Triproline analogues of Pro-Leu-Gly-NH2 with Pro/Leu and Pro/Phe chimeric amino acids in position 2

Article abstract of DOI:10.1016/S0040-4020(00)00887-5

Synthesis of the cis- and trans-isomers of the proline-leucine (3-i-Pr-Pro) and proline-phenylalanine (3-Ph-Pro) chimeric amino acids was accomplished by intramolecular reductive-cycloalkylation of the appropriate azido-olefin. These chimeric amino acids were incorporated into the triprolyl analogues of Pro-Leu-Gly-NH₂: Pro-cis-3-i-Pr-Pro-Pro-NH₂ (3), Pro-trans-3-i-Pr-Pro-Pro-NH₂ (4), Pro-cis-3-Ph-Pro-Pro-NH₂ (5), Pro-trans-3-Ph-Pro-Pro-NH₂ (6). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00887-5

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9801±9808
Triproline Analogues of Pro-Leu-Gly-NH2 with Pro/Leu and
Pro/Phe Chimeric Amino Acids in Position 2
*
Margaret C. Evans and Rodney L. Johnson
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
Received 29 April 2000; accepted 2 August 2000
AbstractÐSynthesis of the cis- and trans-isomers of the proline-leucine (3-i-Pr-Pro) and proline-phenylalanine (3-Ph-Pro) chimeric amino
acids was accomplished by intramolecular reductive-cycloalkylation of the appropriate azido-ole®n. These chimeric amino acids were
incorporated into the triprolyl analogues of Pro-Leu-Gly-NH2: Pro-cis-3-i-Pr-Pro-Pro-NH2 (3), Pro-trans-3-i-Pr-Pro-Pro-NH2 (4), Pro-cis-
3-Ph-Pro-Pro-NH2 (5), Pro-trans-3-Ph-Pro-Pro-NH2 (6). q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
the second prolyl residue is substituted with a phenyl group
were also made.
Pro-Leu-Gly-NH2 (1, PLG) modulates dopamine D2 recep-
tors within the central nervous system. One manifestation of
this modulation in vivo is the ability of PLG to potentiate the
contralateral rotational behavior induced by apomorphine in
rats with unilateral 6-hydroxydopamine lesions of the
nigrostriatal pathway.1±4 PLG brings about this modulation
by increasing the percentage of D2 receptors that exist in the
high-af®nity state, as well as by increasing the af®nity of the
high-af®nity state for dopamine receptor agonists.5±8
Several attempts have been made to de®ne the bioactive
conformation of PLG through the design and synthesis
of conformationally constrained analogues of this tripep-
tide.8±11 In one early study, replacement of the glycinamide
residue with either l- or d-prolinamide yielded active ana-
logues of PLG.12 More recently we found that simul-
taneously replacing both the leucyl and glycinamide
residues of PLG with prolyl residues gave triprolyl ana-
logues with biological activity. In particular, the triprolyl
analogue Pro-Pro-Pro-NH2 (2) possessed good dopamine
receptor modulating activity both in vitro and in vivo.3,13
Synthesis
3-Aryl-prolines, including 3-phenyl-proline, and 3-alkyl-
prolines have been synthesized previously.15±18 Typically,
the approach used has been one involving the initial conden-
sation of diethyl acetamidomalonate with an a,b-unsatu-
rated aldehyde to give the substituted pyrrolidine ring
system. Subsequent 5-deoxygenation followed by saponi®-
cation and decarboxylation provides the 3-substituted
proline. Although this approach is quite versatile it suffers
from the fact that there is no control over stereochemistry
and thus separation of diastereoisomers and resolution of
enantiomers is necessary.
In the present study, the synthesis of triprolyl PLG ana-
logues 3 and 4 in which the second prolyl residue is substi-
tuted at the 3-position with an isopropyl group to mimic the
leucyl side chain has been carried out. Also, since confor-
mationally constrained PLG analogues with isobutyl and
benzyl groups have shown different selectivities with regard
to modulation of the different dopamine receptor subtypes,14
triprolyl PLG analogues 5 and 6 in which the 3-position of
For the synthesis of the 3-isopropyl-prolines (3-i-Pr-Pro,
15a and 15b) and 3-phenyl-prolines (3-Ph-Pro, 16a and
16b) required in this work, we utilized an intramolecular
reductive-cycloalkylation of azido-ole®ns procedure
reported by Waid et al.19 This route allowed us to control
the stereochemistry of the 2-position in the synthesis. The
Keywords: chimeric amino acids; triproline analogue; proline-leucine.
* Corresponding author. Fax: 11-612-624-0139;
e-mail: johns022@tc.umn.edu
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00887-5

9802
M. C. Evans, R. L. Johnson / Tetrahedron 56 (2000) 9801±9808
Scheme 1.
synthesis of these chimeric amino acids and their incorpora-
tion into the triprolyl peptides is outlined in Scheme 1.
of diastereoisomers and 12a and 12b each as single diaster-
eoisomers. The chiral auxiliary of 11 was removed with
LiOH and the a-azido acid which was formed was treated
with MeOH and HCl gas to afford an HPLC separable 1:1
mixture of diastereomeric methyl esters 13a and 13b. Like-
wise, the chiral auxiliaries of 12a and 12b were removed to
give 14a and 14b, respectively. Hydroboration±cycloalky-
lation25 of the azido-ole®ns 13a, 13b, 14a, and 14b
with dicyclohexylborane26 furnished the corresponding
3-substituted prolines 15a, 15b, 16a, and 16b in 95, 83,
55, and 65% yields, respectively.
The 3-substitited 4-pentenoic acids 7 and 820 were converted
to their corresponding mixed anhydrides with pivaloyl
chloride. Reaction of the mixed anhydride in each case
with lithiated S-4-benzyl-2-oxazolidinone21 afforded a 1:1
mixture of diastereomeric N-acyloxazolidinones. In the case
of N-acyloxazolidinones 9, the diastereoisomers were not
separable at this stage. In contrast, N-acyloxazolidinones
10 were chromatographically separable giving 10a and
10b. The chiral imide potassium enolates of 9, 10a
and 10b were formed and quenched by electrophilic
azide transfer22 using 2,4,6-triisopropylbenzenesulfonyl
azide23,24 to afford a-azido carboximides 11 as a mixture
The relative stereochemistries for 15a and 15b were
assigned based on H NMR GOESY (gradient NOE) data,
which are summarized in Fig. 1. Irradiation of the a-CH
1
Figure 1. 1H NMR GOESY data for chimeric amino acids 15b, 16a, and 16b.

