Synthetic Fragments and Analogues of Elastin. 1. The Synthesis V. G U A N T I E R I * , S. G R A N D O * , L. P A N D O L F O * , and A. M. TAMBURRO+,
* D q ) a r t n i e n t of Inorganic Metallorganic and Analytical Chemistry, University of Padova, Italy of Chemistry, University of Basilicata, Potenza, Italy
' Dcy)artment
SY NOPSlS
The synthesis of some repetitive sequences of elastin and their simplified analogues, all comprising the structural unit Gly-X-Gly (X = Val, Leu, Ala), is described. In particular, the following peptides and polypeptides were synthesized and characterized: Boc-Gly-ValGly-Gly-Leu-OMe, Boc-Gly-Leu-Gly-Gly-Val-OMe, Boc-(Gly-Val-Gly-Gly-Leu),-OMe, Boc-(Gly-Val-Gly-Gly-Leu),i-OMe, Boc-Gly-Val-Gly-Gly-OEt, Boc-Leu-Gly-Gly-Leu-OMe, Boc-Val-Gly-Gly-Val-OMe, poly(A1a-Gly-Gly),poly(Va1-Gly-Gly), and poly(Leu-Gly-Gly). In every case, the synthesis w a s accomplished by classical procedures in solution, by using the p-nitrophenyl ester method for the polycondensation step, and the mixed anhydride or the a i d e methods for the coupling steps.
I NTRO DUCT I 0N Elastin, the protein responsible for tissue resiliency, is characterized by a unique primary structure consisting of hydrophobic residues frequently arranged in repeating sequences. As representatives of these regions, the prolinecontaining polytripeptide, (Val-Pro-Gly),,'f2 polytetrapeptide, (Val-Pro-Gly-Gly),, polypentapeptide, (Val-Pro-Gly-Val-Gly),, and polyhexapeptide, (Val-Ala-Pro-Gly-Val-Gly), were synthesized and extensively studied., In all cases the prominent conformational feature of these sequences appeared to be the so-called p-turn, the stability of this particular conformation being strongly enhanced under conditions of low water activity. More recently, additional repeating sequences have been deduced from gene ~ t r u c t u r e ~ comprising -~ Gly-X-Gly triplets, where X = Ala, Leu, or Val, suggesting the possibility that these triplets may have originated from single-base mutations and tandem duplications from a common ancestor. Also
c 1990 John Wiley & Sons, Inc.
CCC OOOS-3525/90/040845-10 $04.00 Biopolymers, Vol. 29, 845-854 (1990)
conserved is the pentapeptide sequence Gly-ValGly-Gly-Leu, of particular interest because it repeats itself twice in the currently known",6 amino acid sequences of beef and pig tropoelastin, and three times those of the sheep and chick. Furthermore, the closely related isomeric sequence GlyLeu-Gly-Gly-Val is also present in different species. Given the unusual abundance of repetitive sequences in elastin, it may be reasonable to assign a priori an important conformational role to them (for a discussion, see Ref. 7). Accordingly, we thought useful to synthesize, as simplified models, the polytripeptides poly(A1a-Gly-Gly), poly(Va1-Gly-Gly), and poly(Leu-Gly-Gly) (general sequence Gly-X-Gly), the two protected pentapeptides Boc-Gly-Val-Gly-Gly-Leu-OMe and Boc-GlyLeu-Gly-Gly-Val-OMe, and the repeating sequences Boc-(Gly-Val-Gly-Gly-Leu),-OMe and Boc-(Gly-Val-Gly-Gly-Leu),-OMe. In addition, short related sequences, such as Boc-Gly-Val-GlyGly-OEt, Boc-Leu-Gly-Gly-Leu-OMe, Boc-ValGly-Gly-Val-OMe, and Boc-Leu-Gly-Gly-Val-OMe were synthesized for comparative purposes. The synthetic procedures are described in the present paper while the related conformational studies in solution are reported in the following one. 845
846
GUANTIERI ET AL.
RESULTS
(Fig. 2) were obtained by mixed anhydride coupling of Boc-X-OH with the above-mentioned dipeptide, saponification of the alkyl esters, formation of the corresponding p-nitrophenylesters, removal of the Boc protecting group, and finally, by polycondensation in the presence of N-methylmorpholine. The solutions were concentrated as much as possible in
The general scheme utilized for the synthesis is shown in Fig. 1. The rationale was to minimize the number of intermediates by selecting as starting sequence the N-deblocked dipeptide C1-H: -GlyGly-OEt. Briefly, the three (X-Gly-Gly), polymers
.-\
(Leu-Gly-Gly)
( Ala-Gly-Gly)
1
/H;-Leu-OMe
BOC-Leu-Gly-Gly-OEt
BOC -A1 a-G1y -G 1 y -0Et
\
\
BOC-Leu-OH
BOC-A1 a-OH
\
/
t
BOC-Leu-Gly-Gly-Val-OMe 1
H -Gly-Gly-OEt
I I
BOC-Gly-Leu-Gly-Gly-Val-OMe
BOC-Val -OH
BOC-Gly-OH
U O C -V a 1 -G1 y -G 1 y - OE t /
H;-Val-OMe
L
2
( V a 1 -G 1 y -G 1 y ) "
BOC-Leu-Gly-Gly-Leu-OMe 7l
*BOC-Gly-Val-Gly-Gly-OEt
\
BOC-Val-Gly-Gly-Leu-OMe
BOC-V a1 -G 1 y-G1 y-V a1 -0Me
I I
BOC-Gly-OH
BOC-Gly-Val-Gly-Gly-Leu-OMe
BOC-(Gly-Val-Gly-Gly-Leu) -0Me 2
i
BOC-(G1y -Val -G1y-G1y -Leu 1 -0Me 3
Figure 1. General scheme for the synthesis of fragments and analogues of elastin. Only the lengthening of the chain is shown, as the detailed schemes for the deblocking and activation steps are reported in the following figures
BOC
OEt
NaOH,Ht
BOC
BOC-
HONp,OCC HC1 o r H C O O H
HS (-
OH
I
NP
0 - - -
~
N i4 M
~-
ONP
I
SYNTHETIC FRAGMEN'I'S. I
aoc
OH
H
OE t
I,aiiH,H
5OC
t
HOhp,OCC
BOC
OH ONP
N M 1-1
BOC BOC
OEt
15C
50C
H
-0N I e -9Me
HCOOH
OH
847
-ORe
-d I 4 e
X
Leu,
Y ~ -V a l
X
'Val.
Y z
1.eu
Figure 3. Scheme for the synthesis of the pentapeptides Boc-Gly-Val-Gly-Gly-Leu-OMe and Boc-Gly-Leu-Gly-Gly-Val-OMe
order t o minimize the formation of cyclic peptides. A rough fractionation of the polymers was achieved by exhaustive washings with water in order to remove the low molecular weight fraction. The synthesis of poly(A1a-Gly-Gly), in which benzyloxycarbonyl was used as the N-protecting group, has been previously published.', The isomeric pentapeptides (Fig. 3 ) were obtained by coupling the N-terminal and C-terminal amino acids, respectively, on the Val-Gly-Gly and Leu-Gly-Gly blocks previously synthesized. On the other hand, the protected deca- and pentadecapeptides were synthesiLed by using the azide coupling method in order t o avoid racemization (Fig. 4). Short reference sequences were obtained according t o the scheme outlined in Fig. 5.
Gly
Vdl
Gly
Gly
Leu
Gly
Val
Wherever the synthetic step could have potentially induced racemization, care was taken to avoid i t by setting glycine as the C-terminal amino acid.
EXPERIMENTAL Boc amino acids were purchased from Novabiochem AG (Laufelfingen, Switzerland) and glycylglycine ethyl ester hydrochloride was purchased from Sigma (St. Louis, MO, USA). The p u i t y of the synthetic products was ascertained by thin layer chromatography on butanolacetic acid-water (3 : 3 : 1) and chloroform-acetone (3 : 2). Further characterization was obtained by
Gly
Gly
Leu
Gly
Val
Gly
BOC
Rnr --Figure 4. Scheme for the synthesis of the decapeptide Boc-(Gly-Val-Gly-Gly-Leu),-OMe and of the pentadecapeptide Boc-(Gly-Val-Gly-Gly-Leu):,-OMe
Gly
Leu
848
GUANTIERI ET AI,.
