Synthesis and 13C NMR equilibria studies of (1-13C)leupeptin and analogs

Full text of DOI:10.1021/jo980070c

5648
J . Org. Chem. 1998, 63, 5648-5655
attributed to the presence of its C-terminal aldehyde.14,15
The C-terminal aldehyde of leupeptin is believed to form
a stabilized hemiacetal with the serine side chain present
in the active site of the enzyme.16 In thiol enzymes a
similar stabilized thiohemiacetal is proposed.17 The
formation of a stabilized hemiacetal as the enzyme-
aldehyde complex is extremely pH dependent.18 The
catalytic activity of serine-type proteases depends on a
histidine in the active site with a pKa between 6.7 and
7.0. The enzyme’s activity at pH 4.0 is therefore expected
to be less than 0.2% of that at pH 8.0. This translates
to a 500-fold weaker binding by the enzyme at lower pH.
However, aldehydes have been shown to be bound only
2-40-fold more weakly at pH 4.0 than 8.0.18 Arguments
have been proposed as to whether the pH dependence of
the stability of the serine protease-aldehyde adducts or
the pH dependence of their rates of formation and
breakdown are of greater significance for the mechanism
of action of these enzymes.14,19 In either case, the pH
dependence of the equilibrium forms of the serine pro-
tease inhibitor leupeptin and the rates of interconversion
of the inactive forms to the aldehyde form greatly
complicate the theories of pH dependence of simple
aldehyde-protease complexes.
Syn th esis a n d 13C NMR Equ ilibr ia Stu d ies
of (1-13C)Leu p ep tin a n d An a logs
Rose Marie McConnell,* Crissy Patterson-Goss, and
Walter Godwin
Division of Math & Sciences, University of Arkansas at
Monticello, Monticello, Arkansas 71656
Brant Stanley
Department of Chemical Engineering, Louisiana Tech
University, Ruston, Louisiana 71272
Received J anuary 14, 1998
In tr od u ction
Leupeptin, N-acetyl-L-leucyl-L-leucyl-L-argininal, 1, is
a well-recognized inhibitor of serine and thiol proteases1-4
similar to trypsin in specificity. Many of the proteolytic
enzymes inhibited by leupeptin are involved in a wide
variety of biological processes. These processes include:
blood pressure regulation,5 coagulation,6 degradation of
proteins,7 virus assembly,8,9 cell proliferation,10 and tumor
generation.11,12 Leupeptin has been frequently used as
a biochemical tool in the study of proteolytic enzymes and
the role they play in biological functions.5-13 The ef-
fectiveness of leupeptin as a protease inhibitor is often
* Corresponding author. (501)460-1066. Fax: (501)460-1316. E-
mail: mcconnellr@uamont.edu.
(1) Schaefer, R. M.; Teschner, M.; Gernot, P.; Leibold, J .; Kulzer,
P.; Heidland, A. Evidence for the Role of Proteases in Uremic
Catabolism. Adv. Exp. Med. Biol. 1988, 240 (Proteases 2), 323-9.
(2) Kim, I. S.; Lee, K. J . Physiological Roles of Leupeptin and
Extracellular Proteases in Mycelium Development of Streptomyces
Exfoliatus SMF13. Microbiology 1995, 141 (4), 1017-25.
(3) Kanai, H. Effects of Protease Inhibitors on Neurite Outgrowth
and Survival of Cultured Neurons. Kitakanto Igaku 1994, 44, 363-
71.
(4) Cuero, A. M.; Palemr, A.; Rivett, J . A.; Knecht, E. Degradation
of Proteasomes by Lysosomes in Rat. Eur. J . Biochem. 1995, 227 (3),
792-800.
(5) Stewart, J . M. Bradykinin Antagonists: Development and Ap-
plications. Biopolymers 1995, 37, 143-55.
Leupeptin and similar argininal compounds have been
shown to exist in three covalent forms in aqueous
solution.19-21 These three covalent forms are the hydrate,
the aldehyde, and the cyclized carbinol amine (Figure 1).
(6) Chi, C. W.; Liu, H.; Liu, C.; Chibber, B.; Baksky, A. K.; Castellino,
F. The Inhibition of Enzymatic Activity of Blood Coagulation and
Fibrinolytic Serine Proteases by New Leupeptin-like Inhibitor from
Streptomyces griseus. J . Antibiot. 1989, 42, 1506-12.
(7) Tyc, F.; Vrbova, G. Stabilization of Neuromuscular J unctions by
Leupeptin Increases Motor Unit Size in Partially Denevated Rat
Muscles. Dev. Brain Res. 1995, 88, 186-93.
(8) Togushi, K.-i.; Majae, J .; Kataoko, T.; Nagal, K. Thiol-Protease
Inhibitors Selectively Inhibit Presentation of Antigen Incorporated
Through B Cell Antigen Receptors. Biochem. Biophys. Res. Commun.
1995, 210 (2), 324-8.
(9) Krusslich, H. G.; Ingraham, R. H.; Skoog, M. T.; Wimmer, E.;
Palla, P. V.; Carter, C. A. Activity of Purified Biosynthetic Proteinase
of Human Immunodeficiency Virus on Natural Substrates and Syn-
thetic Peptides. Natl. Acad. Sci. U.S.A. 1989, 86, 807-11.
(10) March, K. L.; Wilensky, R. L.; Roeske, R. W.; Hathaway, D. R.
Effects of Thiol Protease Inhibitors on Cell Cycle and Proliferation of
Vascular Smooth Muscle Cells in Culture. Circ. Res. 1993, 72, 413-
23.
(11) Kawada, M.; Umezawa, K. Suppression of in Vitro Invasion of
Human Fibrosarcoma Cells by Leupeptin Analog Inhibiting Urokinase-
Plasmin System. Biochem. Biophys. Res. Commun. 1994, 209 (1), 25-
30.
(14) Kennedy, W. P.; Schultz, R. M. Presteady-State and Steady-
State Kinetic Binding of Unhydrated Peptidyl Aldehyde Inhibitors.
Biochemistry 1979, 18, 349-56.
(15) Schroder, E.; Phillips, C.; Garmon, E.; Harlos, K.; Crawford,
C. X-ray Crystallographic Structure of a Papain-Leupeptin Complex.
FEBS Lett. 1993, 315, 38-42.
(16) Ortiz, C.; Tellier, C.; Williams, H.; Stolowich, N. J .; Scott, A. I.
Diastereotopic Covalent Binding of the Natural Inhibitor Leupeptin
to Trypsin. Detecting the Two Interconverting Hemiacetals by Solution
and Solid Phase NMR Spectroscopy. Biochemistry 1991, 30, 10026-
34.
(17) Schultz, R. M.; Varma-Nelson, P.; Ortiz, R.; Kozlowski, K. A.;
Orawski, A. T.; Pagast, P.; Frankfater, A. Active and Inactive Forms
of the Transition-State Analog Protease Inhibitor Leupeptin: Observed
Slow Binding of Leupeptin to Cathepsin B and Papain. J . Biol. Chem.
1989, 264, 1497-1507.
(18) Kuramochi, H.; Nakata, H.; Ishii, S. I. pH Dependence of
Peptide Aldehyde Binding to Trypsin to Form a Neutral Hemiacetal
Transition-State Analog. J . Biochem. (Tokoyo) 1979, 86, 1403-10.