M. C. Evans, R. L. Johnson / Tetrahedron 56 (2000) 9801±9808
9803
(d 4.00) in 15a showed no detectable NOE enhancement,
while irradiation of the a-CH (d 4.36) in 15b showed a large
NOE enhancement (10%) of the CH(CH3)2 protons (d 0.87).
Also, an NOE enhancement of 2.5% was seen for the a-CH
of 15b when the CH(CH3)2 protons were irradiated. These
observed NOEs suggest that in 15b the relationship between
the ester and isopropyl group is trans, while for 15a the
relationship between these two groups is cis. Similar
GOESY experiments were carried out on 16a and 16b
(Fig. 1). Thus for 16a, irradiation of the a-CH (d 4.63)
resulted in a 2% enhancement of the b-CH (d 3.91), while
a 2.4% enhancement was observed for the a-CH (d 4.63)
when the 3-Ph-Pro b-CH was irradiated (d 3.91). In
contrast, irradiation of the a-CH (d 4.32) of 16b showed a
1.7% enhancement of the ortho-aromatic protons (d 7.24).
These observations indicated a cis relationship between the
ester and phenyl groups in 16a and a trans relationship
between these groups in 16b.
Figure 2. x1-Torsional angles of the chimeric amino acid residue in tripro-
lines 3±6.
The 3-substituted proline chimeras 15a, 15b, 16a, and 16b
were coupled to Boc-Pro-OH with EDC´HCl to afford
diprolyl peptides 17a, 17b, 18a, and 18b, respectively.
These methyl esters were saponi®ed with LiOH and the
resulting acids were coupled to prolinamide to afford the
respective protected triprolyl peptides 19a, 19b, 20a, and
20b. The step to afford tripeptide 19b, which contains the
trans proline-leucine chimera, was accomplished in a rather
poor yield (15%) compared to those of the other three
compounds (55±77%). In contrast, the yield observed for
the trans proline-phenylalanine chimera case in which a ¯at
phenyl ring is present instead of the rather bulky isopropyl
substituent was quite good. This suggests that steric
hindrance is playing a key role in the trans proline-leucine
chimera case. The tert-butoxycarbonyl groups of 19a, 19b,
20a, and 20b were removed by HCl/dioxane to give the ®nal
triprolyl peptides 3±6, respectively.
peptides are interacting with the PLG binding site in the
same manner as PLG. Also, since conformationally
constrained PLG analogues with isobutyl and benzyl groups
have shown different selectivities with regard to modulation
of the different dopamine receptor subtypes,14 a comparison
of the dopamine receptor subtype selectivity of 3±6
should help shed light on the structural basis behind such
selectivities.
Experimental
3-(3(R,S)-Isopropyl-4-ene-1-oxopentyl)-4(S)-(benzyl)-2-
oxazolidinone (9). 3(R,S)-Isopropyl-pent-4-enoic acid (7,
285 mg, 2.0 mmol) was dissolved in THF and this solution
was cooled to 2788C. Triethylamine (0.31 mL, 2.2 mmol)
was added via syringe followed by trimethylacetyl chloride
(0.27 mL, 2.2 mmol). The resulting white suspension was
stirred at 2788C for 15 min, then at 08C for 1 h, and ®nally
at 2788C for 15 min. A slurry of lithiated 4(S)-benzyl-2-
oxazolidinone at 2788C (prepared 10 min in advance at
2788C by the addition of 1.6 M n-butylithium in hexanes
(1.4 mL, 2.2 mmol) into the solution of 4(S)-benzyl-2-
oxazolidinone (394 mg, 2.2 mmol) in THF at 2788C) was
added via a cannula to the suspension of the mixed anhy-
dride. The resulting slurry was stirred at 2788C for 15 min,
then for 4 h at room temperature. The reaction was
quenched with saturated ammonium chloride (3 mL) and
the volatiles were removed by rotary evaporation. Water
was added to the mixture, which was then extracted with
CH2Cl2 (3£). The combined organic extracts were dried
over MgSO4. Evaporation of the ®ltrate gave a yellow oil
which was puri®ed by silica gel ¯ash chromatography
(hexanes/EtOAc, 10:1!7.5:1!5:1) to yield 473 mg
(78%) of an inseparable mixture of diastereoisomers: mp
Discussion
The triprolyl compounds 3±6 represent novel conformation-
ally constrained analogues of PLG. The pyrrolidine ring of
each prolyl residue results in the restriction of each f
torsional angle in the peptide to a value around 2608. For
the chimeric amino acid residues in the second position, the
x1 torsional angle also is constrained. Preliminary MM2
molecular modeling of 3±6 using Chem 3D gave the x1
values shown in Fig. 2. In each isomeric case, a trans
conformation for x1 was observed, although with opposite
signs. The values were similar to those seen by Mosberg et
al.27 with their chimeric proline-tyrosine amino acids. On
the basis of their work, a gauche1 conformation also is
possible for the trans isomers 4 and 6, while a gauche2
conformation is possible for the cis isomers 3 and 5.
The triprolyl PLG analogues 3 and 4 with the proline-
leucine chimeric amino acid in the second position have
been designed such that the isopropyl group at position 3
could potentially mimic the leucyl side chain of PLG. Since
previous structure-activity relationship studies on PLG have
shown that the presence of the leucyl side chain results in
enhanced binding of PLG analogues, it is expected that
either 3 or 4 should show enhanced activity if the triproline
1
51±548C. H NMR (CDCl3) d 7.21±7.37 (m, 5H), 5.64±
5.78 (m, 1H), 5.01±5.10 (m, 2H), 4.63±4.69 (m, 1H), 4.13±
4.20 (m, 2H), 2.91±3.34 (m, 3H), 2.49±2.75 (m, 2H), 1.70±
1.77 (m, 1H), 0.89±0.97 (m, 6H) 13C NMR (CDCl3) d 19.6,
21.0, 32.3, 32.4, 38.4, 38.6, 38.7, 38.8, 47.2, 47.4, 55.9,
56.0, 66.6, 66.8, 116.9, 117.0, 128.0, 129.6, 130.1, 136.1,
139.6, 139.7, 154.1, 154.2, 173.2, 173.3. FAB MS m/zˆ302

9804
M. C. Evans, R. L. Johnson / Tetrahedron 56 (2000) 9801±9808
(60%) [M1H]1. Anal. Calcd for C18H23NO3: C, 71.73; H,
7.69; N, 4.65. Found: C, 72.00; H, 7.49; N, 4.68.