BOC Val
BOC
HCOOH
OH
OE t UE t
BOC
BOC
Val
NaOH, ti
BOC
0ble
Y V I l
" I I C
Figure 5. Scheme for the synthesis of the tetrapeptides Boc-Val-Gly-Gly-Val-OMe, Boc-Gly-Val-Gly-Gly-OEt, and Boc-Leu-Gly-Gly-Leu-OMe
'H-nmr spectra recorded on a 60-MHz Varian EM 360A spectrometer. Synthesis of compounds
Boc-X-Gly-Gly-OEt (X = Ala, Val, Leu) Isobutyl chloroformate (0.2 moles) was added a t - 15°C to a solution of Boc-X-OH (0.2 moles) and N-methylmorpholine (NMM; 0.2 moles) in chloroform (800 mL). The temperature was kept a t -15°C for 1 min, then Cl-H,i-Gly-Gly-OEt (0.2 moles) and NMM (0.2 moles) were added. The mixture was kept at room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from the appropriate solvent. The physical properties, nmr parameters, and elemental analyses of these intermediate compounds are reported in Tables I and 11. Boc-X- Gly-Gly-OH
To a solution of Boc-X-Gly-Gly-OEt (187 mmoles) in acetone (500 mL), 1 N sodium hydroxide (205
mL) was added. The solution was stirred at room temperature for 3 h, then the organic solvent was evaporated under reduced pressure and water was added. The unreacted product was extracted with ethyl acetate, and the aqueous solution was cooled a t 4°C and neutralized with dropwise addition of 1N hydrochloric acid (205 mL). The crude material, which precipitated immediately, was extracted with ethyl acetate and dried over sodium sulphate. The solution was evaporated to dryness and the residue was crystallized from the appropriate solvent. The physical properties, nmr parameters, and elemental analyses of these intermediate compounds are reported in Tables I and 11. Boc-X- Gly-Gly- 0Np N,N'-dicyclohexylcarbodiimide (DCC; 43 mmoles) was added to an ice-cold solution of Boc-X-GlyGly-OH (43 mmoles) and p-nitrophenol (HONp; 43 mmoles) in chloroform (200 mL). The mixture was stirred at 0°C for 1 h and a t room temperature for 12 h. The N,N'-dicyclohexylurea was filtered, and the organic solution was washed with 5% sodium bicarbonate, water, dried over sodium sulphate,
SYNTHETIC FRAGMENTS. I
849
Table I Phvsical ProDerties and Elemental Analyses of Intermediate Compounds Elemental Analvsis (%) Compound
Melting Point ( O C) (Crystallization Solvent)
Yield
Boc-Leu-Gly-Gly-OEt
86-87 Diethyl ether 98-99 Boc-Ala-Gly-Gly-OEt Petroleum ether Boc-Val-Gly -Gly-OEt 79-80 Petroleum ether Boc-Leu-Gly-Gly-OH 118- 120 Petroleum ether Boc-Ala-Gly-Gly-OH 161-162 Petroleum ether Boc-Val-Gly-Gly-OH 148-150 Diethyl ether Boc- Leu-Gly-Gly-ONp 93-95 Diethyl ether/petroleum ether Boc-Ala-Gly-Gly-ONp 111- 112 Petroleum ether Boc-Val-Gly-Gly-ONp 108-109 Diethyl ether C1- H l -Leu-Gly-Gly-ONp 192-194 Diethyl ether C 1 H; ~ -Val-Gly-Gly-ONp 204-206 Diethyl ether HCOO H i -Val-Gly-Gly-OEt Oil
and evaporated to dryness. Trituration of the oil residue with petroleum ether or diethyl ether gave a solid product. The physical properties, nmr parameters, and elemental analyses of these intermediate compounds are reported in Tables I and 11. CI - H,+-X-Gly-Gly-ONp
Boc-X-Gly-Gly-ONp (76 mmoles) was dissolved in 3 N hydrochloric acid in dioxane (640 mL). After stirring for 1 h a t room temperature, the peptide precipitation was completed by addition of diethyl ether. The physical properties, nmr parameters, and elemental analyses of these intermediate compounds are reported in Tables I and 11. HCOO - H,+ -Ala-Gly-Gly- ONp Five grams (12 mmoles) of Boc-Ala-Gly-Gly-ONp were dissolved in 98% formic acid (59 mL). After stirring for 3 h a t room temperature, the solution was evaporated to dryness, giving 10.6 g of an uncrystallizable oil (yield 95%), which was directly used in the following step.
k'ound
CalC.
[.I$'
(%)
(c, 1.0, DMF)
94
-9.9
53.38 8.45 10.98 53.42 8.85 10.87
57
-1.2
50.74 7.62 12.67 50.79 7.69 12.65
91
+4.0
53.46 8.15 11.69 53.44 8.16 11.67
77
-11.5
52.16 7.89 12.16 52.04 7.84 12.13
50
-2.1
47.52 6.99 13.85 47.63 7.26 13.68
84
+0.7
50.74 7.62 12.67 50.69 7.50 12.62
89
-6.9
54.07 6.49 12.00 54.20 6.56 11.98
54
-1.9
50.94 5.71 13.19 51.00 5.81 13.08
74
+2.2
53.09 6.25 12.38 53.16 6.17 12.25
93
+4.8
47.70 5.77 13.90 47.52 5.70 13.77
96
+52.1
46.33 5.45 14.40 46.19 5.49 14.15
99
-
C
-
H
-
N
-
C
-
H
-
N
-
Poly(X- Gly-Gly) To a solution of Cl-H,+-X-Gly-Gly-ONp or HCOO-Hl -Ala-Gly-Gly-ONp (70 mmoles) in anhydrous N,N'-dimethylformamide (DMF; 80 mL), NMM (99 mmoles) was added. After stirring for 3 days a t room temperature, the polypeptide precipitation was completed by addition of diethyl ether, repeatedly washed with water, and dried in vacuo over P,O,. The physical properties and nmr parameters of these compounds are reported in Table 111. HCOO - H,f - Val-Gly-Gly-OEt
Boc-Val-Gly-Gly-OEt (61.6 g, 171 mmoles) was dissolved in 98% formic acid (800 mL). After stirring for 2 h a t room temperature, the solution was evaporated to dryness, giving an uncrystallizable oil (52.0 g), which was directly used in the following step (yield 99%). 'H-nmr (CDC1,): yCH,, 1.05 (d, 6H), CH,(OEt) 1.26 (t, 3H), PCH 2.12 (b, lH), aCH, 4.02 (b, 4H), CH,(OEt) 4.12 (9, 2H), aCH 4.33 (b, lH), NH(G1y) 7.90 (bt, lH), HCOO- 8.18 (s, lH), NH(G1y) 8.45 (b, lH), NH; 9.50 (bs, 3H).
850
GUANTIERI ET AI,.
Table I1
H-nmr Data of Intermediate Compounds in CIICI.,
Boc-Leu-Gly-Gly-OEt
1.25 ( t , 3H) 1.25 (t, ,3H) 1.26 ( t , 3H)
Hoe-Ala-Gly-Gly-OEt Boc-Val-Gly-Gly-OEt
0.88
- 3H)
(d,
1.28 (m, 3H)
Boc-Leu-Gly-Gly-OH
Roc-Val-GigGly-OH"
0.83 (d, 6H)
1.43 (s, 9H) 1 .:18 (s, 9H) 1.40 (s, 9H) 1.39 (s, 9H)
(s,
0.92
C1- H i -1,eu-Gly-Gly-ONp"
1.38 (bin, .3H) I .87 (bin, 1 H ) 1 .38 (hm, 9 H )
9H) 2.05
1.43 (s,9H)
- 2H)
(d,
2.08 IH)
(Ill,
1.:18 (s,9H) 1 .:19
Hoc-Leu-Gly-Gly-ONp Boc-Val-Gly-Gly-ONp
1.52 (hm, RH)
(hm, 1 tl) 1.58 .1H)
(tllll,
CI H i -Val-Gly-Gly-ONp'
0.88
(d,
2.0.3
- 2.5H)
(nl, I H )
HCOO H i -Val-Gly-
Gly-OEt Compound Hoc-Ifiu-Gly-Gly-OEt Boc-Ah-Gly-Gly-OEt Boc-Val-Gly-Gly-OEt Bo~-Leu-Gly-Gly-OH
Boc-Val-Gly-Gly-OH" Boc-Leu-Gly-Gly-OIp Roc-Val-Gly-Gly-ONp
C1- H,-I,eu-GlyGly-ONp' C1 H;-Val-GlyGly-ONg HCOO- H,i-Val-GlyGly-OEt
1.05 (d, 6H) aCH,
aCH
1.26 (t, tjH)
CH,(OEt) OCONH
NH;
2.12
(h I H ) NH(Gly 2) o-ONp NH(G1y 1) ni-ONp
4.03 (bm, 1H) 3.93 (bm, 1H) 3.98 (bm, 1H) 4.17 (bm, 1H) 3.87 (bm, 1H) 4.17 (bm, 1H) 4.25 (bd, 3H)
3.78 (d, 2H)
4.10 (bd, 3H)
8.40 (bs, 3H)
8.73 (t, 1H)
7.33 (d, 2H)
8.98 ( t , 1H)
8.23 (d, 2H)
3.83 (d, 2H)
4.13 (bd, 3H)
8.38 (b, 3H)
8.78 (t, 1H)
7.38 (d, 2H)
8.93 (t, 1H)
(d, 2H)
9.50 (bs, 3H)
7.90 (bt, 1H)
4.02
4.33
(b, 4H)
(b, 1H)
4.12 (q, 2H)
HCOO
r'
4.03 (bm, 4H) 3.93 (bm, 4H) 3.98 (bm, 4H) 4.00 (bm, 4H) 3.66 (bm, 4H) 4.00 (hm, 4H) 4.05 (d, 2H)
4.18 (q, 2H) 4.15 (q,2W 4.15 (q,2H)
OH
-.dt3
5.50 (d, 1H) 5.45 (d, 1H) 5.24 (d, 1H) 5.55 (d, 1H) 6.63 (d, 1H) 5.27 (4 1H) 5.21 (d, 1H)
I
(hm, 2H) 7.28 (bm, 2H) 7.16 (bm, 2H) 7.63 8.92 (bm, 2H) (hs, 1H) 8.05 (hm, 2H) 6.83 7.30 8.04 (d, 2H) (bm, 2H) (d, 2H) 7.15 7.28 7.53 8.25 (bm, 1H) (d, 2H) (bm, 1H) (d, 2H)
8.45 (b, 1H)
8.25 8.18 (s, 1H)
" I n Me,SO-d,;.
Table I11 Properties of Polytripeptides Polycondensation Yield ( W )
[ 71Ia (dL/g)
'H-nmr (Me,SO-d,)
Poly(Leu-Gly-Gly)
90
0.21
Poly(Ala-Gly-Gly)
91
0.14
Poly(Va1-Gly-Gly)
80
0.23
SCH,, 0.87 (b, 6H), BCH, + yCH 1.51 (b, 3H), aCH, 3.67 (b, 4H), olCH 4.17 (b, lH), NH 7.88 (b, 2H), NH 8.07 (b, 1H) PCH,, 1.23 (bd, 3H), aCH, 3.83 (b, 4H), aCH 4.26 (b, 1H), NH 8.13 (b, 3H) Y C H ,0.93 ~ (b, 6H), PCH 1.87 (b, lH), aCH, 3.72 (b, 4H), aCH 4.07 (b, lH), NH 7.45 (b), 7.91 (b), 8.26 (b), 8.58 (b) (3H)
Compound
"Intrinsic viscosity in dichioroacetic acid ( c
=
1%) obtained according to the "single-point" method."'