(19) El-Thaher, T. S.; Bailey, G. S.; Wilson, M. T.; Osborn, M.; Moore,
G. A 1H NMR Study of the Interactions Between Rat Tissue Kallikrein
and Two Peptide Inhibitors. Biochim. Biophys. Acta 1992, 1160, 235-
8.
(12) Funahashi, T.; Shimamura, M.; Kocha, T.; Fukuda, T.; Aoyagi,
T. Proportionality of Protease Activity in Malignant Cells to their
Metastic Potentials. Biol. Pharm. Bull. 1994, 17, 1118-20.
(13) Osmak, M.; Korbelik, M.; Suhar, A.; Skrk, J .; Turk, V. The
Influence of Cathepsin B and Leupeptin on Potentially Lethal Radia-
tion Damage Repair in Mammalian Cells. Int. J . Radiat. Oncol. Biol.
Phys. 1989, 16, 707-14.
(20) McConnell, R. M.; Barnes, G. E.; Hoyng, C. F.; Gunn, J . M.
New Leupeptin Analogues. Synthesis and Inhibition Data. J . Med.
Chem. 1990, 33, 86-93.
(21) McConnell, R. M.; York, J . L.; Frizzell, D.; Ezell, C. Inhibition
Studies of Serine and Thiol Proteases by New Leupeptin Analogues.
J . Med. Chem. 1993, 36, 1084-89.
S0022-3263(98)00070-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/17/1998

Notes
J . Org. Chem., Vol. 63, No. 16, 1998 5649
insoluble in traditional solvents used with reducing
agents. Methyl NG-Cbz-4-guanidinobutanoate was dis-
solved in diglyme (2-methoxyethyl ether), chilled in a dry
ice/alcohol bath, and treated with diisobutylaluminum
hydride (DIBAL) to form the aldehyde NG-Cbz-4-guani-
dinobutanal, 3. The aldehyde was stirred with [13C]-
potassium cyanide and ammonia for 2 days. Acidification
and purification then resulted in (1-13C)-NG-Cbz-DL-
arginine hydrochloride, 4. The carbobenzoxy protecting
group was removed by mild catalytic hydrogenolysis (1
atm, 20 °C, 3 h) to give 1-13C-DL-arginine, 5. The (13C)-
DL-arginine was resolved by a method described by
Greenstein and Winitiz,22 to give (1-13C)-L-arginine, 5a .
F igu r e 1. Equilibria forms of leupeptin.
Syn th esis of 1-13C Leu p ep tin a n d An a logs. The
preparation of (1-13C)leupeptin and analogs from (1-13C)-
L-arginine is shown in Scheme 2. First, the R-amine was
protected with a tert-butoxycarbonyl (BOC) group, and
then the guanidino group was blocked by reaction with
excess benzyl chloroformate. (1-13C)-NR-BOC-NG-Cbz-L-
arginine, 6, was cyclized to the δ-lactam by reaction with
1,1′-carbonyldiimidazole and immediately reduced to the
carbinol amine form of the aldehyde. The aldehyde was
converted into the semicarbazone derivative, 7, for
protection and to facilitate purification. Once purified,
the BOC protecting group was removed by treatment
with 30% trifluoroacetic acid in chloroform. The trifluo-
roacetate salt of (1-13C)-NG-Cbz-L-argininal semicarba-
zone was coupled to the appropriate carbobenzoxy-
protected dipeptide acid (Sigma) to form the fully protected
(1-13C)leupeptin analog, 8a -j. Both the N-terminal Cbz
group and the guanidino protecting group were removed
simultaneously by very mild catalytic hydrogenolysis to
give the dihydrochloride salt. The N-terminal amino
group was then selectively reprotected by careful treat-
ment with acetic anhydride and pyridine. The guanidino
group remained protonated in the presence of the pyri-
dine and was therefore unreactive. (1-13C)-NR-Acetyl-
dipeptide-L-argininal semicarbazone hydrochloride, 9a -
j, was then reacted with formaldehyde and aqueous HCl.
The transimination reaction resulted in (1-13C)leupeptin
and analogs, 10a -j. The products were purified by
Sephasorb HP chromatography and gave both a positive
Sakaguchi22 test (for guanidino) and 2,4-DNP24 test (for
aldehydes). The products were then characterized by 1H
NMR, TLC, and combustion analysis.
This slow equilibrium has been documented by two-
dimensional thin-layer chromatography, H NMR, and
1
slightly broadened 13C NMR signals corresponding of
each of the three equilibrium forms.20,21 While frequently
described as a “slow equilibrium”,19-21 the rate of inter-
conversion among the three forms has not been estab-
lished in the literature. Schultz and coworkers14 have
suggested that this equilibrium is pH dependent and may
be responsible for the slow observed binding of leupeptin
to cathepsin B and papain under acidic pH conditions.
However, most trypsin-like serine proteases are typically
assayed under slightly basic pH conditions (pH 7.5-9.0).
And, no previous investigations have been made of the
leupeptin equilibrium under basic pH conditions. There-
fore, a 13C NMR study of this equilibrium of leupeptin
among the three covalent forms under a variety of pH
conditions was undertaken. Due to the poor solubility
of leupeptin in aqueous solutions, a synthesis of leupeptin
with a 13C label on the aldehyde carbon became neces-
sary. The synthesis of (1-13C)-L-arginine, (1-13C)leupep-
tin, and nine analogs is described below. Also provided
is the resulting 13C NMR data of the labeled protease
inhibitors.
Resu lts a n d Discu ssion
Syn th esis of (1-13C)-L-Ar gin in e. By special request
from ICN, (1-13C)arginine can be synthesized and pur-
chased at a quoted cost of $4800 per 25 mg. Due to
budget restraints, we chose to seek an economical prepa-
ration of (1-13C)-L-arginine. Traditionally arginine has
been synthesized by reaction of cyanamide on orni-
thine.22,23 However, we found the cost of commerically
available (1-13C)-DL-ornithine (nearly $1500 per 100 mg)
to also be prohibitive. Therefore, we set out to synthesize
(1-13C)-L-arginine from a less expensive source of 13C-
labeled carbon. The preparation of (1-13C)arginine is
shown in Scheme 1. In this synthetic approach 4-guani-
dinobutanoic acid (Sigma) was esterified by treatment
with thionyl chloride and methanol, and then the guani-
dino group was protected with a carbobenzoxy (Cbz)
group to give methyl NG-Cbz-4-guanidinobutanoate, 2.
Due to the high basicity and weak nucleophilicity of the
guanidino group, a strong base, sodium methoxide, and
a 20-fold excess of benzyl chloroformate was necessary.
No evidence of the di-Cbz product was observed. The
presence of the carbobenzoxy group enhanced the solubil-
ity of 2 somewhat. However, compound 2 was still
NMR Equ ilibr iu m Stu d ies. The NMR samples were
prepared by utilizing phosphoric acid-d3, sodium formate-
d, or sodium acetate-d3, in deuterium oxide (D2O) with
the pH adjusted with either DCl (37%) in D2O or sodium
deuteroxide (40%) in D2O. The pH of each buffer was
measured using an Orion combination electrode with pH
values in D2O corrected for the different response of a
pH electrode toward deuterium and protium25 (meter
reading +0.41 for D2O solutions). Due to limited solubil-
ity, each NMR sample contained 5 mg (0.043-0.048 mM)
of the appropriate 13C-labeled compound in 0.25 mL of
buffered D2O in a 5 mm NMR tube. NMR spectroscopy
1
was performed at 500 and 100.62 MHz for H and 13C on
a Bruker AMX-500 NMR with an ASPECT STATION
(24) Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Engle, R. G.