NMR (CDCl3) d 16.8, 20.6, 21.1, 21.8, 28.1, 29.5, 38.2,
38.5, 53.0, 53.9, 55.9, 56.7, 60.2, 63.5, 67.0, 67.4, 119.5,
120.9, 128.2, 129.7, 129.7, 130.1, 133.4, 135.5, 136.1,
153.5, 170.6, 172.0. FAB HRMS (NBAL matrix) m/zˆ
349.1867 (C18H22N4O31Li1 requires 349.1852).
3-(3(R)-Phenyl-4-ene-1-oxopentyl)-4(S)-(benzyl)-2-oxazoli-
dinone (10a) and 3-(3(S)-phenyl-4-ene-1-oxopentyl)-4(S)-
(benzyl)-2-oxazolidinone (10b). The same conditions
described for the synthesis of 9 were used to convert
3(R,S)-phenyl-pent-4-enoic acid (8, 1.63 g, 9.25 mmol) to
10a and 10b. Separation of these diastereoisomers was
accomplished by two silica gel ¯ash chromatography runs
(hexanes/EtOAc, 10:1!7.5:1).
3-(2(S)-Azido-3(R)-phenyl-4-ene-1-oxopentyl)-4(S)-(benzyl)-
2-oxazolidinone (12a). Compound 10a (282 mg, 0.84 mmol)
was converted to 12a by the same procedure described
above for 11. The material was puri®ed by silica gel ¯ash
chromatography (hexanes/EtOAc, 5:1) to give 215 mg
(68%). An analytical sample was obtained by HPLC with
a Waters Spherisorbw 4.6£150 mm column (hexanes/
EtOAc, 5:1; tRˆ2.5 min): [a]Dˆ1161.3 (c 0.6, MeOH).
1H NMR (CDCl3) d 7.17±7.36 (m, 10H), 6.23 (ddd, Jˆ
10a: Yield 713 mg (46%); mp 102±1058C; [a]Dˆ189.3
1
(c 1.0, CH2Cl2). TLC Rfˆ0.47 (hexanes/EtOAc, 5:2); H
NMR (CDCl3) d 7.20±7.38 (m, 10H), 6.10 (ddd, Jˆ17.4,
10.2, 7.2 Hz, 1H), 5.17 (dd, Jˆ10.2, 1.2 Hz, 2H), 4.54±4.61
(m, 1H), 4.02±4.14 (m, 3H), 3.44, 3.51 (2dd, Jˆ16.2,
7.5 Hz, 2H), 3.27 (dd, Jˆ9.9, 3.3 Hz, 1H), 2.72 (dd,
Jˆ13.2, 9.9 Hz, 1H). 13C NMR (CDCl3) d 38.5, 41.2,
46.0, 55.9, 66.8, 115.6, 127.4, 128.0, 128.5, 129.3, 129.6,
130.1, 136.0, 141.1, 143.2, 154.1, 172.1; FAB MS m/z 336
(75%) [M1H]1. Anal. Calcd for C21H21NO3: C, 75.20;
H, 6.31; N, 4.18. Found: C, 75.30; H, 6.33; N, 4.16.
16.8, 8.4 Hz, 1H), 5.51 (d, Jˆ9.9 Hz, 1H), 5.38 (d, Jtrans
ˆ
16.8 Hz, 1H), 5.33 (d, Jcisˆ8.1 Hz, 1H), 4.13±4.19 (m, 1H),
3.89±3.99 (m, 2H), 3.55 (dd, Jˆ8.1 Hz, 1H), 3.22 (dd,
Jˆ13.5, 3.0 Hz, 1H), 2.72 (dd, Jˆ13.5, 9.9 Hz, 1H); 13C
NMR (CDCl3) d 38.3, 53.2, 56.2, 62.4, 66.9, 119.3, 128.1,
128.3, 129.0, 129.4, 129.7, 130.1, 135.5, 136.6, 138.9,
153.2, 170.7; FAB HRMS (NBAL matrix) m/zˆ383.1689
(C21H20N4O31Li1 requires 383.1695).
10b: Yield 775 mg (50%); mp 70±738C; [a]Dˆ165.5
3-(2(S)-Azido-3(S)-phenyl-4-ene-1-oxopentyl)-4(S)-(benzyl)-
2-oxazolidinone (12b). Compound 10b (250 mg, 0.75 mmol)
was converted to 12b by the same procedure described
above for 11. The material was puri®ed by silica gel ¯ash
chromatography (hexanes/EtOAc, 5:1) to give 191 mg
(68%). An analytical sample was obtained by HPLC with
a Waters Spherisorbw 4.6£150 mm column (hexanes/
EtOAc, 5:1; tRˆ2.7 min): [a]Dˆ188.4 (c 0.56, MeOH).
1H NMR (CDCl3) d 7.10±7.44 (m, 10H), 6.03 (ddd,
Jˆ17.1, 8.1 Hz, 1H), 5.60 (d, Jˆ10.5 Hz, 1H), 5.21
(d, Jtransˆ17.1 Hz, 1H), 5.12 (d, Jcisˆ8.7 Hz, 1H), 4.66±
4.72 (m, 1H), 4.18±4.26 (m, 2H), 3.91 (dd, Jˆ9.6 Hz,
1H), 3.34 (dd, Jˆ13.5, 3.3 Hz, 1H), 2.88 (dd, Jˆ13.5,
9.3 Hz, 1H); 13C NMR (CDCl3) d 38.4, 53.8, 55.9, 63.0,
67.2, 118.8, 128.2, 128.4, 128.7, 129.8, 130.2, 135.4,
137.2, 139.6, 153.9, 170.8; FAB HRMS (NBAL matrix)
m/zˆ383.1692 (C21H20N4O3 1 Li1 requires 383.1695).
1
(c 1.0, CH2Cl2); TLC Rfˆ0.40 (hexanes/EtOAc, 5:2); H
NMR (CDCl3) d 7.22±7.43 (m, 8H), 7.09±7.14 (m, 2H),
6.09 (ddd, Jˆ17.4, 10.2, 7.5 Hz, 1H), 5.14 (dd, Jˆ10.2 Hz,
2H), 4.62±4.70 (m, 1H), 4.03±4.20 (m, 3H), 3.58 (dd,
Jˆ8.4, 7.5 Hz, 1H), 3.35 (dd, Jˆ8.4, 7.5 Hz, 1H), 3.12
(dd, Jˆ13.5, 3.3 Hz, 1H), 2.63 (dd, Jˆ13.5, 9.3 Hz, 1H);
13C NMR (CDCl3) d 38.3, 41.4, 46.0, 55.7, 66.7, 115.7,
127.4, 128.0, 128.5, 129.3, 129.6, 130.1, 135.8, 141.1,
143.2, 154.1, 172.1. FAB MS m/z 336 (20%) [M1H]1.