SYNTHZ'I'IC FIIAGMES'I'S. I
BOC- Gly- Val-Gly- Gly- Oft Isobutyl chloroformate (22.14 mL, 170 mmoles) was added a t - 15°C t o a solution of Boc-Gly-OH (29.8 g, 170 mmoles) and NMM (18.7 mL, 170 mmoles) in chloroform (600 mL). The temperature was kept a t - 15°C for 1 min, then HCOO-H,C-Val-Gly-GlyOEt (52.0 g, 170 mmoles) and NMM (18.7 mL, 170 mmoles) were added. The mixture was kept a t room temperature for 24 h. The organic solution w a s washed with 5% sodium bicarbonate, water, 5 5 citric acid, water, and dried over sodium sulphate. The solution was evaporated t o dryness, and the residue was crystallized from chlorofonndiethyl ether. Yield: 20.0 g (as%)), mp 145-146"C, [a]f:I= t 5 . 6 (c, 1.0; DMF).
,
I H-nmr (CDCI !): y CH 0.92 (d, 6H), CH (OEt) 1.24 (t, 3H), CH (Boc) 1.43 (s, 9H), PCH 2.05 (bm, lH), aCH, 4.02 ( b , 6H), CH, (OEt) 4.19 (4,2H), aCH 4.48 (bm, lH), NH ( G l v , ) 5.77 (b, lH), NH 7.70 (b, 3H).
851
mmoles) in chloroform (50 mL). The temperature was kept a t -15°C for 1 min, then C1 H;-LeuOMe (2.6 g, 14.5 mmoles) and NMM (1.6 mL, 14.5 mmoles) were added. The mixture was kept a t room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from petroleum ether. Yield: 6.0 g (88%), mp 78-80"C, [.If:' = -23.2 (c, 1.0; DMF). 'H nmr (CDCI,): SCH, 0.94 (b, 12H), CH (Hoc) 1.43 (s, 9H), PCH, + CH 1.63 (b, 3H), CH,(OMe) 3.72 (s, 3H), a C H , 3.98 (b, 4H), aCH 4.42 (b, 2H), N H (I,eu,) 5.47 (d, lH), NH 7.17 (b, 1H), NH 7.52 (b, 2H). Anal. calc. for: C,,H,,,N,O,: C 55.91 H 8.53 N 11.86. Found: C 55.82 H 8.57 N 11.74.
Boc-Leu-Gly-Gly-Val-OMe Anal. calc. for C,,H,,,N,07 . 1/3H,O: C 51.17 H 7.81 N 13.25. Found: C 51.24 H 7.90 N 13.15.
Boc- Val-Gly-Gly- Val-OMe
Isobutyl chloroformate (1.6 mL, 12 mmoles) was added a t - 15°C t o a solution of Boc-Val-Gly-GlyOH (4.0 g, 12 mmoles) and NMM (1.3 mL, 12 mmoles) in DMF (40 mL). The temperature was kept at -15°C for 1 min, then C1-Hl-Val-OMe (2.2 g, 12 mmoles) and NMM (1.3 mL, 12 mmoles) were added. The mixture was kept a t room temperature for 24 h, then the solvent was evaporated to dryness and the residue was dissolved in chloroform. The obtained solution was washed with 5% sodium bicarbonate, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated t o dryness, and the residue was crystallized from chloroform-diethyl ether. Yield: 5.0 g (94%,),mp 120 122"C, [a]:; = -7.0 (c, 1.0; DMF). 'H-rimr (CIICI $): yCH 0.97 (d, 12H), C H , (Boc) 1.43 (s, 9H), PCH 2.06 (m, lH), CH,(OMe) 3.72 (s, 3H), aCH, 4.03 (hd, 4H), a C H 4.56 (d, lH), N H (Val,) 5.48 (d, 1H), NH (Val ,) 7.23 (d, lH), NH (Gly) 7.43, 7.53 (bt, 2H). Anal. calc. for C,,,H,,,N,07: C 54.04 H 8.41 N 12.60. Found: C 54.10 H 8.40 N 12.56.
Boc- Leu-Gly-Gly- Leu-OMe
Isobutyl chloroformate (1.9 mL, 14.5 mmoles) was added a t - 15°C to a solution of Boc-Leu-Gly-GlyOH (5.0 g, 14.5 mmoles) and NMM (1.6 mL, 14.5
Isobutyl chloroformate (13.1 mL, 100 mmoles) was added a t - 15°C to a solution of Boc-Leu-Gly-GlyOH (35.0 g, 100 mmoles) and NMM ! L1 mL, 100 mmoles) in chloroform (350 mL). The temperature was kept a t -15°C for 1 min, then C1 Hi-ValOMe (16.8 g, 100 mmoles) and NMM ( 11 mL, 100 mnoles) were added. The mixture was kept a t room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid. water, and dried over sodium sulpnate. The solution was evaporated to dryness, and the residue was crystallized from chlorofoi-mdiethyl ether. Yield: 41.8 g (91%),mp 144&145"C, = -18.0 (c, 1.0; DMF).
$I.[
'H-nmr (CDCI j ) : yCH, + SCH, 0.88, 0.97 (b, 12H), CH ,(Boc) 1.42 (s, 9H), PCH, 1.60 (bm, 2H), fiCH + yCH 2.15 (b, ZH), CH,(OMe) 3.68 (s, 3H), aC'H, 3.97 (bm, 4H), aCH 4.42 (b, 2H), NH(Leu) 5.37 (d, IH), NH 7.32 (b, 3H). Anal. calc. for C,,H,,N,O, 1/3H,O: C 34.29 H 8.39 N 12.05. Found: C 54.16 H 8.30 N 12.00.
HCOO - H2+-Leu - Gly- Gly- Val- O M e Boc-Leu-Gly-Gly-Val-OMe (41.2 g, 89 mmoles) was dissolved in 98% formic acid (450 mL). After stirring for 2 h a t room temperature, the solution was evaporated t o dryness, and the residue was crystallized from chloroform-diethyl ether. Yield: 36.0 g (99%),mp 121-122"C, [a]:: = - 10.0 ( c , 1.0; DMF). 'H-nmr (dimethylsulphoxide : M e l S O - d
):
yCH
,+
852
GUANTlERI ET AL.
SCH, 0.90 (b, 12H), PCH, 1.48 (bm, 2H), PCH + yCH 1.88 (b, 2H), CH,,(OMe) 3.62 (s, 3H), aCH, 3.73 (b, 4H), aCH 4.15 (b, 2H), NH; 6.75 (b, 3H), HCOO-+ NH 8.15 (b, 4H). Anal. calc. for C,,H,sN,O,: C 50.48 H 7.99 N 13.85. Found: C 50.47 H 8.24 N 13.50.
Boc-Gly-Leo-Gly-Gly- Val-OMe
Isobutyl chloroformate (5.8 mL, 44 mmoles) was added a t - 15°C to a solution of Boc-Gly-OH (7.8 g, 44 mmoles) and NMM (4.89 mL, 44 mmoles) in chloroform (200 mL). The temperature was kept a t - 15°C for 1 min, then HCOO-Hl-Leu-Gly-GlyVal-OMe (18.0 g, 44 mmoles) and NMM (4.89 mL, 44 mmoles) were added. The mixture was kept at room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from chloroformdiethyl ether. Yield: 20.0 g (88%), mp 17O-17l0C, [a];; = -15.8 (c, 1.0; DMF).
+
'H-nmr (Me,SO - dti): yCH,, SCH:, 0.91 (b, 12H), CH,,(Boc) 1.43 (s, 9H), PCH, 1.54 (m, 2H), yCH 1.65 (bm, lH), PCH 2.09 (m, lH), aCH,(Gly,) 3.62 (d, 2H), CH,(OMe) 3.68 (s, 3H), aCH,(GIy,) 3.73 (d, 2H), aCH,(Gly,) 3.81 (d, 2H), aCH(Va1) 4.21 (pt, lH), aCH(Leu) 4.35 (bm, lH), NH(Gly,) 7.01 (t, lH), NH(Leu) 7.96 (d, lH), NH(Gly,) 8.06 (bt, lH), NH(Va1) 8.15 (d, lH), NH(Gly,) 8.33 (bt, 1H).
Anal. calc. for C,,,H,,N,O,: C 53.57 H 8.03 N 13.58. Found: C 53.65 H 8.16 N 13.74.
Boc- Val-Gly-Gly- Leu-OMe
NMM (4.05 mL, 36 mmoles) was added to a solution of Cl-H,'-Leu-OMe (6.7 g, 36 mmoles) in chloroform (50 mL). Then Boc-Val-Gly-Gly-ONp (15.0 g, 33 mmoles) was added. The solution was stirred at room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from chloroformdiethyl ether. Yield: 14.8 g (go%), mp 120-122"C, [a];; = - 12.4 (c, 1.0; DMF). 'H-nmr (Me,SO-d,): yCH,, + SCH,, 0.93 (b, 12H), CH,(Boc) 1.43 (s, 9H), PCH, 1.65 (bm, 2H), PCH + yCH 2.02 (b, 2H), CH,,(OMe) 3.67 (s, 3H), aCH, 3.76 (b, 4H), aCH 4.42 (b, 2H), NH(Va1) 6.75 (d, 1H), NH 8.13 (b, 3H).
Anal. calc. for C,,H,,N,O,: C 55.00 H 8.37 N 12.21. Found: C 54.92 H 8.40 N 12.11. HCOO - H2+-Val-Gly-Gly-Leu-OMe
Boc-Val-Gly-Gly-Leu-OMe (10.2 g, 22 mmoles) was dissolved in 98% formic acid (110 mL). After stirring for 2 h a t room temperature, the solution was evaporated to dryness, and the residue was crystallized from chloroform-diethyl ether. Yield: 8.2 g (91%), mp 148-149"C, [(w]2,0 = -16.1 (c, 1.0; DMF).
,
'H-nmr (Me,SO-d,): Y C H ,+ ~ 6CH 0.92 (b, 12H), PCH, 1.53 (b, 2H), PCH + yCH 1.83 (b, 2H), CH,(OMe) 3.62 (s, 3H), aCH, 3.78 (b, 4H), aCH 4.28 (b, 2H), NH; 7.32 (b, 3H), HCOO- 8.26 (s, lH), NH 8.57 (b, 3H). C 50.48 H 7.99 N 13.85. Anal. calc. for C,,H,,,N,O,: Found: C 50.46 H 7.95 N 13.58.