Aldehydes and Ketones, In Organic Laboratory Techniques. A Micros-
cale Approach, 2nd ed.; Saunders College Publishing: Fort Worth, TX,
1995; p 496.
(25) McDougall, A. O.; Long, F. A. Relative Hydrogen Bonding of
Deuterium. II. Acid Ionization Constants in H2O and D2O. J . Phys.
Chem. 1962, 66, 429-33.
(22) Greenstein, J . P.; Winitz, M. Arginine In Chemistry of the Amino
Acids; Wiley Publishing: New York; 1961; Reprint 1986, Robert E.
Krieger Publishing: Malabar, FL, Vol. 3, p 1841-1855.
(23) Ramadas, K.; Srinivasan, N. An Expedient Synthesis of Sub-
stituted Guanidines. Tetrahedron Lett. 1995, 36 (16), 2841-4.

5650 J . Org. Chem., Vol. 63, No. 16, 1998
Notes
Sch em e 1
Sch em e 2
data system. Spectra were recorded at 25 °C. Chemical
shifts were referenced to the internal sodium 3-(trimeth-
ylsilyl)propane sulfonate set at δ 0.0. Spectral widths
and the number of data points were adjusted to obtain
digital resolution in the frequency domain better than
um forms of compound 10a were observed at δ 92.5 for
the hydrate, δ 78.4 and 78.0 for the two forms of the
cyclized carbinol amine, and 202.3 for the free aldehyde.
At pH 9.5 the signal for the hydrate, the carbinol amine,
and the aldehyde equilibria forms were shifted further
downfield (the hydrate and carbinol amine by 2-3 ppm,
and the aldehyde by 0.3 ppm). This assignment of the
slightly broadened 13C NMR signals is in agreement with
those made by Scott and coworkers,16 as well as our
earlier work with leupeptin.20,21 The 13C NMR spectra
of the other 13C-labeled analogs, 10b-j, showed similar
chemical shifts. Peaks area ratios of the hydrate, free
aldehyde, and cyclized carbinol amine for each 13C-labeled
leupeptin analog, at pH values ranging from 3.0 to 9.5,
are shown in Table 1. Both 1H and 13C NMR data of
compounds 10a -j indicate that the hydrated aldehyde
1
0.8 or 0.6 Hz/data point for H and 13C NMR, respectively.
Broad band decoupled 13C NMR spectra, utilizing 12 s
pulse delays, were obtained for each compound at eight
different pH values. Off-resonance decoupled spectra
were also obtained to corroborate peak assignments.
The 13C-labeled carbonyl carbon of argininal in com-
pound 10a in its three equilibrium forms was observed
in acidic solutions (pH 3.0-6.5) at δ 90.3 for the hydrate,
δ 77.1 and 76.6 for the R and S forms of the carbinol
amine, and 202.1 for the free aldehyde. In neutral and
slightly basic solutions (pH 7.0-8.5) the three equilibri-

Notes
J . Org. Chem., Vol. 63, No. 16, 1998 5651
Ta ble 1. Ra tio of NMR Sign a l In ten sities (Hyd r a te/F r ee
Ald eh yd e/Cyclized Ca r bin ol Am in e) a t Va r iou s p H
Va lu esa
phase with a half-time of several minutes. These authors
suggest that the lag phase for inhibition is evidence for
a transient intermediate that proceeds tight binding by
leupeptin. However, Schultz coworkers17 propose that
the slow approach to steady-state inhibition of cathepsin
B and papain at nanomolar concentrations of leupeptin
is due to the stability of the thiohemiacetal adduct, as
well as to a low concentration of the active form of the
inhibitor. Since thiol proteases are normally assayed
under acidic pH conditions, our data confirming the lack
of a significant amount of the free aldehyde form of
leupeptin at these pH conditions may help explain the
observed slow binding of leupeptin by cathepsin B and
papain.
Biochemical assay of our synthetic (1-13C)leupeptin and
analogs as protease inhibitors will be conducted as soon
as an available standard enzyme assay technique can be
modified for use with the labeled compounds. Due to
apparent kinetic isotope effects, we have not been able
to establish accurate Ki values for (1-13C)leupeptin and
analogs because the rates of inhibition for these com-
pounds are outside the parameters for standard spectro-
photometric enzyme assay techniques. Once valid Ki
values are established, an investigation and comparison
of the rates of inhibition with the relative amounts of free
aldehyde at specific pHs will be reported.
compd
no.
3.0
5.0
6.0
6.5
7.0
7.5
8.5
9.5
10a
10b
10c
10d
10e
10f
10g
10h
10i
9/0/1
9/0/1 7/1/2 6/2/1 2/5/3 3/4/3 1/1/8 1/0/9
10/0/0 9/0/1 8/1/1 7/1/2 3/4/3 3/3/4 1/0/9 1/0/9
9/0/1
9/0/1
9/0/1
9/0/1
9/0/1
9/0/1
9/0/1
9/0/1
9/0/1 8/1/1 6/2/1 2/5/3 2/4/4 1/0/9 1/0/9
9/0/1 6/3/1 5/4/1 2/6/2 2/5/3 2/1/7 1/0/9
9/0/1 7/2/1 6/2/1 3/5/2 2/4/4 1/1/8 1/0/9
9/0/1 8/1/1 6/2/1 3/4/3 3/1/6 1/0/9 1/0/9
9/0/1 9/0/1 8/1/1 5/3/2 4/4/2 1/0/9 1/0/9
9/0/1 5/4/2 4/5/1 1/7/2 1/6/3 2/4/4 1/0/9
9/0/1 6/3/1 5/4/1 1/8/1 1/7/2 1/6/3 1/3/6
9/0/1 8/1/1 7/2/1 1/6/3 2/4/4 1/0/9 1/0/9
10j
a
13C NMR broad band decoupled spectra with 12 s pulse delays.
Exp er im en ta l Section
Ma ter ia ls. All solvents used were of ACS purity and were
purchased either from Fisher Scientific or Aldrich. The starting
material, 4-guanidinobutanoic acid, and the carbobenzoxy-
protected dipeptides were purchased from Sigma Chemical Co.
(St. Louis, MO). The [13C]potassium cyanide was purchased
from Cambridge Isotope Laboratories. All other reagents used
were purchased from Aldrich. The purity of each major inter-
mediate or product was checked by TLC (silica gel 60-F254
plates, E. Merck) and in some cases HPLC (Perkin Elmer LCI-
100 TriDet, reversed phase C18 analytical column, 5 µm). Unless
otherwise indicated, the intermediates and product were homo-
geneous by TLC and HPLC. The structures of the final product
and each intermediate were verified by 1H NMR (Hitachi R-1200,
60 MHz). Aqueous solutions were evaporated under the vacuum
of an oil pump using a rotary evaporator equipped with a low-
temperature circulating bath (-40 °C). Organic solvents were
removed on a rotary evaporator under the vacuum of a water
aspirator with bath temperatures of 40 °C or lower.
F igu r e 2. Molecular modeling of 10i showing hydrogen
bonding of guanidinium NH of argininal P1 side chain to the
nitro oxygens of the P2 p-nitrophenylalanyl side chain (light
blue ) C, dark blue ) N, red ) O, gray ) H, and hydrogen
bonds are shown in yellow).