Anal. Calcd for C21H21NO3: C, 75.20; H, 6.31; N, 4.18.
Found: C, 75.41; H, 6.48; N, 4.26.
3-(2(S)-Azido-3(R,S)-isopropyl-4-ene-1-oxopentyl)-4(S)-
(benzyl)-2-oxazolidinone (11). To 3 mL of dry THF at
2788C under N2 was added 0.5 M potassium hexamethyl-
disilazide in toluene (3.07 mL, 1.53 mmol). To this solution
was added via cannula a precooled (2788C) solution of 9
(420 mg, 1.39 mmol) in 3 mL of dry THF. The reaction was
stirred at 2788C for 30 min. To this solution of potassium
enolate was added via cannula a precooled (2788C) solution
of trisyl azide (567 mg, 1.74 mmol) in 3 mL of THF. The
reaction was quenched with acetic acid (0.37 mL,
6.4 mmol) after 1±2 min. The mixture was warmed imme-
diately to 308C and maintained at this temperature for 1.5 h.
The solution was partitioned between CH2Cl2 and dilute
brine. The aqueous phase was washed with CH2Cl2 (3£).
The organic phases were combined and washed with 1 M
NaHCO3, dried over MgSO4, ®ltered and then concentrated
to a yellow oil. The crude product was puri®ed by silica gel
¯ash chromatography (hexanes/EtOAc, 5:1) to yield
299 mg (63%). An analytical sample of the inseparable
mixture of diastereoisomers was obtained by HPLC with a
Waters Spherisorbw 4.6£150 mm column (hexanes/EtOAc,
Methyl 2(S)-azido-3(R)-isopropyl-4-pentenoate (13a) and
methyl 2(S)-azido-3(S)-isopropyl-4-pentenoate (13b).
Compound 11 (1.9 g, 5.55 mmol) was dissolved in THF/
H2O (3:1, 25 mL) and this solution was placed in an ice
bath. Solid LiOH (266 mg, 11.1 mmol) was added and the
solution was allowed to stir at 08C for 0.5 h. The solution
was diluted with 1 M NaHCO3 and then the THF was
removed under aspirator pressure. The solution was washed
with EtOAc (3£) and acidi®ed to pHˆ3 with solid citric
acid. The aqueous solution was extracted with EtOAc
(3£) and the combined organic extracts were dried over
MgSO4, ®ltered and concentrated to afford the diastereoiso-
meric mixture of free acids. The mixture was dissolved in
MeOH and an excess of anhydrous HCl gas was bubbled
into the solution, which was then capped and stirred over-
night at room temperature. Excess HCl and the MeOH were
removed under aspirator pressure by forming an azeotrope
with CH2Cl2 (3£). A yield of 543 mg (50%) of the mixture
of diastereoisomers was obtained which were separated on a
Waters PrepLCe 25£100 mm silica PrepPak cartridge
(hexanes/EtOAc, 60:1).
1
5:1; tRˆ2.1 min). H NMR (CDCl3) d 7.22±7.39 (m, 5H),
5.60±5.82 (m, 1H), 5.00±5.34 (m, 3H), 4.58±4.66 (m, 1H),
4.21±4.28 (m, 2H), 3.30±3.41 (m, 1H), 2.80±2.89 (m, 1H),
2.53±2.61 (m, 0.5H), 2.31±2.39 (m, 0.5H), 2.16±2.22
(m, 0.5H), 1.75±1.82 (m, 0.5H), 0.88±1.05 (m, 6H). 13C

M. C. Evans, R. L. Johnson / Tetrahedron 56 (2000) 9801±9808
9805
1
13a: Yieldˆ202 mg; [a]Dˆ26.4 (c 1.07, CH2Cl2); 1H
NMR (CDCl3) d 5.61 (ddd, Jˆ16.8, 9.9, 9.9 Hz, 1H), 5.10
(dd, Jcisˆ9.0, 1.8 Hz, 1H), 5.05 (dd, Jtransˆ15.6, 1.8 Hz,
1H), 4.08 (d, Jˆ5.7 Hz, 1H), 3.73 (s, 3H), 2.26 (ddd,
Jˆ9.9, 7.8, 5.4 Hz, 1H), 1.64±1.73 (m, 1H), 0.95
(d, Jˆ6.3 Hz, 3H), 0.85 (d, Jˆ6.6 Hz, 3H); 13C NMR
(CDCl3) d 20.4, 21.2, 29.0, 53.0, 53.9, 64.7, 119.7, 135.7,
171.2; EI HRMS (4% NH3 in CH4) m/zˆ215.1506
(C9H15N3O21NH14 requires 215.1550).
(c 0.17, H2O); H NMR (D2O) d 4.19 (d, Jˆ7.2 Hz, 1H),
3.81 (s, 3H), 3.28±3.47 (m, 2H), 2.33±2.41 (m, 1H), 2.09±
2.20 (m, 1H), 1.76±1.89 (m, 2H), 0.96 (d, Jˆ6.6 Hz, 3H),
0.90 (d, Jˆ6.9 Hz, 3H); 13C NMR (D2O) d 18.3, 20.0, 27.1,
30.0, 46.7, 49.4, 54.4, 62.2, 171.5; GOESY NMR (D2O,
500 MHz) irradiation at d 4.00 (3-i-Pr-Pro a-CH) showed
no NOE enhancement; FAB HRMS (MNBA matrix) m/zˆ
172.1344 (C9H17NO21H1 requires 172.1337).
trans-3-Isopropyl-l-proline methyl ester hydrochloride
(15b). This material was obtained from azide 13b
(142 mg, 0.72 mmol) through use of the same procedures
as those described above for 15a. A yield of 124 mg (83%)
was obtained and HPLC analysis on a Waters Spherisorbw
4.6£150 mm column (CHCl3/MeOH, 5:1) gave a single
peak (tRˆ1.0 min): mp 98±1028C; [a]Dˆ127.5 (c 0.16,
1
13b: Yieldˆ157 mg; [a]Dˆ171.1 (c 1.22, CH2Cl2); H
NMR (CDCl3) d 5.57 (ddd, Jˆ16.8, 10.2, 10.2 Hz, 1H),
5.12 (dd, Jcisˆ10.2, 1.8 Hz, 1H), 5.05 (dd, Jtransˆ16.8,
1.8 Hz, 1H), 3.71 (s, 3H), 3.70 (d, Jˆ9.6 Hz, 1H), 2.38
(ddd, Jˆ9.6, 4.5 Hz, 1H), 1.97±2.04 (m, 1H), 0.91
(d, Jˆ6.6 Hz, 3H), 0.85 (d, Jˆ6.9 Hz, 3H); 13C NMR
(CDCl3) d 17.6, 21.7, 28.0, 52.5, 52.7, 64.2, 120.4, 133.8,
171.2; EI HRMS (4% NH3 in CH4) m/zˆ215.1510
(C9H15N3O2 1 NH14 requires 215.1550).
