 5-Valine Angioteasin Amino Acid
Asp. Arg . Val . Tyr . Ile . His . Pro . Phe . Leu.
Synthetic 5-Isoleucine-(10- 1~C(U)Leucine) Angioteasin I
CIRA Standard 5-Valine Angiotensin 2
Theor.
Found
Theor .
Found
1 1 1 1 1 2 1 1 1
1.0
1 1 2 1
0 .7 0 .9 1 .8
0 .6
1 .0
0 .7 0 .9
2 .0 1 .1 1 .0 1 .0
-
2 1 1 1
0 .4
1 .9 0 .9 1 .0 1 .1
Vol. 7, No. 12
ANGIOTENSIN SYNTHESIS
645
References
412 (1962) .
1.
Merrifield, R. B., Federation Proc . 21,
2.
Marshall, G . R. and Merrifield, R . B., Biochem.
3.
Schwyzer, R ., Sieber, P ., and Kappeler, H ., Helv Chim . Acta ,
4.
Khosla, M. C ., Smeby, R . R.,
5.
Greenstein, J . P., and Winitz, M., Chemistr, Vol . 3, John Wiley and Sons, Inc ., New York
4, 2394 (1965) .
(1959) .
and Bumpus, F . M ., Biochem .
42, 2622
6, 754 (1967) .
of the Amino Acids, p . 181,
19 2 .