(see Figure 1) is the major form at lower pH (3.0-6.0).
At basic pH (8.5 and 9.5), most of the 13C-labeled
compounds showed the cyclized carbinol amine as the
dominant form, with the hydrated aldehyde as a second-
ary form. Very little free aldehyde was observed under
extremely basic conditions. For (1-13C)leupeptin, 10a ,
and similar analogs, a narrow pH range of 7.0-7.5
showed the greatest proportion of the biologically active
free aldehyde form. Acetyl-leucyl-cyclohexylalanyl-(1-
13C)argininal, 10j, as well as compounds 10b,c,f,g that
have only stearic alterations show little variation from
10a . The pH range of dominant free aldehyde was
significantly wider in the NMR studies of acetyl-leucyl-
phenylalanyl-(1-13C)argininal, 10e, and wider still for
acetyl-leucyl-p-chlorophenylalanyl-(1-3C)argininal, 10h ,
and acetyl-leucyl-p-nitrophenylalanyl-(1-13C)argininal, 10i.
This widening of the pH range of available free aldehyde
is more likely to be due to an electronic rather than a
stearic affect. However, hydrogen bonding of the guani-
dinium arginine side chain to the π system of the
p-nitrophenylalanine side chain in compound 10i, as
shown in Figure 2, also appears to prevent the cyclization
of the guanidino nitrogen with the C-terminal aldehyde.
Meth yl 4-Gu a n id in obu ta n oa te Hyd r och lor id e. A solu-
tion of 4-guanidinobutanoic acid (5 g, 34.4 mmol) in 50 mL of
thionyl chloride (Aldrich) was stirred at room temperature for
18 h. The solution was then chilled in a dry ice/acetone bath to
-78 °C. Methanol (200 mL, precooled in a dry ice/acetone bath)
was slowly (over 5 min) and carefully added to the cold thionyl
chloride solution. (This step should always be performed in a
fume hood with an open system to avoid the pressure build-up
of escaping gases!) The mixture was allowed to slowly (over 3
h) warm to room temperature while being stirred. The solution
was then concentrated under reduced pressure to about one-
fourth of its original volume. The concentrated solution was
triturated with diethyl ether to produce a crude solid. The solid
material was then recrystallized four times from methanol/
diethyl ether to give methyl 4-guanidinobutanoate‚HCl (5.5 g,
93% yield): HPLC Rv ) 3.75 mL (acetonitrile/water, 7/3, v/v,
C18, 5 µm); 1H NMR (D2O) δ 3.7 (s, 3H), 2.9-3.2 (t, 2H, J ) 7
Hz), 1.7-2.0 (m, 2H). Anal. Calcd for C6H15N3O2Cl: C, 36.65;
H, 7.69; N, 21.37. Found: C, 36.63; H, 7.68; N, 21.38.
Meth yl NG-Ca r boben zoxy-4-gu a n id in obu ta n oa te (2). A
solution of methyl 4-guanidinobutanoate hydrochloride (5.0 g,
25.6 mmol) in 100 mL of methanol was mixed with a solution of
Biaci and Gyger-Marazzi26 reported that the inhibition
of cathepsin B by leupeptin occurs after a significant lag
(26) Biaci, A.; Gyger-Marazzi, M. Eur. J . Biochem. 1982, 129, 33-
41.

5652 J . Org. Chem., Vol. 63, No. 16, 1998
Notes
13.8 g (256 mmol) of sodium methoxide in 100 mL of methanol.
The combined mixture was chilled in an ice/water bath. Benzyl
chloroformate (72 mL total, 512 mmol) was added, in 10 mL
increments every 20 min, to the cold, rapidly stirring solution.
After each 10 mL addition of benzyl chloroformate, 15 mL of a
precooled solution of sodium methoxide (20 g, 370 mmol) in 100
mL of methanol was also added. The reaction mixture was
stirred in an ice/water bath for an additional 2 h and then at
room temperature for 18 h. The reaction mixture was then
neutralized with 100 mL of 1.0 M aqueous HCl. A 1 mL portion
of the mixture was removed and diluted with 5 mL of distilled
water. The pH of this portion was tested (pH ) 6.5). The
reaction mixture was then concentrated to about 100 mL under
reduced pressure. The concentrated mixture was diluted with
ethyl acetate/isopropyl alcohol (300 mL of 6/4, v/v) and stirred
for 2 h at room temperature. The undissolved sodium chloride
byproduct was removed by filtration. The solvent was removed
by evaporation under reduced pressure and the crude product
was redissolved in 200 mL of the ethyl acetate/isopropyl alcohol
(6/4, v/v) solution. The mixture was filtered again and the
solvent was evaporated under reduced pressure. The product
was redissolved in 20 mL of isopropyl alcohol and then precipi-
tated with the addition of 200 mL of ethyl ether. The crude solid
was recrystallized four times from isopropyl alcohol/ether to give
2 (4.9 g, 65% yield): TLC Rf 0.13 (12% ethanol/ethyl acetate,
silica gel F254); HPLC Rv ) 6.25 mL (acetonitrile/water, 7/3,
v/v, C18, 5 µm); 1H NMR (DMSO-d6) δ 7.4 (s, 5H), 5.1 (s, 2H),
3.9-4.3 (m, 2H), 3.6 (s, 3H), 2.9-3.3 (t, 3H, J ) 7 Hz), 2.2-2.7
(m, 2H), 1.6-2.1 (m, 2H). Anal. Calcd for C14H19N3O4: C, 57.33;
H, 6.53; N, 14.33. Found: C, 57.29; H, 6.55; N, 14.36.
13CC13H21N4O4Cl: C, 48.92; H, 6.12; N, 16.20. Found: C, 49.15;
H, 6.09; N, 16.25.
(1-13C)-DL-Ar gin in e Dih yd r och lor id e (5). A solution of 2.0
g (5.8 mmol) of NG-carbobenzoxy-1-13C-DL-arginine hydrochloride
(4) in 200 mL of methanol and 0.5 mL of 1 M aqueous HCl,
containing 0.7 g of prewetted 10% palladium on carbon, was
stirred under hydrogen (1 atm) at 20 °C for 3 h. The catalyst
was removed by filtration and the solvent evaporated under
reduced pressure. The Sakaguchi22-positive product was tritu-
rated with acetonitrile to give 1.3 g (93% yield) of 5: mp ) 224-
227 °C; HPLC Rv ) 3.55 mL (0.1% TCA in acetonitrile/water,
1
6/4, v/v, C18, 5 µm); H NMR (deuterium oxide) δ 4.1-4.4 (m, 1
H), 3.2-3.6 (m, 2H), 1.8-2.2 (m, 4H). Anal. Calcd for
13CC5H16N4O2Cl2: C, 29.45; H, 6.50; N, 22.58. Found: C, 29.59;
H, 6.48; N, 22.62.
(1-13C)-L-Ar gin in e Dih yd r och lor id e (5a ). (1-13C)-DL-argi-
nine hydrochloride (2.5 g) was resolved as its acetyl derivative
by the method described by Greenstein and Winitz22 to give 0.88
g (70% yield) of the title compound: mp ) 227-230 °C; [R]25
)
D
+24° (c ) 2, 1 M HCl); HPLC Rv ) 3.54 mL (0.1% TCA in
acetonitrile/water, 6/4, v/v, C18, 5 µm); 1H NMR (10% DCl in D2O)
δ 4.3-4.6 (m, 1H), 3.3-3.7 (m, 2H), 1.7-2.1 (m, 4H). Anal. Calcd
for 13CC5H16N4O2Cl2: C, 29.45; H, 6.50; N, 22.58. Found: C,
29.57; H, 6.51; N, 22.56.