Boc-Gly- Val-Gly-Gly-Leo-OMe
Isobutyl chloroformate (2.6 mL, 20 mmoles) was added at - 15°C to a solution of Boc-Gly-OH (3.5 g, 20 mmoles) and NMM (2.19 mL, 20 mmoles) in chloroform (60 mL). The temperature was kept a t - 15°C for 1 min, then HCOO-H,+-Val-Gly-GlyLeu-OMe (8.2 g, 20 mmoles) and NMM (2.19 mL, 20 mmoles) were added. The mixture was kept a t room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from chloroformdiethyl ether. Yield: 5.0 g (50%), mp 105-107"C, [ a ] E = -11.5 (c, 1.0; DMF). 'H-nmr (Me,SO-d,): yCH, + SCH, 0.89 (bm, 12H), CH,(Boc) 1.45 (s, 9H), PCH, 1.61 (bm, 2H), yCH 1.68 (bm, lH), PCH 2.03 (m, lH), aCH,(Gly,) 3.64 (bd, 2H), CH,(OMe) 3.66 (s, 3H), aCH,(Gly, + Gly,) 3.79 (b, 4H), aCH(Va1) 4.23 (bm, lH), aCH(Leu) 4.35 (bm, lH), NH(Gly,) 7.06 (bt, lH), NH(Va1) 7.73 (d, lH), NH(Gly,) 8.12 (bt, lH), NH(Leu) 8.25 (d, lH), NH(Gly,) 8.37 (bt, 1H). Anal. calc. for C,,,H,,N,O,: C 53.57 H. 8.03 N 13.58. Found: C 53.54 H 8.06 N 13.54.
Boc- Gly- Val- Gly- Gly-Leu- NHNH,
To a solution of Boc-Gly-Val-Gly-Gly-Leu-OMe (10.0 g, 19.3 mmoles) in methanol (130 mL), hydrazine monohydrate (7.8 mL) was added. The reaction mixture was kept under stirring for 4 days a t room temperature, then evaporated to dryness, and
SYNTHETIC E'IIAGMENTS. I
853
the residue was crystallized from acetone-diethyl ether. Yield: 4.4 g (88%), mp 189-191°C, [ a ] : = - 8.7 (c, 1.0; DMF).
Anal. calc. for C,,,H70N,00,,1:C 53.43 H 7.86 N 15.57. Found: C 53.15 H 7.84 N 15.64.
'H-nmr (Me,SO-d,) yCH,, + 6CH,? 0.88 (b, 12H), CH,,(Boc) 1.38 (s, 9H), PCH, 1.49 (b, 2H), PCH + yCH 1.71 (b, 2H), aCH, + NH, 3.72 (b, 8H), aCH 4.05 (b, 2H), NH(Gly,) 6.95 (b, lH), NH 7.98 (b, 4H), NH(hydr) 10.06 (b, IH).
Boc-(Gly-Val-Gly-Gly-Leu),OMe (3 g, 3.33 mmoles) was dissolved in a 3 N solution of hydrochloric acid in ethyl acetate. After stirring for 2 h a t room temperature, the solution was evaporated to dryness and the residue was crystallized from diethyl ether. Yield: 2.6 g (93%),mp 205-207°C.
CI - H,+ -(Gly- Val-Gly-Gly-Leu),- O M e
Anal. calc. for C,,H,,NiOT: C 51.25 H 8.01 N 19.02. Found: C 51.29 H 8.16 N 18.81.
CI - H,'
- Gly- Val- Gly- Gly-Leu- O M e
Boc-Gly-Val-Gly-Gly-Leu-OMe (7.8 g, 15 mmoles) was dissolved in a 3 N solution of hydrochloric acid in ethyl acetate. After stirring for 1 h a t room temperature, the solution was evaporated to dryness and the residue was crystallized from diethyl ether. Yield: 6.5 g (95%), mp 166-168"C, [ a ] ; ; = - 15.7 (c, 1.0; DMF). H-nmr (Me,So-d,,): yCH + 6CH,, 0.92 (b, 12H), PCH, CH 1.60 (b, 4H), CH,(OMe) 3.57 (s, 3H), a C H , 3.70 (b, 6H), aCH 4.20 (b, 2H), NH; + NH 8.30 (b, 7H).
+ yCH + j3
Anal. calc. for C,,H,,IN-,O,jCl . 1/2H,O: C 46.90 H 7.65 N 15.19. Found: C 46.99 H 7.65 N 14.98.
Boc-(Gly- Val- Gly-Gly-Leu),-OMe t-Butyl nitrite (0.65 mL, 6.00 mmoles) was added t o a stirred solution, at -15"C, of Boc-Gly-Val-
Gly-Gly-Leu-NHNH, (2.5 g, 4.77 mmoles) in DMF (47 mL) containing 4 mL (19.11 mmoles) of 4.8 N hydrochloric acid in dioxane. The solution was stirred for 10 min a t - 15"C, then cooled to - 60°C and neutralized with NMM (2.12 mL, 19.11 mmoles). T o this azide solution, an ice-cold solution of 2.0 g (4.4 mmoles) of ClVH,t-Gly-Val-GlyLeu-OMe and 0.49 mL (4.4 mmoles) of NMM in D M F (47 mL) were added. The reaction mixture was kept stirring a t 5°C for 5 days and a t room temperature for 1 day, then concentrated and the product precipitated with water. The solid material was filtered and washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over P,O-). Yield: 2.7 g (68%), mp 238-239"C, [a];: = -- 16.0 ( c , 1.0; DMF). 'H-nmr (MeLSO-d,,): yCH, + SCH, 0.83 (b, 24H), CH ((Boc) 1.38 ( s , 9H), PCH, 1.55 (b, 4H), PCH + yCH 2.03 (b, 4H), CH {(OMe)3.62 (s, 3H), aCH, 3.75 (b, 12H), aCH 4.22 (b, 4H), NH(Gly,) 6.93 (b, lH), NH 7.70 (b, 3H), N H 8.25 (b, 6H).
'H-nmr (Me,SO-d,): yCH + 6CH I 0.86 (b. 24H), PCH, 1.57 (b, 4H), PCH + yCH 1.97 (b, 4H), CH ,((>Me)3.60 (s, 3H), aCH, 3.73 (b, 12H), aCH 4.23 (b, 4H), N H ; + NH 8.18 (b, 12H). Anal. Calc. for C,,,H,,,N,,,O,,Cl . H,O: C 49.25 H 7.69 N 16.40. Found C 49.10 H 7.77 N 16.00.
Boc-(Gly- Val-Gly-Gly-Leu),- O M e
t-Butyl nitrite (0.53 mL, 4.9 mmoles) b a s added to a stirred solution, a t - 15"C, of Boc-Gly-Val-GlyGly-Leu-NHNH, (2.0 g, 3.9 mmoles) in DMF (33 mL) containing 3.25 mL (15.6 mmoles) of 4.8 N hydrochloric acid in dioxane. The solution was kept stirring for 10 min a t - 15"C, then cooled to -60°C and neutralized with NMM (1.74 mL, 15.6 mmoles). To this azide solution, an ice-cold solution of C1 -H,' -(Gly-Val-Gly-Gly-Leu)2-OMe(3.0 g, 3.6 mmoles) and 0.44 mL (3.6 mmoles) of NMM in DMF (5 mL) were added. The reaction mixture was kept stirring a t 5°C for 5 days and a t room temperature for 1 day, then concentrated and the product precipitated with water. The solid material was filtered and washed with 5% sodium bicarbonate, water, 5% citric acid, water and dried over P,05. Yield: 3.2 g (70%),mp 245-248"".
,
I H-nmr (Me,SO-d,): yCH + SCH 0 &1 (h, 36H), CH,(Boc) 1.38 ( s , 9H), PCH, 1.52 (b, 6H). PCH + yCH 1.97 (b, 6H), CH,(OMe) 3.63 (s, 3H), aCH, 1.77 (b, 18H), aCH 4.07 (b, 6H), NH(Gly,) 7.02 (h, 1H). N H 7.77 (b, 5H), NH 8.30 (b, 9H).
Anal. calc. for C,7H,,,N,r,0,, 3/2H,O: C 52.23 H 7.87 N 16.04. Found: C 52.33 H 7.88 N 15.94.
REFERENCES 1. Tamburro, A. M. & Guantieri, V. (1986) Int. J . Biol.
Macromol. 8, 62-63. 2. Guantieri, V., Tamburro, A. M., Cabroi, I)., Hroch, H. & Vasilescu, D. (1987) Znt. J . Pepti& Protein Res. 29, 216-230.
854
GUANTIERI ET AL.
3. Urry, D. W. (1982) Methods Enzymol. 82, 673-716, and references cited therein. 4. Cicila, G., May, M., Omstein-Goldstein, N., Indik, Z., Morrow, S., Yeh, H. S., Rosenbloom, J. C., Boyd, C., Rosenbloom J. C. & Yoon, K. (1985) Biochemistry 24, 3075-3080. 5. Bressan, G., Argos, P. & Stanley, K. K. (1987) Biochemistry 26, 1497-1503. 6 . Raju, K. & Anwar, R. A. (1987) J . Biol. Chem. 262, 5755-5762. 7. Guantieri, V., Jaques, A. M., Serafmi Fracassini, A. & Tamburro, A. M. (1987) Biopolymers 26, 1957-
1963. 8. Brach, A. & Spach, G. (1968) Peptzdes. North-Holland, Amsterdam, pp. 45-49. 9. Anderson, J. M., Rippon, W. H. & Walton, A. G. (1970) Biochem. Biophys. Res. Commun. 39, 802808. 10. Solomon, 0. F. & Ciuta, I. %. (1962) J . Appl. Polym. Sci. 24, 683-686.