1
H2O); H NMR (D2O) d 4.53 (d, Jˆ7.2 Hz, 1H), 3.81
(s, 3H), 3.58±3.66 (m, 1H), 3.29±3.39 (m, 1H), 2.15±2.27
(m, 2H), 1.72±1.80 (m, 1H), 1.44±1.51 (m, 1H), 1.02
(d, Jˆ6.6 Hz, 3H), 0.90 (d, Jˆ6.6 Hz, 3H); 13C NMR
(D2O) d 21.2, 22.0, 27.3, 28.9, 46.0, 51.0, 53.9, 62.4,
170.5; GOESY NMR (D2O, 500 MHz) irradiation at d
4.36 (3-i-Pr-Pro a-CH) showed an NOE enhancement of
10% at d 0.87 (CH(CH3)3) while irradiation at d 0.87
(CH(CH3)3) showed an NOE enhancement of 2.5% at d
4.36 (3-i-Pr-Pro a-CH); FAB HRMS (MNBA matrix)
m/zˆ172.1346 (C9H17NO21H1 requires 172.1337).
Methyl 2(S)-azido-3(R)-phenyl-4-pentenoate (14a). Com-
pound 12a (1.2 g, 3.18 mmol) was converted to 14a
(yieldˆ495 mg (67%)) by the same method as that
described above for the formation of 13a: [a]Dˆ26.1
1
(c 0.59, CH2Cl2); H NMR (CDCl3) d 7.26±7.38 (m, 5H),
6.17 (ddd, Jˆ17.4, 9.0, 9.0 Hz, 1H), 5.28 (d, Jtransˆ15.9 Hz,
1H), 5.27 (d, Jcisˆ11.4 Hz, 2H), 4.13 (d, Jˆ7.2 Hz, 1H),
3.93 (dd, Jˆ8.1, 7.5 Hz, 1H); 13C NMR (CDCl3) d 52.3,
53.1, 66.9, 119.2, 128.2, 128.8, 129.4, 136.3, 139.6, 170.4;
FAB MS m/zˆ232 (30%) [M1H]1. Anal. Calcd for
C16H27N3O4: C, 62.33; H, 5.67; N, 18.17. Found: C,
62.42; H, 6.00; N, 18.02.
cis-3-Phenyl-l-proline methyl ester hydrochloride (16a).
This material was obtained from azide 14b (340 mg,
1.47 mmol) through use of the same procedures as those
described above for 15a. A yield of 197 mg (55%) was
obtained and HPLC analysis on a Waters Spherisorbw
4.6£150 mm column (CHCl3/MeOH, 5:1) gave a single
peak (tRˆ1.5 min): mp 59±648C; [a]Dˆ161.4 (c 0.5,
MeOH); 1H NMR (D2O) d 7.21±7.40 (m, 5H), 4.72
(d, Jˆ9.0 Hz, 1H), 3.96±4.04 (m, 1H), 3.74±3.82 (m,
1H), 3.42±3.52 (m, 1H), 3.33 (s, 3H), 2.35±2.48 (m, 2H).
13C NMR (D2O) d 29.6, 46.5, 46.9, 53.8, 64.3, 128.6, 129.0,
129.6, 136.6, 169.7; GOESY NMR (D2O, 500 MHz) irradi-
ation at d 4.63 (3-Ph-Pro a-CH) showed an NOE enhance-
ment (2.0%) at d 3.91 (PhCH) while irradiation at d 3.91
(PhCH) showed NOE enhancement (2.4%) at d 4.63 (3-Ph-
Pro a-CH); FAB HRMS (MNBA matrix) m/zˆ206.1181
(C12H16NO21H1 requires 206.1181).
Methyl 2(S)-azido-3(S)-phenyl-4-pentenoate (14b). Com-
pound 12b (855 mg, 2.27 mmol) was converted to 14b
(yieldˆ365 mg (70%)) by the same method as that
described above for the formation of 13a: [a]Dˆ125.0
1
(c 0.86, CH2Cl2); H NMR (CDCl3) d 7.27±7.41 (m, 5H),
6.06 (ddd, Jˆ18.0, 9.9, 8.4 Hz, 1H), 5.21 (d, Jtransˆ18.3 Hz,
1H), 5.20 (d, Jcisˆ9.9 Hz, 1H), 4.16 (d, Jˆ9.0 Hz, 1H), 3.90
(dd, Jˆ8.4, 8.4 Hz, 1H), 3.76 (s, 3H); 13C NMR (CDCl3) d
52.6, 53.0, 66.6, 118.5, 128.3, 128.9, 129.5, 137.1, 139.3,
170.5; FAB MS m/zˆ232 (5%) [M1H]1. Anal. Calcd for
C16H27N3O4: C, 62.33; H, 5.67; N, 18.17. Found: C, 62.13;
H, 5.90; N, 17.97.
cis-3-Isopropyl-l-proline methyl ester hydrochloride
(15a). In a dry ¯ask under Ar was placed cyclohexene
(0.59 mL, 5.8 mmol) and anhydrous Et2O (5 mL). The solu-
tion was cooled to 08C. Borane dimethylsul®de complex
(0.5 M in Et2O, 0.58 mL, 2.9 mmol) was added and the
reaction was allowed to stir at 08C for 3 h. The Et2O was
blown off with a stream of Ar and the resulting dicyclo-
hexylborane was dried under vacuum for 10 min and then
suspended in freshly distilled CH2Cl2. Azide 13a (191 mg,
0.97 mmol), which had been dissolved in CH2Cl2, was
transferred via a cannula at 08C into the white suspension.
The reaction mixture was stirred for 2 d at room tempera-
ture. The CH2Cl2 was extracted with 1 M HCl and then the
aqueous phase was evaporated to afford a 191 mg (95%) of
a white solid. HPLC analysis of this material on a Waters
Spherisorbw 4.6£150 mm column (CHCl3/MeOH, 5:1) gave
a single peak (tRˆ1.4 min): mp 74±788C; [a]Dˆ117.0
trans-3-Phenyl-l-proline methyl ester hydrochloride (16b).