Nr-BOC-NG-Cbz-(1-13C)-L-a r gin in e (6). A solution of 5a
(1.0 g, 4.1 mmol) in 20 mL of distilled water was treated with
3.0 mL of triethyl amine and stirred at room temperature for
20 min. A solution of 4.0 g (16.2 mmol) of BOC-ON (2-tert-
butoxycarbonyloxyimino-2-phenylacetonitrile, Aldrich) in 20 mL
of 1,4-dioxane was added to the aqueous solution of 5a . The
mixture was warmed to 40 °C to complete dissolution and then
stirred overnight at room temperature. The solvent was then
evaporated under reduced pressure and the product triturated
with ether. The NR-BOC-(1-13C)-L-arginine hydrochloride was
purified by reverse phase prep plate chromatography (Whatman
PLKC18F Linear-K) using 95% ethanol as a mobile phase. 1H
NMR verified the presence of only one BOC group and a positive
Sakaguchi test verified the presence of the free guanidinium
group. NR-BOC-(1-13C)-L-arginine hydrochloride (0.95 g, 3.1
mmol) was dissolved in 50 mL methanol and treated with 15
mL of 1 M sodium methoxide in methanol and chilled in an ice-
water bath. Benzyl chloroformate (8.0 mL, 57 mmol) was added
in 1 mL increments every 20 min to the cold, rapidly stirring
methanolic solution. After each 1 mL addition of benzyl chlo-
roformate, 1 mL of a precooled 1 M sodium methoxide in
methanol was also added. The reaction mixture was stirred in
an ice bath for an additional 2 h and then at room temperature
for 18 h. The reaction mixture was then neutralized by the
addition of 80 mL of 0.5 M aqueous HCl. A 5-drop aliquot of
the mixture was removed and diluted with 2 mL of distilled
water, and the pH was tested (pH ) 6.5). Aqueous NaOH (5
mL, 1.0 M) was then added to the reaction mixture with stirring.
Another 5-drop aliquot was removed and diluted with 2 mL of
distilled water, and the pH was tested (pH ) 7.6). The reaction
mixture was then concentrated under reduced pressure. The
resulting concentrate was diluted with 150 mL of 10% aceto-
nitrile/ethyl acetate and stirred for 1 h. The undissolved sodium
chloride was removed by filtration. The solvent was then
removed under reduced pressure and the crude NR-BOC-NG-Cbz-
(1-13C)-L-arginine was purified by silica gel chromatography
using 5% methanol/ethyl acetate as a mobile phase to give 6
(1.5 g, 64%): TLC Rf ) 0.27 (ethyl acetate/methanol, 9/1, v.v);
HPLC Rv ) 9.95 mL (acetonitrlle/water, 8/2, v/v, C18, 5 µm); 1H
NMR (DMSO-d6) δ 8.5 (m, 2H), 7.2 (s, 5H), 5.2 (s, 2H), 4.4-4.5
(m, 1H), 3.4-3.6 (t, J ) 7 Hz, 2H), 1.8-2.2 (m, 4H), 1.1 (s, 9H).
Anal. Calcd for 13CC18H28N4O6: C, 55.98; H, 6.89; N, 13.68.
Found: C, 56.19; H, 6.91; N, 13.65.
NG-Ca r boben zoxy-4-gu a n id in obu ta n a l (3). A solution of
4.5 g (15.3 mmol) of methyl NG-carbobenzoxy-4-guanidino-
butanoate (2) in 100 mL of anhydrous diglyme (2-methoxy ethyl
ether) was chilled in a dry ice/ethanol-water (2/3, v/v) bath to
-30 °C under a N2 atmosphere, vigorously stirred, and slowly
treated with 60 mL of 1.0 M diisobutyl aluminum hydride
solution in tetrahydrofuran (Aldrich) over 30 min. The reaction
mixture was stirred at -30 °C under N2 for an additional 2 h
and then quenched with 100 mL of 0.1 M aqueous HCl. The
reaction mixture was then partitioned between the phases of
400 mL of ethyl acetate, 100 mL of 1-butanol, and 400 mL of
10% sodium potassium tartarate. The organic layer was washed
with 100 mL of distilled water and dried over anhydrous
magnesium sulfate. The organic layer was then concentrated
under reduced pressure and the remainder of the solvent was
evaporated in vacuo. The crude product was purified by silica
gel chromatography (60 cm × 2 cm, 1 × d) using 95/5 ethyl
acetate/ethanol as the mobile phase to give 2.8 g (69% yield) of
1
3: TLC Rf 0.18 (12% ethanol/ethyl acetate, silica gel F254); H
NMR (DMSO-d6) δ 9.5 (s, 0.8H, CHO), 7.5 (s, 5H), 5.8-6.1 (m,
0.2H, carbinol amine), 5.2 (s, 2H), 3.9-4.4 (m, 2H), 3.0-3.3 (t,
J ) 7 Hz, 2H), 2.1-2.6 (m, 2H), 1.5-2.0 (m, 2H). Anal. Calcd
for C13H17N3O3: C, 59.30; H, 6.51; N, 15.96. Found: C, 59.27;
H, 6.53; N, 15.99.
NG-Ca r boben zoxy-[1-13C]-DL-a r gin in e Hyd r och lor id e (4).
A solution of 2.5 g (9.5 mmol) of NG-carbobenzoxy-4-guanidi-
nobutanal (3) in 10 mL of 5% aqueous methanol was saturated
with ammonia. (NH3(g) was vigorously bubbled through the
solution for 1 h at room temperature.) The solution was then
mixed with a solution of 650 mg [13C]potassium cyanide (ICN
Stable Isotopes) and 1.1 g of ammonium chloride in 10 mL of
saturated aqueous ammonia. The mixture was stirred at room
temperature for 48 h. The solvent was then removed in vacuo
and 20 mL of cold 12 M HCl was added. The mixture was
refluxed for 5 h and then stirred for 24 h at room temperature.
The solvent was evaporated in vacuo and the product was
purified on Dowex 50W X-4 (90 cm × 3 cm, 1 × d) using 2 M
aqueous HCl. The ninhydrin-positive product was dissolved in
100 mL of methanol, mixed with Darco G-60 decolorizing carbon,
filtered, and concentrated to about 25 mL under reduced
pressure. Crystallization was induced by the addition of 200
mL of cold diethyl ether, to give 2.1 g (65% yield) of 4: HPLC
Rv ) 4.7 mL (acetonitrile/water, 6/4, v/v, C18, 5 µm); 1H NMR
(methanol-d4) δ 11.5 (m, 1H), 7.3 (s, 5H), 5.8-6.0 (m, 2H), 5.2
(s, 2H), 4.9-5.1 (m, 2H), 4.0-4.2 (m, 1H), 3.0-3.3 (t, J ) 7 Hz,
2H), 2.2-2.6 (m, 2H), 1.5-2.1 (m, 2H). Anal. Calcd for
Nr-BOC-NG-Cbz-(1-13C)-L-Ar gin in a l Sem ica r ba zon e (7).