Received February 16, 1988 Accepted March 22, 1989
* D q ) a r t n i e n t of Inorganic Metallorganic and Analytical Chemistry, University of Padova, Italy of Chemistry, University of Basilicata, Potenza, Italy
' Dcy)artment
SY NOPSlS
The synthesis of some repetitive sequences of elastin and their simplified analogues, all comprising the structural unit Gly-X-Gly (X = Val, Leu, Ala), is described. In particular, the following peptides and polypeptides were synthesized and characterized: Boc-Gly-ValGly-Gly-Leu-OMe, Boc-Gly-Leu-Gly-Gly-Val-OMe, Boc-(Gly-Val-Gly-Gly-Leu),-OMe, Boc-(Gly-Val-Gly-Gly-Leu),i-OMe, Boc-Gly-Val-Gly-Gly-OEt, Boc-Leu-Gly-Gly-Leu-OMe, Boc-Val-Gly-Gly-Val-OMe, poly(A1a-Gly-Gly),poly(Va1-Gly-Gly), and poly(Leu-Gly-Gly). In every case, the synthesis w a s accomplished by classical procedures in solution, by using the p-nitrophenyl ester method for the polycondensation step, and the mixed anhydride or the a i d e methods for the coupling steps.
I NTRO DUCT I 0N Elastin, the protein responsible for tissue resiliency, is characterized by a unique primary structure consisting of hydrophobic residues frequently arranged in repeating sequences. As representatives of these regions, the prolinecontaining polytripeptide, (Val-Pro-Gly),,'f2 polytetrapeptide, (Val-Pro-Gly-Gly),, polypentapeptide, (Val-Pro-Gly-Val-Gly),, and polyhexapeptide, (Val-Ala-Pro-Gly-Val-Gly), were synthesized and extensively studied., In all cases the prominent conformational feature of these sequences appeared to be the so-called p-turn, the stability of this particular conformation being strongly enhanced under conditions of low water activity. More recently, additional repeating sequences have been deduced from gene ~ t r u c t u r e ~ comprising -~ Gly-X-Gly triplets, where X = Ala, Leu, or Val, suggesting the possibility that these triplets may have originated from single-base mutations and tandem duplications from a common ancestor. Also
c 1990 John Wiley & Sons, Inc.
CCC OOOS-3525/90/040845-10 $04.00 Biopolymers, Vol. 29, 845-854 (1990)
conserved is the pentapeptide sequence Gly-ValGly-Gly-Leu, of particular interest because it repeats itself twice in the currently known",6 amino acid sequences of beef and pig tropoelastin, and three times those of the sheep and chick. Furthermore, the closely related isomeric sequence GlyLeu-Gly-Gly-Val is also present in different species. Given the unusual abundance of repetitive sequences in elastin, it may be reasonable to assign a priori an important conformational role to them (for a discussion, see Ref. 7). Accordingly, we thought useful to synthesize, as simplified models, the polytripeptides poly(A1a-Gly-Gly), poly(Va1-Gly-Gly), and poly(Leu-Gly-Gly) (general sequence Gly-X-Gly), the two protected pentapeptides Boc-Gly-Val-Gly-Gly-Leu-OMe and Boc-GlyLeu-Gly-Gly-Val-OMe, and the repeating sequences Boc-(Gly-Val-Gly-Gly-Leu),-OMe and Boc-(Gly-Val-Gly-Gly-Leu),-OMe. In addition, short related sequences, such as Boc-Gly-Val-GlyGly-OEt, Boc-Leu-Gly-Gly-Leu-OMe, Boc-ValGly-Gly-Val-OMe, and Boc-Leu-Gly-Gly-Val-OMe were synthesized for comparative purposes. The synthetic procedures are described in the present paper while the related conformational studies in solution are reported in the following one. 845
846
GUANTIERI ET AL.
RESULTS
(Fig. 2) were obtained by mixed anhydride coupling of Boc-X-OH with the above-mentioned dipeptide, saponification of the alkyl esters, formation of the corresponding p-nitrophenylesters, removal of the Boc protecting group, and finally, by polycondensation in the presence of N-methylmorpholine. The solutions were concentrated as much as possible in
The general scheme utilized for the synthesis is shown in Fig. 1. The rationale was to minimize the number of intermediates by selecting as starting sequence the N-deblocked dipeptide C1-H: -GlyGly-OEt. Briefly, the three (X-Gly-Gly), polymers
.-\
(Leu-Gly-Gly)
( Ala-Gly-Gly)
1
/H;-Leu-OMe
BOC-Leu-Gly-Gly-OEt
BOC -A1 a-G1y -G 1 y -0Et
\
\
BOC-Leu-OH
BOC-A1 a-OH
\
/
t
BOC-Leu-Gly-Gly-Val-OMe 1
H -Gly-Gly-OEt
I I
BOC-Gly-Leu-Gly-Gly-Val-OMe
BOC-Val -OH
BOC-Gly-OH
U O C -V a 1 -G1 y -G 1 y - OE t /
H;-Val-OMe
L
2
( V a 1 -G 1 y -G 1 y ) "
BOC-Leu-Gly-Gly-Leu-OMe 7l
*BOC-Gly-Val-Gly-Gly-OEt
\
BOC-Val-Gly-Gly-Leu-OMe
BOC-V a1 -G 1 y-G1 y-V a1 -0Me
I I
BOC-Gly-OH
BOC-Gly-Val-Gly-Gly-Leu-OMe
BOC-(Gly-Val-Gly-Gly-Leu) -0Me 2
i
BOC-(G1y -Val -G1y-G1y -Leu 1 -0Me 3
Figure 1. General scheme for the synthesis of fragments and analogues of elastin. Only the lengthening of the chain is shown, as the detailed schemes for the deblocking and activation steps are reported in the following figures
BOC
OEt
NaOH,Ht
BOC
BOC-
HONp,OCC HC1 o r H C O O H
HS (-
OH
I
NP
0 - - -
~
N i4 M
~-
ONP
I
SYNTHETIC FRAGMEN'I'S. I
aoc
OH
H
OE t
I,aiiH,H
5OC
t
HOhp,OCC
BOC
OH ONP
N M 1-1
BOC BOC
OEt
15C
50C
H
-0N I e -9Me
HCOOH
OH
847
-ORe
-d I 4 e
X
Leu,
Y ~ -V a l
X
'Val.
Y z
1.eu
Figure 3. Scheme for the synthesis of the pentapeptides Boc-Gly-Val-Gly-Gly-Leu-OMe and Boc-Gly-Leu-Gly-Gly-Val-OMe
order t o minimize the formation of cyclic peptides. A rough fractionation of the polymers was achieved by exhaustive washings with water in order to remove the low molecular weight fraction. The synthesis of poly(A1a-Gly-Gly), in which benzyloxycarbonyl was used as the N-protecting group, has been previously published.', The isomeric pentapeptides (Fig. 3 ) were obtained by coupling the N-terminal and C-terminal amino acids, respectively, on the Val-Gly-Gly and Leu-Gly-Gly blocks previously synthesized. On the other hand, the protected deca- and pentadecapeptides were synthesiLed by using the azide coupling method in order t o avoid racemization (Fig. 4). Short reference sequences were obtained according t o the scheme outlined in Fig. 5.
Gly
Vdl
Gly
Gly
Leu
Gly
Val
Wherever the synthetic step could have potentially induced racemization, care was taken to avoid i t by setting glycine as the C-terminal amino acid.
EXPERIMENTAL Boc amino acids were purchased from Novabiochem AG (Laufelfingen, Switzerland) and glycylglycine ethyl ester hydrochloride was purchased from Sigma (St. Louis, MO, USA). The p u i t y of the synthetic products was ascertained by thin layer chromatography on butanolacetic acid-water (3 : 3 : 1) and chloroform-acetone (3 : 2). Further characterization was obtained by
Gly
Gly
Leu
Gly
Val
Gly
BOC
Rnr --Figure 4. Scheme for the synthesis of the decapeptide Boc-(Gly-Val-Gly-Gly-Leu),-OMe and of the pentadecapeptide Boc-(Gly-Val-Gly-Gly-Leu):,-OMe
Gly
Leu
848
GUANTIERI ET AI,.