This material was obtained from azide 14a (482 mg,
2.08 mmol) through use of the same procedures as those
described above for 15a. A yield of 326 mg (65%) was
obtained and HPLC analysis on a Waters Spherisorbw
4.6£150 mm column (CHCl3/MeOH, 5:1) gave a single peak
(tRˆ1.7 min): mp 65±698C; [a]Dˆ131.5 (c 0.71, MeOH); 1H
NMR (D2O) d 7.32±7.44 (m, 5H), 4.48 (d, Jˆ9.9 Hz, 1H), 3.71
(s, 3H), 3.45±3.70 (m, 3H), 2.49±2.66 (m, 1H), 2.17±2.26 (m,
1H); 13C NMR (D2O) d 33.7, 46.6, 48.4, 54.4, 65.3, 128.2,
128.8, 129.9, 138.6, 170.2; GOESY NMR (D2O, 500 MHz)
irradiation at d 4.32 (3-Ph-Pro a-CH) showed an NOE
enhancement (1.7%) at d 7.24 (Ar-H); FAB HRMS (MNBA
matrix) m/zˆ206.1185 (C12H16NO21H1 requires 206.1181).
N-tert-Butoxycarbonyl-l-prolyl-cis-3-isopropyl-l-proline
methyl ester (17a). Boc-l-Pro-OH (118 mg, 0.55 mmol),

9806
M. C. Evans, R. L. Johnson / Tetrahedron 56 (2000) 9801±9808
15a (100 mg, 0.48 mmol) and HOBt´H2O (75 mg,
0.55 mmol) were dissolved in CH2Cl2 (10 mL) under N2.
The mixture was cooled to 2788C after which was added
NEt3 (0.13 mL, 0.97 mmol), followed by EDC´HCl
(106 mg, 0.55 mmol). The mixture was stirred at a low
temperature for 30 min and then warmed to room tempera-
ture where it was kept for two days. The organic layer was
extracted with 1 M NaHCO3, 10% citric acid, water and
brine. The organic layer was dried over MgSO4, ®ltered
and concentrated under aspirator pressure. The crude
product was puri®ed by silica gel ¯ash chromatography
(EtOAc/hexanes, 2:1) to give 52 mg (29%) of product:
for C22H30N2O5: C, 65.65; H, 7.51; N, 6.96. Found: C,
65.64; H, 7.47; N, 7.03.
N-tert-Butoxycarbonyl-l-prolyl-trans-3-phenyl-l-proline
methyl ester (18b). Boc-l-Pro-OH (102 mg, 0.47 mmol)
and 16b (100 mg, 0.41 mmol) were coupled together by
the same method as that described above for 17a to
give 44 mg (26%) of product after silica gel ¯ash
chromatography (EtOAc/hexanes, 2:1). Analytical HPLC
analysis on a Waters Spherisorbw 4.6£150 mm column
(CHCl3/MeOH, 5:1) gave a single peak (tRˆ1.25 min):
1
[a]Dˆ110.9 (c 0.54, CH2Cl2); H NMR (CDCl3, rotomers
1
[a]Dˆ250.4 (c 0.91, CH2Cl2); H NMR (CDCl3, rotomers
observed) d 7.22±7.36 (m, 5H), 4.57±4.62 (m, 1.5H), 4.46
(dd, Jˆ8.1 and 3.6 Hz, 0.5H), 4.05±4.13, 3.80±3.90
(m, 1H), 3.67, 3.70 (2s, 3H), 3.35±3.75 (m, 4H), 1.83±
2.50 (m, 6H), 1.40, 1.46 (2s, 9H); 13C NMR (CDCl3, roto-
mers observed) d 24.1, 24.8, 29.1, 29.2, 29.8, 30.8, 33.9,
34.7, 46.6, 47.1, 47.4, 47.4, 47.9, 48.8, 52.8, 53.0, 58.1,
58.4, 65.8, 65.9, 80.1, 80.2, 127.5, 127.8, 127.9, 128.0,
129.4, 129.5, 140.8, 141.0, 154.4, 155.2, 172.0, 172.2,
173.0, 173.2; FAB HRMS (MNBA matrix) m/zˆ403.2241
(C22H30N2O5 1 H1 requires 403.2233).
observed in a ratio of 4:3) d 4.49 (dd, Jˆ8.4, 2.7 Hz,
0.57H), 4.37 (dd, Jˆ8.1, 3.6 Hz, 0.43H), 4.25 (dd, Jˆ6.0,
2.7 Hz, 1H), 3.82±3.86 (m, 1H), 3.67, 3.68 (2s, 3H), 3.33±
3.58 (m, 3H), 1.66±2.16 (m, 8H), 1.36, 1.41 (s, 9H), 0.97
(dd, Jˆ6.6 Hz), 0.91 (dd, Jˆ6.9 Hz, 3H); 13C NMR (CDCl3,
rotomers observed) d 20.2, 21.6, 21.8, 24.1, 24.7, 29.0, 29.1,
29.3, 29.4, 30.5, 30.8, 30.9, 46.8, 47.0, 47.3, 47.4, 49.7,
50.1, 52.7, 52.8, 58.1, 58.3, 63.0, 63.2, 80.0, 154.4, 155.1,
171.7, 172.1, 174.0, 174.3; FAB MS (MNBA matrix)
m/zˆ369 (40%) [M1H]1. Anal. Calcd for C19H32N2O5: C,
61.93; H, 8.75; N, 7.60. Found: C, 61.87; H, 8.50; N, 7.39.
N-tert-Butoxycarbonyl-l-prolyl-cis-3-isopropyl-l-prolyl-
l-prolinamide (19a). Dipeptide 17a (41 mg, 0.11 mmol)
was reacted with 3N NaOH (0.06 mL, 0.17 mmol)/MeOH/
THF (1:1:5) for 2 d at room temperature. The MeOH and
THF were removed under aspirator pressure and the remain-
ing aqueous residue was diluted with distilled water. The
solution was washed with EtOAc (3£) and acidi®ed to
pHˆ3 with solid citric acid. The aqueous solution was
extracted with EtOAc (3£) and the combined organic layers
were washed with brine, dried over MgSO4 and ®ltered. The
®ltrate was concentrated under aspirator pressure to afford
the dipeptide free acid (20 mg, 0.13 mmol). This material
was dissolved in CH2Cl2 (10 mL) along with l-prolin-
amide´HCl (40 mg, 0.11 mmol) and HOBt´H2O (18 mg,
0.13 mmol). The solution under nitrogen was cooled to
2788C and then NEt3 (0.03 mL, 0.23 mmol) and EDC´HCl
(25 mg, 0.13 mmol) were added. The mixture was stirred at
a low temperature for 30 min and then warmed to room
temperature where the reaction was allowed to proceed
for two days. The organic layer was extracted with 1 M
NaHCO3, 10% citric acid, water and brine. The organic
layer was dried over MgSO4, ®ltered and concentrated
under aspirator pressure and the crude product
obtained was puri®ed by silica gel ¯ash chromatography
(CH2Cl2/MeOH, 30:1!20:1) to give 29 mg (57%) of
material, which yielded a single peak (tRˆ1.3 min) by
HPLC analysis on a Waters Spherisorbw 4.6£150 mm
column (CHCl3/MeOH, 20:1): [a]Dˆ284.3 (c 0.19,
CH2Cl2); FAB HRMS (MNBA matrix) m/zˆ451.2926
(C22H30N2O5 1 H1 requires 451.2922).