NR-BOC-NG-Cbz-(1-13C)-L-arginine (5.8 g, 14.2 mmol) was dis-
solved in 100 mL of freshly distilled tetrahydrofuran (THF) and
stirred under N2 for 1 h. The mixture was chilled in ice/H2O
bath and treated with 2.8 g of 1,1′-carbonyldiimidazole. The
mixture was stirred at 5 °C for 1 h and then chilled in a dry
ice-alcohol/H2O, 3/1 by vol, (-40 °C) bath under N2. LiAlH4
(35 mL, 1 M) in THF (Aldrich) was added dropwise over 20 min.
The reaction mixture was allowed to stir at -40 °C for an
additional 20 min under N2. The reaction was then quenched

Notes
J . Org. Chem., Vol. 63, No. 16, 1998 5653
by the addition of 100 mL of 0.1 M aqueous HCl. The mixture
was then partitioned between the phases of dichloromethane
(400 mL) and 5% sodium potassium tartarate (400 mL). The
organic phase was washed with distilled water (100 mL), dried
over anhydrous MgSO4, and evaporated under reduced pressure.
The crude product was dissolved in 100 mL of 95% ethanol and
treated with 100 mL of a solution containing 2.7 g semicarbazide
hydrochloride in 3% aqueous sodium bicarbonate. The mixture
was stirred for 18 h at 25 °C and then partitioned between the
phases of ethyl acetate (200 mL) and 5% sodium potassium
tartarate (200 mL). The organic phase was dried over anhydrous
MgSO4 and evaporated under reduced pressure. The crude
semicarbazone was purified by silica gel column chromatography
(75 × 2.5 cm) using 3% methanol/ethyl acetate as the mobile
phase to produce 2.2 g (34%) of 7: TLC Rf ) 0.36 (5% methanol
in ethyl acetate); HPLC Rv ) 4.05 mL (acetonitrile/water, 8/2,
v/v, C18, 5 µm); 1H NMR (DMSO-d6) δ 9.4-9.6 (m, 1H), 8.2-8.5
(m, 2H), 7.2 (s, 5H), 6.9 (d, J ) 7 Hz, 1H), 5.8-6.1 (m, 3H), 5.2
(s, 2H), 4.0-4.2 (m, 1H), 3.1-3.3 (t, J ) 7 Hz, 2H), 1.8-2.2 (m,
4H), 1.1 (s, 9H). Anal. Calcd for 13CC19H30N7O5: C, 53.66; H,
6.73; N, 21.81. Found: C, 53.87; H, 6.71; N, 21.86.
) 7 Hz, 2H), 1.3-1.7 (m, 14H), 0.9-1.1 (m, 9H). Anal. Calcd
for 13CC34H51N9O7: C, 59.28; H, 7.22; N, 17.74. Found: C, 59.52;
H, 7.20; N, 17.73.
Cbz-L-leu cyl-L-p h en yla la n yl-NG-Cbz-(1-13C)-L-a r gin in a l
sem ica r ba zon e (8d ): TLC Rf ) 0.32 (ethyl acetate/methanol,
9/1, v/v); HPLC Rv ) 7.05 mL (methanol/water, 7/3, v/v, C18, 5
µm); 1H NMR (DMSO-d6) δ 9.4-9.6 (m, 1H), 7.8-8.1 (m, 2H),
7.4 (s, 5H), 7.2 (s, 5H), 7.0 (s, 5H), 6.8 (d, J ) 7 Hz, 1H), 5.9-6.1
(m, 3H), 5.3 (s, 2H), 5.1 (s, 2H), 4.6-4.8 (m, 3H), 3.9-4.2 (m,
3H), 3.0-3.4 (m, 4H), 1.4-1.7 (m, 7H), 0.9-1.1 (d, J ) 7 Hz,
6H). Anal. Calcd for 13CC37H49N9O7: C, 61.41; H, 6.63; N, 16.92.
Found: C, 61.71; H, 6.66; N, 16.94.
Cbz-L-p h en yla la n yl-L-leu cyl-NG-Cbz-(1-13C)-L-a r gin in a l
sem ica r ba zon e (8e): TLC Rf ) 0.25 (ethyl acetate/methanol,
9/1, v/v); HPLC Rv ) 7.15 mL (methanol/water, 7/3, v/v, C18, 5
µm); 1H NMR (DMSO-d6) δ 9.4-9.5 (m, 1H), 7.9-8.1 (m, 2H),
7.4 (s, 5H), 7.2 (s, 5H), 6.9 (s, 5H), 6.8 (d, J ) 7 Hz, 1H), 5.8-6.0
(m, 3H), 5.3 (s, 2H), 5.1 (s, 2H), 4.5-4.9 (m, 3H), 3.8-4.1 (m,
3H), 2.9-3.3 (m, 4H), 1.3-1.7 (m, 7H), 0.8-1.0 (d, J ) 7 Hz,
6H). Anal. Calcd for 13CC37H49N9O7: C, 61.41; H, 6.63; N, 16.92.
Found: C, 61.69; H, 6.65; N, 16.90.
NG-Cb z-(1-13C)-L-Ar gin in a l Sem ica r b a zon e Tr iflu or o-
a ceta te. A solution of 2.0 g of 7 in 40 mL of trifluoroacetic acid/
chloroform (50/50 by volume) was chilled in an ice/H2O bath and
stirred at 5 °C for 3 h. The solution was concentrated under
reduced pressure. The crude product was triturated with
anhydrous ether and recrystallized from methanol/ether to give
2.1 g of NG-Cbz-(1-13C)-L-argininal semicarbazone trifluoroac-
etate: TLC Rf ) 0.09, ninhydrin positive, Sakaguchi negative
(ethyl acetate/methanol, 9/1, v/v); 1H NMR (D2O) δ 7.1 (s 5H),
6.9 (d, J ) 7 Hz, 1H), 5.2 (s, 2H), 4.0-4.2 (m, 1H), 3.0-3.2 (t, J
) 7 Hz, 2H), 1.7-2.1 (m, 4H).
Cou p lin g P r oced u r e. A solution of 1 mmol of the appropri-
ate carbobenzoxy-protected dipeptide (Sigma) in 10 mL of
anhydrous N,N-dimethylformamide (DMF) was chilled to -15
°C with vigorous stirring. The solution was treated with 0.140
mL (1 mmol) of triethylamine. After 30 min, 0.097 mL (1 mmol)
of ethyl chloroformate was added to the cold reaction mixture.
The reaction mixture was stirred at -15 °C for an additional
45 min. A precooled solution containing 0.464 g (1 mmol) of NG-
Cbz-(1-13C)-L-argininal semicarbazone trifluoroacetate (7) and
0.280 g (2 mmol) of triethylamine in 10 mL of anhydrous DMF
was then added to the reaction mixture. The resulting solution
was stirred at 0 °C for 4 h and then overnight at room
temperature. The reaction mixture was then partitioned be-
tween the phases of ethyl acetate (200 mL) and 0.1 M aqueous
NaOH (200 mL). The organic layer was extracted with 10%
aqueous NaHCO3 (200 mL), distilled water (200 mL), 0.1 M
aqueous HCl (200 mL), and distilled water again (200 mL). The
organic layer was dried over anhydrous MgSO4 and concentrated
under reduced pressure. The tripeptide was solidified by the
addition of anhydrous ether and then recrystallized from
methanol/ether.