BOC Val
BOC
HCOOH
OH
OE t UE t
BOC
BOC
Val
NaOH, ti
BOC
0ble
Y V I l
" I I C
Figure 5. Scheme for the synthesis of the tetrapeptides Boc-Val-Gly-Gly-Val-OMe, Boc-Gly-Val-Gly-Gly-OEt, and Boc-Leu-Gly-Gly-Leu-OMe
'H-nmr spectra recorded on a 60-MHz Varian EM 360A spectrometer. Synthesis of compounds
Boc-X-Gly-Gly-OEt (X = Ala, Val, Leu) Isobutyl chloroformate (0.2 moles) was added a t - 15°C to a solution of Boc-X-OH (0.2 moles) and N-methylmorpholine (NMM; 0.2 moles) in chloroform (800 mL). The temperature was kept a t -15°C for 1 min, then Cl-H,i-Gly-Gly-OEt (0.2 moles) and NMM (0.2 moles) were added. The mixture was kept at room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from the appropriate solvent. The physical properties, nmr parameters, and elemental analyses of these intermediate compounds are reported in Tables I and 11. Boc-X- Gly-Gly-OH
To a solution of Boc-X-Gly-Gly-OEt (187 mmoles) in acetone (500 mL), 1 N sodium hydroxide (205
mL) was added. The solution was stirred at room temperature for 3 h, then the organic solvent was evaporated under reduced pressure and water was added. The unreacted product was extracted with ethyl acetate, and the aqueous solution was cooled a t 4°C and neutralized with dropwise addition of 1N hydrochloric acid (205 mL). The crude material, which precipitated immediately, was extracted with ethyl acetate and dried over sodium sulphate. The solution was evaporated to dryness and the residue was crystallized from the appropriate solvent. The physical properties, nmr parameters, and elemental analyses of these intermediate compounds are reported in Tables I and 11. Boc-X- Gly-Gly- 0Np N,N'-dicyclohexylcarbodiimide (DCC; 43 mmoles) was added to an ice-cold solution of Boc-X-GlyGly-OH (43 mmoles) and p-nitrophenol (HONp; 43 mmoles) in chloroform (200 mL). The mixture was stirred at 0°C for 1 h and a t room temperature for 12 h. The N,N'-dicyclohexylurea was filtered, and the organic solution was washed with 5% sodium bicarbonate, water, dried over sodium sulphate,
SYNTHETIC FRAGMENTS. I
849
Table I Phvsical ProDerties and Elemental Analyses of Intermediate Compounds Elemental Analvsis (%) Compound
Melting Point ( O C) (Crystallization Solvent)
Yield
Boc-Leu-Gly-Gly-OEt
86-87 Diethyl ether 98-99 Boc-Ala-Gly-Gly-OEt Petroleum ether Boc-Val-Gly -Gly-OEt 79-80 Petroleum ether Boc-Leu-Gly-Gly-OH 118- 120 Petroleum ether Boc-Ala-Gly-Gly-OH 161-162 Petroleum ether Boc-Val-Gly-Gly-OH 148-150 Diethyl ether Boc- Leu-Gly-Gly-ONp 93-95 Diethyl ether/petroleum ether Boc-Ala-Gly-Gly-ONp 111- 112 Petroleum ether Boc-Val-Gly-Gly-ONp 108-109 Diethyl ether C1- H l -Leu-Gly-Gly-ONp 192-194 Diethyl ether C 1 H; ~ -Val-Gly-Gly-ONp 204-206 Diethyl ether HCOO H i -Val-Gly-Gly-OEt Oil
and evaporated to dryness. Trituration of the oil residue with petroleum ether or diethyl ether gave a solid product. The physical properties, nmr parameters, and elemental analyses of these intermediate compounds are reported in Tables I and 11. CI - H,+-X-Gly-Gly-ONp
Boc-X-Gly-Gly-ONp (76 mmoles) was dissolved in 3 N hydrochloric acid in dioxane (640 mL). After stirring for 1 h a t room temperature, the peptide precipitation was completed by addition of diethyl ether. The physical properties, nmr parameters, and elemental analyses of these intermediate compounds are reported in Tables I and 11. HCOO - H,+ -Ala-Gly-Gly- ONp Five grams (12 mmoles) of Boc-Ala-Gly-Gly-ONp were dissolved in 98% formic acid (59 mL). After stirring for 3 h a t room temperature, the solution was evaporated to dryness, giving 10.6 g of an uncrystallizable oil (yield 95%), which was directly used in the following step.
k'ound
CalC.
[.I$'
(%)
(c, 1.0, DMF)
94
-9.9
53.38 8.45 10.98 53.42 8.85 10.87
57
-1.2
50.74 7.62 12.67 50.79 7.69 12.65
91
+4.0
53.46 8.15 11.69 53.44 8.16 11.67
77
-11.5
52.16 7.89 12.16 52.04 7.84 12.13
50
-2.1
47.52 6.99 13.85 47.63 7.26 13.68
84
+0.7
50.74 7.62 12.67 50.69 7.50 12.62
89
-6.9
54.07 6.49 12.00 54.20 6.56 11.98
54
-1.9
50.94 5.71 13.19 51.00 5.81 13.08
74
+2.2
53.09 6.25 12.38 53.16 6.17 12.25
93
+4.8
47.70 5.77 13.90 47.52 5.70 13.77
96
+52.1
46.33 5.45 14.40 46.19 5.49 14.15
99
-
C
-
H
-
N
-
C
-
H
-
N
-
Poly(X- Gly-Gly) To a solution of Cl-H,+-X-Gly-Gly-ONp or HCOO-Hl -Ala-Gly-Gly-ONp (70 mmoles) in anhydrous N,N'-dimethylformamide (DMF; 80 mL), NMM (99 mmoles) was added. After stirring for 3 days a t room temperature, the polypeptide precipitation was completed by addition of diethyl ether, repeatedly washed with water, and dried in vacuo over P,O,. The physical properties and nmr parameters of these compounds are reported in Table 111. HCOO - H,f - Val-Gly-Gly-OEt
Boc-Val-Gly-Gly-OEt (61.6 g, 171 mmoles) was dissolved in 98% formic acid (800 mL). After stirring for 2 h a t room temperature, the solution was evaporated to dryness, giving an uncrystallizable oil (52.0 g), which was directly used in the following step (yield 99%). 'H-nmr (CDC1,): yCH,, 1.05 (d, 6H), CH,(OEt) 1.26 (t, 3H), PCH 2.12 (b, lH), aCH, 4.02 (b, 4H), CH,(OEt) 4.12 (9, 2H), aCH 4.33 (b, lH), NH(G1y) 7.90 (bt, lH), HCOO- 8.18 (s, lH), NH(G1y) 8.45 (b, lH), NH; 9.50 (bs, 3H).
850
GUANTIERI ET AI,.
Table I1
H-nmr Data of Intermediate Compounds in CIICI.,
Boc-Leu-Gly-Gly-OEt
1.25 ( t , 3H) 1.25 (t, ,3H) 1.26 ( t , 3H)
Hoe-Ala-Gly-Gly-OEt Boc-Val-Gly-Gly-OEt
0.88
- 3H)
(d,
1.28 (m, 3H)
Boc-Leu-Gly-Gly-OH
Roc-Val-GigGly-OH"
0.83 (d, 6H)
1.43 (s, 9H) 1 .:18 (s, 9H) 1.40 (s, 9H) 1.39 (s, 9H)
(s,
0.92
C1- H i -1,eu-Gly-Gly-ONp"
1.38 (bin, .3H) I .87 (bin, 1 H ) 1 .38 (hm, 9 H )
9H) 2.05
1.43 (s,9H)
- 2H)
(d,
2.08 IH)
(Ill,
1.:18 (s,9H) 1 .:19
Hoc-Leu-Gly-Gly-ONp Boc-Val-Gly-Gly-ONp
1.52 (hm, RH)
(hm, 1 tl) 1.58 .1H)
(tllll,
CI H i -Val-Gly-Gly-ONp'
0.88
(d,
2.0.3
- 2.5H)
(nl, I H )
HCOO H i -Val-Gly-
Gly-OEt Compound Hoc-Ifiu-Gly-Gly-OEt Boc-Ah-Gly-Gly-OEt Boc-Val-Gly-Gly-OEt Bo~-Leu-Gly-Gly-OH
Boc-Val-Gly-Gly-OH" Boc-Leu-Gly-Gly-OIp Roc-Val-Gly-Gly-ONp
C1- H,-I,eu-GlyGly-ONp' C1 H;-Val-GlyGly-ONg HCOO- H,i-Val-GlyGly-OEt
1.05 (d, 6H) aCH,
aCH
1.26 (t, tjH)
CH,(OEt) OCONH
NH;
2.12
(h I H ) NH(Gly 2) o-ONp NH(G1y 1) ni-ONp
4.03 (bm, 1H) 3.93 (bm, 1H) 3.98 (bm, 1H) 4.17 (bm, 1H) 3.87 (bm, 1H) 4.17 (bm, 1H) 4.25 (bd, 3H)
3.78 (d, 2H)
4.10 (bd, 3H)
8.40 (bs, 3H)
8.73 (t, 1H)
7.33 (d, 2H)
8.98 ( t , 1H)
8.23 (d, 2H)
3.83 (d, 2H)
4.13 (bd, 3H)
8.38 (b, 3H)
8.78 (t, 1H)
7.38 (d, 2H)
8.93 (t, 1H)
(d, 2H)
9.50 (bs, 3H)
7.90 (bt, 1H)
4.02
4.33
(b, 4H)
(b, 1H)
4.12 (q, 2H)
HCOO
r'
4.03 (bm, 4H) 3.93 (bm, 4H) 3.98 (bm, 4H) 4.00 (bm, 4H) 3.66 (bm, 4H) 4.00 (hm, 4H) 4.05 (d, 2H)
4.18 (q, 2H) 4.15 (q,2W 4.15 (q,2H)
OH
-.dt3
5.50 (d, 1H) 5.45 (d, 1H) 5.24 (d, 1H) 5.55 (d, 1H) 6.63 (d, 1H) 5.27 (4 1H) 5.21 (d, 1H)
I
(hm, 2H) 7.28 (bm, 2H) 7.16 (bm, 2H) 7.63 8.92 (bm, 2H) (hs, 1H) 8.05 (hm, 2H) 6.83 7.30 8.04 (d, 2H) (bm, 2H) (d, 2H) 7.15 7.28 7.53 8.25 (bm, 1H) (d, 2H) (bm, 1H) (d, 2H)
8.45 (b, 1H)
8.25 8.18 (s, 1H)
" I n Me,SO-d,;.
Table I11 Properties of Polytripeptides Polycondensation Yield ( W )
[ 71Ia (dL/g)
'H-nmr (Me,SO-d,)
Poly(Leu-Gly-Gly)
90
0.21
Poly(Ala-Gly-Gly)
91
0.14
Poly(Va1-Gly-Gly)
80
0.23
SCH,, 0.87 (b, 6H), BCH, + yCH 1.51 (b, 3H), aCH, 3.67 (b, 4H), olCH 4.17 (b, lH), NH 7.88 (b, 2H), NH 8.07 (b, 1H) PCH,, 1.23 (bd, 3H), aCH, 3.83 (b, 4H), aCH 4.26 (b, 1H), NH 8.13 (b, 3H) Y C H ,0.93 ~ (b, 6H), PCH 1.87 (b, lH), aCH, 3.72 (b, 4H), aCH 4.07 (b, lH), NH 7.45 (b), 7.91 (b), 8.26 (b), 8.58 (b) (3H)
Compound
"Intrinsic viscosity in dichioroacetic acid ( c
=
1%) obtained according to the "single-point" method."'