N-tert-Butoxycarbonyl-l-prolyl-trans-3-isopropyl-l-proline
methyl ester (17b). Boc-l-Pro-OH (72 mg, 0.33 mmol) and
15b (60 mg, 0.29 mmol) were coupled together by the same
method as that described above for 17a to give 36 mg (34%)
of product after silica gel ¯ash chromatography (EtOAc/
1
hexanes, 2:1): [a]Dˆ223.2 (c 1.08, CH2Cl2); H NMR
(CDCl3, rotomers observed in a ratio of 3:2) d 4.69
(d, Jˆ7.5 Hz, 0.6H), 4.64 (d, Jˆ7.2 Hz, 0.4H), 4.43 (dd,
Jˆ8.1, 3.3 Hz, 0.6H), 4.32 (dd, Jˆ9, 4.2 Hz, 0.4H), 3.69,
3.67 (2s, 3H), 3.35±3.65 (m, 4H), 1.78±2.17 (m, 7H), 1.41,
1.43 (2s, 9H), 1.06 (apparent dd, Jˆ6.0, 10.8 Hz, 3H), 0.92
(apparent t, Jˆ6.6 Hz, 3H); 13C NMR (CDCl3, rotomers
observed) d 22.2, 22.8, 22.9, 24.2, 24.7, 29.0, 29.1, 29.5,
29.6, 29.7, 30.4, 46.7, 46.8, 47.3, 47.6, 49.9, 50.2, 52.2,
52.4, 57.9, 58.1, 62.0, 80.0, 80.1, 155.2, 171.8, 172.3,
172.7, 173.0; LCQ MS (Direct Infusion Electrospray)
m/zˆ391.1 (100%) [M1Na]1. Anal. Calcd for
C19H32N2O5: C, 61.93; H, 8.75; N, 7.60. Found: C, 62.15;
H, 8.60; N, 7.39.
N-tert-Butoxycarbonyl-l-prolyl-cis-3-phenyl-l-proline
methyl ester (18a). Boc-l-Pro-OH (102 mg, 0.47 mmol)
and 16a (100 mg, 0.41 mmol) were coupled together by
the same method as that described above for 17a to give
85.5 mg (52%) of product after silica gel ¯ash chroma-
tography (EtOAc/hexanes, 2:1): mp 149±1518C; [a]Dˆ2
1
8.0 (c 0.57, CH2Cl2); H NMR (CDCl3, rotomers observed
in a 9:5 ratio) d 7.19±7.34 (m, 5H), 4.82 (d, Jˆ8.7 Hz,
0.65H), 4.77 (d, Jˆ9.0 Hz, 0.35H), 4.51 (dd, Jˆ8.4,
3.0 Hz, 0.65H), 4.41 (dd, Jˆ8.4, 3.7 Hz, 0.35H), 3.83±
3.93 (m, 1H), 3.30±3.71 (m, 4H), 3.22, 3.20 (2s, 3H),
2.62±2.70 (m, 1H), 1.81±2.27 (m, 5H), 1.45, 1.43 (2s,
9H); 13C NMR (CDCl3, rotomers observed) d 24.7, 24.8,
29.1, 29.2, 29.4, 29.5, 29.7, 30.5, 46.5, 46.7, 46.8, 46.8,
47.3, 47.6, 52.0, 52.1, 58.0, 58.2, 64.3, 80.1, 80.2, 128.2,
128.3, 128.4, 128.5, 129.0, 129.1, 136.7, 137.1, 154.4,
155.2, 171.7, 171.9, 172.0, 172.4; LCQ MS (Direct Infusion
Electrospray) m/zˆ425.1 (100%) [M1Na]1. Anal. Calcd
N-tert-Butoxycarbonyl-l-prolyl-trans-3-isopropyl-l-prolyl-
l-prolinamide (19b). Dipeptide 17b (224 mg, 0.61 mmol)
was converted to 19b by the same procedures as those
described above for the synthesis of 19a. A yield of
35 mg (15%) of material was obtained, which yielded a
single peak (tRˆ1.2 min) by HPLC analysis on a Waters
Spherisorbw 4.6£150 mm column (CHCl3/MeOH, 5:1):
[a]Dˆ250 (c 0.15, CH2Cl2); FAB HRMS (MNBA matrix)
m/zˆ451.2943 (C23H38N4O5 1 H1 requires 451.2922).

M. C. Evans, R. L. Johnson / Tetrahedron 56 (2000) 9801±9808
9807
N-tert-Butoxycarbonyl-l-prolyl-cis-3-phenyl-l-prolyl-l-
prolinamide (20a). Dipeptide 18a (70 mg, 0.16 mmol) was
converted to 20a by the same procedures as those described
above for the synthesis of 19a. A yield of 46 mg (55%) of
material was obtained, which yielded a single peak
(tRˆ1.1 min) by HPLC analysis on a Waters Spherisorbw
4.6£150 mm column (CHCl3/MeOH, 20:1): [a]Dˆ254.3
(c 0.96, CH2Cl2); FAB HRMS (MNBA matrix)
m/zˆ485.2767 (C22H30N2O5 1 H1 requires 485.2764).
by HPLC analysis on a Waters Spherisorbw 4.6£150 mm
column eluting with 5:1 CHCl3/MeOH: [a]Dˆ227.4
1
(c 0.31, MeOH); H NMR (D2O) d 7.32±7.44 (m, 5H),
4.58±4.71 (m, 1H), 4.07±4.17 (m, 1H), 3.85±3.97
(m, 1H), 3.59±3.82 (m, 3H), 3.24±3.49 (m, 3H), 3.02±
3.08 (m, 1H), 1.46±2.68 (m, 10H); 13C NMR (D2O) d
21.6, 24.5, 24.6, 24.9, 29.1, 29.2, 29.3, 29.4, 31.0, 31.1,
46.3, 47.1, 47.2, 47.3, 47.5, 48.8, 59.4, 59.4, 60.8, 61.3,
62.4, 63.4, 128.0, 128.9, 129.0, 129.1, 129.2, 129.3, 129.8,
129.9, 136.6, 136.8, 168.6, 168.8, 170.8, 171.3, 176.5,
176.7; FAB HRMS (MNBA matrix) m/zˆ385.2241
(C22H30N2O5 1 H1 requires 385.2240).