Cb z-L-leu cyl-L-a la n yl-NG-Cb z-(1-13C)-L-a r gin in a l sem i-
ca r ba zon e (8f): TLC Rf ) 0.16 (ethyl acetate/methanol, 9/1, v/v);
HPLC Rv ) 3.25 mL (methanol/water, 6/4, v/v, C18, 5 µm); 1H
NMR (DMSO-d6) δ 9.4-9.6 (m, 1H), 7.9-8.1 (m, 2H), 7.4 (s, 5H),
7.2 (s, 5H), 6.8 (d, J ) 7 Hz, 1H), 5.8-6.0 (m, 3H), 5.3 (s, 2H),
5.1 (s, 2H), 4.6-4.9 (m, 3H), 3.7-4.1 (m, 3H), 3.0-3.3 (t, J ) 7
Hz, 2H), 2.0-2.2 (d, J ) 7 Hz, 3H), 1.2-1.7 (m, 7H), 0.9-1.1 (d,
J ) 7 Hz, 6H). Anal. Calcd for 13CC31H45N9O7: C, 57.62; H,
6.78; N, 18.85. Found: C, 57.84; H, 6.76; N, 18.81.
Cb z-L-leu cyl-â-a la n yl-NG-Cb z-(1-13C)-L-a r gin in a l sem i-
ca r ba zon e (8g): TLC Rf ) 0.14 (ethyl acetate/methanol, 9/1,
v/v); HPLC Rv ) 3.45 mL (methanol/water, 6/4, v/v, C18, 5 µm);
1H NMR (DMSO-d6) δ 9.4-9.6 (m, 1H), 7.9-8.2 (m, 2H), 7.4 (s,
5H), 7.2 (s, 5H), 6.8 (d, J ) 7 Hz, 1H), 5.7-6.0 (m, 3H), 5.3 (s,
2H), 5.1 (s, 2H), 4.6-4.9 (m, 3H), 3.6-4.1 (m, 2H), 2.9-3.4 (m,
6H), 1.2-1.8 (m, 7H), 0.9-1.1 (d, J ) 7 Hz, 6H). Anal. Calcd
for 13CC31H45N9O7: C, 57.62; H, 6.78; N, 18.85. Found: C, 57.87;
H, 6.75; N, 18.80.
Cb z-L-leu cyl-L-p -ch lor op h en yla la n yl-NG-Cb z-(1-13C)-L-
a r gin in a l sem ica r ba zon e (8h ): TLC Rf ) 0.22 (acetonitrile/
ethyl acetate, 1/2, v/v); HPLC Rv ) 5.88 mL (methanol/water,
8/2, v/v, C18, 5 µm); 1H NMR (DMSO-d6) δ 9.4-9.6 (m, 1H), 8.0-
8.3 (m, 2H), 7.1-7.7 (m, 14H), 6.8 (d, J ) 7 Hz, 1H), 5.7-6.0
(m, 3H), 5.3 (s, 2H), 5.1 (s, 2H), 4.6-4.9 (m, 3H), 3.9-4.2 (m,
3H), 3.0-3.5 (m, 4H), 1.4-1.7 (m, 7H), 0.9-1.1 (d, J ) 7 Hz,
6H). Anal. Calcd for 13CC37H48N9O7Cl: C, 58.70; H, 6.21; N,
16.18. Found: C, 58.96; H, 6.19; N, 16.15.
Cbz-L-leu cyl-L-p-n itr oph en ylalan yl-NG-Cbz-(1-13C)-L-ar gin -
in a l sem ica r ba zon e (8i): TLC Rf ) 0.14 (acetonitrile/ethyl
acetate, 1/2, v/v); HPLC Rv ) 4.22 mL (methanol/water, 8/2, v/v,
C18, 5 µm); 1H NMR (DMSO-d6) δ 9.4-9.7 (m, 1H), 8.0-8.3 (m,
2H), 7.0-7.9 (m, 14H), 6.8 (d, J ) 7 Hz, 1H), 5.7-6.0 (m, 3H),
5.3 (s, 2H), 5.1 (s, 2H), 4.5-4.9 (m, 3H), 3.8-4.2 (m, 3H), 2.8-
3.5 (m, 4H), 1.4-1.7 (m, 7H), 0.9-1.1 (d, J ) 7 Hz, 6H). Anal.
Calcd for 13CC37H48N10O9: C, 57.91; H, 6.13; N, 17.73. Found:
C, 58.19; H, 6.11; N, 17.70.
Cb z-L-leu cyl-L-leu cyl-NG-Cb z-(1-13C)-L-a r gin in a l sem i-
ca r ba zon e (8a ): TLC Rf ) 0.55 (ethyl acetate/methanol, 9/1,
v/v); HPLC Rv ) 6.33 mL (methanol/water, 7/3, v/v, C18, 5 µm);
1H NMR (DMSO-d6) δ 9.4-9.6 (m, 1H), 8.2-84 (m, 2H), 7.4 (s,
5H), 7.0 (s, 5H), 6.8 (d, J ) 7 Hz, 1H), 5.8-6.0 (m, 3H), 5.3 (s,
2H), 5.1 (s, 2H), 4.6-4.8 (m, 3H), 4.1-4.4 (m, 3H), 3.4-3.6 (t, J
) 7 Hz, 2H), 1.5-1.8 (m, 10H), 0.9-1.0 (d, J ) 7 Hz, 12H). Anal.
Calcd for 13CC34H51N9O7: C, 59.28; H, 7.22; N, 17.74. Found:
C, 59.54; H, 7.19; N, 17.75.
Cbz-L-leu cyl-L-cycloh exyla la n yl-NG-Cbz-(1-13C)-L-a r gin i-
n a l sem ica r ba zon e (8j): TLC Rf ) 0.18 (acetonitrile/ethyl
acetate, 1/2, v/v); HPLC Rv ) 6.77 mL (acetonitrile/water, 7/3,
v/v, C18, 5 µm); 1H NMR (DMSO-d6) δ 9.4-9.7 (m, 1H), 8.0-
8.4 (m, 2H), 7.4 (s, 5H), 7.1 (s, 5H), 6.8 (d, J ) 7 Hz, 1H), 5.6-
6.0 (m, 3H), 5.3 (s, 2H), 5.1 (s, 2H), 4.6-4.9 (m, 3H), 3.7-4.1
(m, 3H), 2.9-3.1 (t, J ) 7 Hz, 2H), 2.0-2.3 (m, 2H), 1.4-1.7 (m,
12H), 0.8-1.2 (m, 12H). Anal. Calcd for 13CC37H55N9O7: C,
60.92; H, 7.38; N, 16.79. Found: C, 61.21; H, 7.36; N, 16.75.
Gen er a l N-Ter m in a l a n d Gu a n id in o Deblock in g P r o-
ced u r e. A dilute solution containing 0.50 mmol of the fully
protected tripeptide (8a -j) in 200 mL of methanol was treated
with a slurry of 10 mg of 10% palladium on carbon in 2.0 mL of
Cbz-L-leu cyl-L-va lyl-NG-Cbz-(1-13C)-L-a r gin in a l sem ica r -
ba zon e (8b): TLC Rf ) 0.42 (ethyl acetate/methanol, 9/1, v/v);
HPLC Rv ) 6.21 mL (methanol/water, 7/3, v/v, C18, 5 µm); 1H
NMR (DMSO-d6) δ 9.5-9.6 (m, 1H), 8.0-8.2 (m, 2H), 7.4 (s, 5H),
7.1 (s, 5H), 6.9 (d, J ) 7 Hz, 1H), 5.8-6.0 (m, 3H), 5.3 (s, 2H),
5.1 (s, 2H), 4.7-4.9 (m, 3H), 4.2-4.4 (m, 3H), 3.4-3.6 (t, J ) 7
Hz, 2H), 1.4-1.7 (m, 8H), 1.0-1.2 (d, J ) 7 Hz, 12H). Anal.