SYNTHZ'I'IC FIIAGMES'I'S. I
BOC- Gly- Val-Gly- Gly- Oft Isobutyl chloroformate (22.14 mL, 170 mmoles) was added a t - 15°C t o a solution of Boc-Gly-OH (29.8 g, 170 mmoles) and NMM (18.7 mL, 170 mmoles) in chloroform (600 mL). The temperature was kept a t - 15°C for 1 min, then HCOO-H,C-Val-Gly-GlyOEt (52.0 g, 170 mmoles) and NMM (18.7 mL, 170 mmoles) were added. The mixture was kept a t room temperature for 24 h. The organic solution w a s washed with 5% sodium bicarbonate, water, 5 5 citric acid, water, and dried over sodium sulphate. The solution was evaporated t o dryness, and the residue was crystallized from chlorofonndiethyl ether. Yield: 20.0 g (as%)), mp 145-146"C, [a]f:I= t 5 . 6 (c, 1.0; DMF).
,
I H-nmr (CDCI !): y CH 0.92 (d, 6H), CH (OEt) 1.24 (t, 3H), CH (Boc) 1.43 (s, 9H), PCH 2.05 (bm, lH), aCH, 4.02 ( b , 6H), CH, (OEt) 4.19 (4,2H), aCH 4.48 (bm, lH), NH ( G l v , ) 5.77 (b, lH), NH 7.70 (b, 3H).
851
mmoles) in chloroform (50 mL). The temperature was kept a t -15°C for 1 min, then C1 H;-LeuOMe (2.6 g, 14.5 mmoles) and NMM (1.6 mL, 14.5 mmoles) were added. The mixture was kept a t room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from petroleum ether. Yield: 6.0 g (88%), mp 78-80"C, [.If:' = -23.2 (c, 1.0; DMF). 'H nmr (CDCI,): SCH, 0.94 (b, 12H), CH (Hoc) 1.43 (s, 9H), PCH, + CH 1.63 (b, 3H), CH,(OMe) 3.72 (s, 3H), a C H , 3.98 (b, 4H), aCH 4.42 (b, 2H), N H (I,eu,) 5.47 (d, lH), NH 7.17 (b, 1H), NH 7.52 (b, 2H). Anal. calc. for: C,,H,,,N,O,: C 55.91 H 8.53 N 11.86. Found: C 55.82 H 8.57 N 11.74.
Boc-Leu-Gly-Gly-Val-OMe Anal. calc. for C,,H,,,N,07 . 1/3H,O: C 51.17 H 7.81 N 13.25. Found: C 51.24 H 7.90 N 13.15.
Boc- Val-Gly-Gly- Val-OMe
Isobutyl chloroformate (1.6 mL, 12 mmoles) was added a t - 15°C t o a solution of Boc-Val-Gly-GlyOH (4.0 g, 12 mmoles) and NMM (1.3 mL, 12 mmoles) in DMF (40 mL). The temperature was kept at -15°C for 1 min, then C1-Hl-Val-OMe (2.2 g, 12 mmoles) and NMM (1.3 mL, 12 mmoles) were added. The mixture was kept a t room temperature for 24 h, then the solvent was evaporated to dryness and the residue was dissolved in chloroform. The obtained solution was washed with 5% sodium bicarbonate, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated t o dryness, and the residue was crystallized from chloroform-diethyl ether. Yield: 5.0 g (94%,),mp 120 122"C, [a]:; = -7.0 (c, 1.0; DMF). 'H-rimr (CIICI $): yCH 0.97 (d, 12H), C H , (Boc) 1.43 (s, 9H), PCH 2.06 (m, lH), CH,(OMe) 3.72 (s, 3H), aCH, 4.03 (hd, 4H), a C H 4.56 (d, lH), N H (Val,) 5.48 (d, 1H), NH (Val ,) 7.23 (d, lH), NH (Gly) 7.43, 7.53 (bt, 2H). Anal. calc. for C,,,H,,,N,07: C 54.04 H 8.41 N 12.60. Found: C 54.10 H 8.40 N 12.56.
Boc- Leu-Gly-Gly- Leu-OMe
Isobutyl chloroformate (1.9 mL, 14.5 mmoles) was added a t - 15°C to a solution of Boc-Leu-Gly-GlyOH (5.0 g, 14.5 mmoles) and NMM (1.6 mL, 14.5
Isobutyl chloroformate (13.1 mL, 100 mmoles) was added a t - 15°C to a solution of Boc-Leu-Gly-GlyOH (35.0 g, 100 mmoles) and NMM ! L1 mL, 100 mmoles) in chloroform (350 mL). The temperature was kept a t -15°C for 1 min, then C1 Hi-ValOMe (16.8 g, 100 mmoles) and NMM ( 11 mL, 100 mnoles) were added. The mixture was kept a t room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid. water, and dried over sodium sulpnate. The solution was evaporated to dryness, and the residue was crystallized from chlorofoi-mdiethyl ether. Yield: 41.8 g (91%),mp 144&145"C, = -18.0 (c, 1.0; DMF).
$I.[
'H-nmr (CDCI j ) : yCH, + SCH, 0.88, 0.97 (b, 12H), CH ,(Boc) 1.42 (s, 9H), PCH, 1.60 (bm, 2H), fiCH + yCH 2.15 (b, ZH), CH,(OMe) 3.68 (s, 3H), aC'H, 3.97 (bm, 4H), aCH 4.42 (b, 2H), NH(Leu) 5.37 (d, IH), NH 7.32 (b, 3H). Anal. calc. for C,,H,,N,O, 1/3H,O: C 34.29 H 8.39 N 12.05. Found: C 54.16 H 8.30 N 12.00.
HCOO - H2+-Leu - Gly- Gly- Val- O M e Boc-Leu-Gly-Gly-Val-OMe (41.2 g, 89 mmoles) was dissolved in 98% formic acid (450 mL). After stirring for 2 h a t room temperature, the solution was evaporated t o dryness, and the residue was crystallized from chloroform-diethyl ether. Yield: 36.0 g (99%),mp 121-122"C, [a]:: = - 10.0 ( c , 1.0; DMF). 'H-nmr (dimethylsulphoxide : M e l S O - d
):
yCH
,+
852
GUANTlERI ET AL.
SCH, 0.90 (b, 12H), PCH, 1.48 (bm, 2H), PCH + yCH 1.88 (b, 2H), CH,,(OMe) 3.62 (s, 3H), aCH, 3.73 (b, 4H), aCH 4.15 (b, 2H), NH; 6.75 (b, 3H), HCOO-+ NH 8.15 (b, 4H). Anal. calc. for C,,H,sN,O,: C 50.48 H 7.99 N 13.85. Found: C 50.47 H 8.24 N 13.50.
Boc-Gly-Leo-Gly-Gly- Val-OMe
Isobutyl chloroformate (5.8 mL, 44 mmoles) was added a t - 15°C to a solution of Boc-Gly-OH (7.8 g, 44 mmoles) and NMM (4.89 mL, 44 mmoles) in chloroform (200 mL). The temperature was kept a t - 15°C for 1 min, then HCOO-Hl-Leu-Gly-GlyVal-OMe (18.0 g, 44 mmoles) and NMM (4.89 mL, 44 mmoles) were added. The mixture was kept at room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from chloroformdiethyl ether. Yield: 20.0 g (88%), mp 17O-17l0C, [a];; = -15.8 (c, 1.0; DMF).
+
'H-nmr (Me,SO - dti): yCH,, SCH:, 0.91 (b, 12H), CH,,(Boc) 1.43 (s, 9H), PCH, 1.54 (m, 2H), yCH 1.65 (bm, lH), PCH 2.09 (m, lH), aCH,(Gly,) 3.62 (d, 2H), CH,(OMe) 3.68 (s, 3H), aCH,(GIy,) 3.73 (d, 2H), aCH,(Gly,) 3.81 (d, 2H), aCH(Va1) 4.21 (pt, lH), aCH(Leu) 4.35 (bm, lH), NH(Gly,) 7.01 (t, lH), NH(Leu) 7.96 (d, lH), NH(Gly,) 8.06 (bt, lH), NH(Va1) 8.15 (d, lH), NH(Gly,) 8.33 (bt, 1H).
Anal. calc. for C,,,H,,N,O,: C 53.57 H 8.03 N 13.58. Found: C 53.65 H 8.16 N 13.74.
Boc- Val-Gly-Gly- Leu-OMe
NMM (4.05 mL, 36 mmoles) was added to a solution of Cl-H,'-Leu-OMe (6.7 g, 36 mmoles) in chloroform (50 mL). Then Boc-Val-Gly-Gly-ONp (15.0 g, 33 mmoles) was added. The solution was stirred at room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from chloroformdiethyl ether. Yield: 14.8 g (go%), mp 120-122"C, [a];; = - 12.4 (c, 1.0; DMF). 'H-nmr (Me,SO-d,): yCH,, + SCH,, 0.93 (b, 12H), CH,(Boc) 1.43 (s, 9H), PCH, 1.65 (bm, 2H), PCH + yCH 2.02 (b, 2H), CH,,(OMe) 3.67 (s, 3H), aCH, 3.76 (b, 4H), aCH 4.42 (b, 2H), NH(Va1) 6.75 (d, 1H), NH 8.13 (b, 3H).
Anal. calc. for C,,H,,N,O,: C 55.00 H 8.37 N 12.21. Found: C 54.92 H 8.40 N 12.11. HCOO - H2+-Val-Gly-Gly-Leu-OMe
Boc-Val-Gly-Gly-Leu-OMe (10.2 g, 22 mmoles) was dissolved in 98% formic acid (110 mL). After stirring for 2 h a t room temperature, the solution was evaporated to dryness, and the residue was crystallized from chloroform-diethyl ether. Yield: 8.2 g (91%), mp 148-149"C, [(w]2,0 = -16.1 (c, 1.0; DMF).
,
'H-nmr (Me,SO-d,): Y C H ,+ ~ 6CH 0.92 (b, 12H), PCH, 1.53 (b, 2H), PCH + yCH 1.83 (b, 2H), CH,(OMe) 3.62 (s, 3H), aCH, 3.78 (b, 4H), aCH 4.28 (b, 2H), NH; 7.32 (b, 3H), HCOO- 8.26 (s, lH), NH 8.57 (b, 3H). C 50.48 H 7.99 N 13.85. Anal. calc. for C,,H,,,N,O,: Found: C 50.46 H 7.95 N 13.58.