N-tert-Butoxycarbonyl-l-prolyl-trans-3-phenyl-l-prolyl-
l-prolinamide (20b). Dipeptide 18b (52 mg, 0.13 mmol)
was converted to 20b by the same procedures as those
described above for the synthesis of 19a. A yield of
48 mg (77%) of material was obtained, which yielded a
single peak (tRˆ1.1 min) by HPLC analysis on a Waters
Spherisorbw 4.6£150 mm column (CHCl3/MeOH, 20:1):
[a]Dˆ254.2 (c 1.1, CH2Cl2); FAB HRMS (MNBA matrix)
m/zˆ485.2759 (C22H30N2O5 1 H1 requires 485.2764).
l-Prolyl-trans-3-phenyl-l-prolyl-l-prolinamide hydro-
chloride (6). Triprolyl peptide 20b (13 mg, 0.03 mmol)
was treated in the same manner as that described above
for 3. Lyophilization of the residue gave 10 mg (85%) of
a ¯uffy white solid, which gave a single peak (tRˆ1.2 min)
by HPLC analysis on a Waters Spherisorbw 4.6£150 mm
column eluting with 5:1 CHCl3/MeOH: [a]Dˆ214.6
1
l-Prolyl-cis-3-isopropyl-l-prolyl-l-prolinamide hydro-
chloride (3). Triprolyl peptide 19a (20 mg, 0.04 mmol)
was dissolved in 4N HCl/dioxane (5 mL) and the resulting
solution was stirred overnight at room temperature. Excess
HCl and the dioxane were removed under aspirator pressure
by forming an azeotrope with CH2Cl2 (3£). Lyophilization
of the residue gave 17 mg (100%) of a ¯uffy white solid,
which gave a single peak (tRˆ1.2 min) by HPLC analysis on
a Waters Spherisorbw 4.6£150 mm column eluting with 5:1
CHCl3/MeOH: [a]Dˆ296.6 (c 0.8, MeOH); 1H NMR
(D2O, rotomers observed) d 4.53±4.60 (m, 1H), 4.31±
4.36 (m, 1H), 3.87±3.94 (m, 1H), 3.49±3.80 (m, 4H),
3.29±3.41 (m, 2H), 2.48±2.55 (m, 2H), 2.16±2.32
(m, 3H), 1.78±2.07 (m, 7H), 0.96 (s, Jˆ6.6 Hz, 3H), 0.90
(s, Jˆ6.6 Hz, 3H); 13C NMR (D2O, rotomers observed) d
18.3, 21.3, 24.5, 25.5, 26.4, 28.8, 29.1, 30.1, 43.9, 47.3,
47.6, 48.9, 49.4, 59.6, 60.9, 61.3, 62.9, 67.3, 70.2, 71.4,
72.2, 168.4, 172.7, 177.3; FAB HRMS (MNBA matrix)
m/zˆ351.2400 (C22H30N2O5 1 H1 requires 351.2398).
(c 0.21, MeOH); H NMR (D2O) d 7.33±7.46 (m, 5H),
4.67±4.74 (m, 1H), 4.29±4.34 (m, 1H), 3.92±3.96
(m, 1H), 3.61±3.80 (m, 5H), 3.36±3.52 (m, 2H), 2.53±
2.58 (m, 2H), 2.29±2.39 (m, 2H), 2.00±2.14 (m, 4H),
1.69±1.77 (m, 2H); 13C NMR (D2O) d 24.5, 25.2, 28.7,
29.9, 34.1, 43.8, 43.9, 47.3, 48.4, 48.6, 49.3, 59.7, 61.0,
66.1, 67.3, 70.2, 71.4, 71.5, 72.3, 128.5, 128.7,
129.7, 129.8, 139.6, 168.2, 171.6, 177.0; FAB HRMS
(MNBA matrix) m/zˆ385.2257 (C22H30N2O51H1 requires
385.2241).
Acknowledgements
This work was supported in part by an NIH grant (NS20036)
to R. L. J. The authors thank Tom Crick for mass spectral
analysis.
References
l-Prolyl-trans-3-isopropyl-l-prolyl-l-prolinamide hydro-
chloride (4). Triprolyl peptide 19b (34 mg, 0.08 mmol)
was treated in the same manner as that described above
for 3. Lyophilization of the residue gave 29 mg (100%) of
a ¯uffy white solid, which gave a single peak (tRˆ1 min) by
HPLC analysis on a Waters Spherisorbw 4.6£150 mm
column eluting with 5:1 CHCl3/MeOH: [a]Dˆ250.5
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1
(c 0.39, MeOH); H NMR (D2O) d 4.48±4.53 (m, 1H),
4.28±4.33 (m, 1H), 4.08±4.16 (m, 1H), 3.60±3.73
(m, 4H), 3.32±3.42 (m, 2H), 2.44±2.55 (m, 1H), 2.21±
2.29 (m, 1H), 1.81±2.18 (m, 7H), 1.48±1.57 (m, 1H), 0.96
(d, Jˆ6.6 Hz, 3H), 0.85 (d, Jˆ6.6 Hz, 3H). 13C NMR (D2O,
rotomers observed) d 21.3, 23.2, 24.6, 25.6, 28.1, 29.1, 29.3,
30.2, 43.9, 47.5, 47.6, 49.5, 50.5, 59.2, 60.9, 60.9, 61.0,
70.6, 71.4, 72.3, 168.6, 171.8, 177.3; FAB HRMS
(MNBA matrix) m/zˆ351.2376 (C22H30N2O5 1 H1 requires
351.2398).
5. Chiu, S.; Paulose, C. S.; Mishra, R. K. Peptides 1981, 2, 105±
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l-Prolyl-cis-3-phenyl-l-prolyl-l-prolinamide hydro-
chloride (5). Triprolyl peptide 20a (28 mg, 0.06 mmol)
was treated in the same manner as that described above
for 3. Lyophilization of the residue gave 24 mg (99%) of
a ¯uffy white solid, which gave a single peak (tRˆ1.2 min)

9808
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