Boc-Gly- Val-Gly-Gly-Leo-OMe
Isobutyl chloroformate (2.6 mL, 20 mmoles) was added at - 15°C to a solution of Boc-Gly-OH (3.5 g, 20 mmoles) and NMM (2.19 mL, 20 mmoles) in chloroform (60 mL). The temperature was kept a t - 15°C for 1 min, then HCOO-H,+-Val-Gly-GlyLeu-OMe (8.2 g, 20 mmoles) and NMM (2.19 mL, 20 mmoles) were added. The mixture was kept a t room temperature for 24 h. The organic solution was washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over sodium sulphate. The solution was evaporated to dryness, and the residue was crystallized from chloroformdiethyl ether. Yield: 5.0 g (50%), mp 105-107"C, [ a ] E = -11.5 (c, 1.0; DMF). 'H-nmr (Me,SO-d,): yCH, + SCH, 0.89 (bm, 12H), CH,(Boc) 1.45 (s, 9H), PCH, 1.61 (bm, 2H), yCH 1.68 (bm, lH), PCH 2.03 (m, lH), aCH,(Gly,) 3.64 (bd, 2H), CH,(OMe) 3.66 (s, 3H), aCH,(Gly, + Gly,) 3.79 (b, 4H), aCH(Va1) 4.23 (bm, lH), aCH(Leu) 4.35 (bm, lH), NH(Gly,) 7.06 (bt, lH), NH(Va1) 7.73 (d, lH), NH(Gly,) 8.12 (bt, lH), NH(Leu) 8.25 (d, lH), NH(Gly,) 8.37 (bt, 1H). Anal. calc. for C,,,H,,N,O,: C 53.57 H. 8.03 N 13.58. Found: C 53.54 H 8.06 N 13.54.
Boc- Gly- Val- Gly- Gly-Leu- NHNH,
To a solution of Boc-Gly-Val-Gly-Gly-Leu-OMe (10.0 g, 19.3 mmoles) in methanol (130 mL), hydrazine monohydrate (7.8 mL) was added. The reaction mixture was kept under stirring for 4 days a t room temperature, then evaporated to dryness, and
SYNTHETIC E'IIAGMENTS. I
853
the residue was crystallized from acetone-diethyl ether. Yield: 4.4 g (88%), mp 189-191°C, [ a ] : = - 8.7 (c, 1.0; DMF).
Anal. calc. for C,,,H70N,00,,1:C 53.43 H 7.86 N 15.57. Found: C 53.15 H 7.84 N 15.64.
'H-nmr (Me,SO-d,) yCH,, + 6CH,? 0.88 (b, 12H), CH,,(Boc) 1.38 (s, 9H), PCH, 1.49 (b, 2H), PCH + yCH 1.71 (b, 2H), aCH, + NH, 3.72 (b, 8H), aCH 4.05 (b, 2H), NH(Gly,) 6.95 (b, lH), NH 7.98 (b, 4H), NH(hydr) 10.06 (b, IH).
Boc-(Gly-Val-Gly-Gly-Leu),OMe (3 g, 3.33 mmoles) was dissolved in a 3 N solution of hydrochloric acid in ethyl acetate. After stirring for 2 h a t room temperature, the solution was evaporated to dryness and the residue was crystallized from diethyl ether. Yield: 2.6 g (93%),mp 205-207°C.
CI - H,+ -(Gly- Val-Gly-Gly-Leu),- O M e
Anal. calc. for C,,H,,NiOT: C 51.25 H 8.01 N 19.02. Found: C 51.29 H 8.16 N 18.81.
CI - H,'
- Gly- Val- Gly- Gly-Leu- O M e
Boc-Gly-Val-Gly-Gly-Leu-OMe (7.8 g, 15 mmoles) was dissolved in a 3 N solution of hydrochloric acid in ethyl acetate. After stirring for 1 h a t room temperature, the solution was evaporated to dryness and the residue was crystallized from diethyl ether. Yield: 6.5 g (95%), mp 166-168"C, [ a ] ; ; = - 15.7 (c, 1.0; DMF). H-nmr (Me,So-d,,): yCH + 6CH,, 0.92 (b, 12H), PCH, CH 1.60 (b, 4H), CH,(OMe) 3.57 (s, 3H), a C H , 3.70 (b, 6H), aCH 4.20 (b, 2H), NH; + NH 8.30 (b, 7H).
+ yCH + j3
Anal. calc. for C,,H,,IN-,O,jCl . 1/2H,O: C 46.90 H 7.65 N 15.19. Found: C 46.99 H 7.65 N 14.98.
Boc-(Gly- Val- Gly-Gly-Leu),-OMe t-Butyl nitrite (0.65 mL, 6.00 mmoles) was added t o a stirred solution, at -15"C, of Boc-Gly-Val-
Gly-Gly-Leu-NHNH, (2.5 g, 4.77 mmoles) in DMF (47 mL) containing 4 mL (19.11 mmoles) of 4.8 N hydrochloric acid in dioxane. The solution was stirred for 10 min a t - 15"C, then cooled to - 60°C and neutralized with NMM (2.12 mL, 19.11 mmoles). T o this azide solution, an ice-cold solution of 2.0 g (4.4 mmoles) of ClVH,t-Gly-Val-GlyLeu-OMe and 0.49 mL (4.4 mmoles) of NMM in D M F (47 mL) were added. The reaction mixture was kept stirring a t 5°C for 5 days and a t room temperature for 1 day, then concentrated and the product precipitated with water. The solid material was filtered and washed with 5% sodium bicarbonate, water, 5% citric acid, water, and dried over P,O-). Yield: 2.7 g (68%), mp 238-239"C, [a];: = -- 16.0 ( c , 1.0; DMF). 'H-nmr (MeLSO-d,,): yCH, + SCH, 0.83 (b, 24H), CH ((Boc) 1.38 ( s , 9H), PCH, 1.55 (b, 4H), PCH + yCH 2.03 (b, 4H), CH {(OMe)3.62 (s, 3H), aCH, 3.75 (b, 12H), aCH 4.22 (b, 4H), NH(Gly,) 6.93 (b, lH), NH 7.70 (b, 3H), N H 8.25 (b, 6H).
'H-nmr (Me,SO-d,): yCH + 6CH I 0.86 (b. 24H), PCH, 1.57 (b, 4H), PCH + yCH 1.97 (b, 4H), CH ,((>Me)3.60 (s, 3H), aCH, 3.73 (b, 12H), aCH 4.23 (b, 4H), N H ; + NH 8.18 (b, 12H). Anal. Calc. for C,,,H,,,N,,,O,,Cl . H,O: C 49.25 H 7.69 N 16.40. Found C 49.10 H 7.77 N 16.00.
Boc-(Gly- Val-Gly-Gly-Leu),- O M e
t-Butyl nitrite (0.53 mL, 4.9 mmoles) b a s added to a stirred solution, a t - 15"C, of Boc-Gly-Val-GlyGly-Leu-NHNH, (2.0 g, 3.9 mmoles) in DMF (33 mL) containing 3.25 mL (15.6 mmoles) of 4.8 N hydrochloric acid in dioxane. The solution was kept stirring for 10 min a t - 15"C, then cooled to -60°C and neutralized with NMM (1.74 mL, 15.6 mmoles). To this azide solution, an ice-cold solution of C1 -H,' -(Gly-Val-Gly-Gly-Leu)2-OMe(3.0 g, 3.6 mmoles) and 0.44 mL (3.6 mmoles) of NMM in DMF (5 mL) were added. The reaction mixture was kept stirring a t 5°C for 5 days and a t room temperature for 1 day, then concentrated and the product precipitated with water. The solid material was filtered and washed with 5% sodium bicarbonate, water, 5% citric acid, water and dried over P,05. Yield: 3.2 g (70%),mp 245-248"".
,
I H-nmr (Me,SO-d,): yCH + SCH 0 &1 (h, 36H), CH,(Boc) 1.38 ( s , 9H), PCH, 1.52 (b, 6H). PCH + yCH 1.97 (b, 6H), CH,(OMe) 3.63 (s, 3H), aCH, 1.77 (b, 18H), aCH 4.07 (b, 6H), NH(Gly,) 7.02 (h, 1H). N H 7.77 (b, 5H), NH 8.30 (b, 9H).
Anal. calc. for C,7H,,,N,r,0,, 3/2H,O: C 52.23 H 7.87 N 16.04. Found: C 52.33 H 7.88 N 15.94.
REFERENCES 1. Tamburro, A. M. & Guantieri, V. (1986) Int. J . Biol.
Macromol. 8, 62-63. 2. Guantieri, V., Tamburro, A. M., Cabroi, I)., Hroch, H. & Vasilescu, D. (1987) Znt. J . Pepti& Protein Res. 29, 216-230.
854
GUANTIERI ET AL.
3. Urry, D. W. (1982) Methods Enzymol. 82, 673-716, and references cited therein. 4. Cicila, G., May, M., Omstein-Goldstein, N., Indik, Z., Morrow, S., Yeh, H. S., Rosenbloom, J. C., Boyd, C., Rosenbloom J. C. & Yoon, K. (1985) Biochemistry 24, 3075-3080. 5. Bressan, G., Argos, P. & Stanley, K. K. (1987) Biochemistry 26, 1497-1503. 6 . Raju, K. & Anwar, R. A. (1987) J . Biol. Chem. 262, 5755-5762. 7. Guantieri, V., Jaques, A. M., Serafmi Fracassini, A. & Tamburro, A. M. (1987) Biopolymers 26, 1957-
1963. 8. Brach, A. & Spach, G. (1968) Peptzdes. North-Holland, Amsterdam, pp. 45-49. 9. Anderson, J. M., Rippon, W. H. & Walton, A. G. (1970) Biochem. Biophys. Res. Commun. 39, 802808. 10. Solomon, 0. F. & Ciuta, I. %. (1962) J . Appl. Polym. Sci. 24, 683-686.
Received February 16, 1988 Accepted March 22, 1989