Calcd for 13CC33H49N9O7: C, 58.75; H, 7.09; N, 18.09. Found:
C, 58.96; H, 7.11; N, 18.07.
0.10 M HCl. The mixture was stirred under
a hydrogen
Cbz-L-leu cyl-L-isoleu cyl-NG-Cbz-(1-13C)-L-a r gin in a l sem i-
ca r ba zon e (8c): TLC Rf ) 0.58 (ethyl acetate/methanol, 9/1,
v/v); HPLC Rv ) 6.45 mL (methanol/water, 7/3, v/v, C18, 5 µm);
1H NMR (DMSO-d6) δ 9.3-9.5 (m, 1H), 8.1-8.3 (m, 2H), 7.4 (s,
5H), 7.2 (s, 5H), 6.8 (d, J ) 7 Hz, 1H), 5.7-6.0 (m, 3H), 5.3 (s,
2H), 5.1 (s, 2H), 4.7-4.9 (m, 3H), 4.0-4.3 (m, 3H), 3.3-3.5 (t, J
atmosphere (1 atm), with hydrogen bubbling through the
mixture, at room temperature for 4 h. The catalyst was removed
by filtration and the solution was concentrated under reduced
pressure. Excess water was removed by the addition and
subsequent evaporation of acetonitrile. The crude soil was
recrystallized from methanol/ether. The Sakaguchi- and nin-

5654 J . Org. Chem., Vol. 63, No. 16, 1998
Notes
hydrin-positive dihydrochloride salt of the tripeptide semicar-
bazone was dissolved in 10 mL of anhydrous N,N-dimethyl-
formamide and chilled to 0 °C. The rapidly stirring solution was
treated with 40 µL (0.50 mmol) of anhydrous pyridine. The
solution was stirred at 0 °C for 30 min and then treated with
47 µL (0.50 mmol) of acetic anhydride. The mixture was stirred
at 0 °C for 3 h and then overnight at room temperature.
Distilled water (100 µL) was added to quench the reaction. Cold
THF (100 mL at 0 °C) was added to precipitate the product. The
crude solid was purified by Sephasorb H-P chromatography (120
× 5 cm column) using methanol as the mobile phase. The
fractions showing both a positive Sakaguchi22 and a negative
ninhydrin test were pooled and concentrated under reduced
pressure.
Acet yl-L-leu cyl-L-leu cyl-(1-13C)-L-a r gin in a l sem ica r b a -
zon e‚HCl (9a ): TLC Rf ) 0.67 (ethanol/water, 6/4, on Whatman
MKC18F reversed phase TLC); HPLC Rv ) 6.21 mL (0.05% TCA
in methanol/water, 7/3, v/v, C18, 5 µm); 1H NMR (DMSO-d6) δ
8.4-8.5 (m, 3H), 7.8-8.0 (m, 3H), 6.9-7.0 (d, J ) 7 Hz, 1H),
5.8-6.0 (m, 3H), 4.6-4.8 (m, 3H), 3.9-4.3 (m, 3H), 3.4-3.6 (t, J
) 7 Hz, 2H), 2.4 (s, 3H), 1.5-1.8 (m, 10H), 0.9-1.1 (d, J ) 7 Hz,
12H). Anal. Calcd for 13CC20H42N9O4Cl: C, 48.60; H, 8.12; N,
24.19. Found: C, 48.79; H, 8.10; N, 24.11.
0.9-1.1 (d, J ) 7 Hz, 6H). Anal. Calcd for 13CC17H36N9O4Cl:
C, 45.35; H, 7.58; N, 26.32. Found: C, 45.52; H, 7.55; N, 26.26.
Acetyl-L-leu cyl-L-p-ch lor op h en yla la n yl-(1-13C)-L-a r gin i-
n a l sem ica r ba zon e‚HCl (9h ): TLC Rf ) 0.71 (ethanol/water,
7/3, on Whatman MKC18F reversed phase TLC); HPLC Rv ) 4.21
mL (0.05% TCA in methanol/water, 8/2, v/v, C18, 5 µm); 1H NMR
(DMSO-d6) δ 8.4-8.8 (m, 3H), 7.6-8.1 (m, 7H), 6.9-7.0 (d, J )
7 Hz, 1H), 5.7-6.0 (m, 3H), 4.6-4.9 (m, 3H), 3.7-4.1 (m, 3H),
3.0-3.5 (m, 4H), 2.5 (s, 3H), 1.4-1.7 (m, 7H), 0.9-1.1 (d, J ) 7
Hz, 6H). Anal. Calcd for 13CC23H39N9O4Cl2: C, 49.07; H, 6.67;
N, 21.38. Found: C, 49.29; H, 6.65; N, 21.34.
Acet yl-L-leu cyl-L-p -n it r op h en yla la n yl-(1-13C)-L-a r gin i-
n a l sem ica r ba zon e‚HCl (9i): TLC Rf ) 0.77 (ethanol/water,
7/3, on Whatman MKC18F reversed phase TLC); HPLC Rv ) 3.98
mL (0.05% TCA in methanol/water, 8/2, v/v, C18, 5 µm); 1H NMR
(DMSO-d6) δ 8.5-8.8 (m, 3H), 7.5-8.2 (m, 7H), 6.9-7.0 (d, J )
7 Hz, 1H), 5.7-6.0 (m, 3H), 4.7-4.9 (m, 3H), 3.7-4.0 (m, 3H),
3.1-3.5 (m, 4H), 2.4 (s, 3H), 1.4-1.7 (m, 7H), 0.9-1.1 (d, J ) 7
Hz, 6H). Anal. Calcd for 13CC23H39N10O6Cl: C, 48.20; H, 6.55;
N, 23.24. Found: C, 48.43; H, 6.52; N, 23.40.
Acet yl-L-leu cyl-L-cycloh exyla la n yl-(1-13C)-L-a r gin in a l
sem ica r ba zon e‚HCl (9j): TLC Rf ) 0.44 (ethanol/water, 6/4,
on Whatman MKC18F reversed phase TLC); HPLC Rv ) 6.56
mL (0.05% TCA in methanol/water, 7/3, v/v, C18, 5 µm); 1H NMR
(DMSO-d6) δ 8.3-8.6 (m, 3H), 7.9-8.2 (m, 3H), 6.9-7.0 (d, J )
7 Hz, 1H), 5.7-6.0 (m, 3H), 4.5-4.9 (m, 3H), 3.7-4.2 (m, 3H),
2.9-3.1 (t, J ) 7 Hz, 2H), 2.5 (s, 3H), 2.0-2.3 (m, 2H), 1.3-1.7
(m, 12H), 0.8-1.2 (m, 12H). Anal. Calcd for 13CC23H46N9O4Cl:
C, 51.55; H, 8.26; N, 22.47. Found: C, 51.79; H, 8.30; N, 22.44.
Gen er a l P r oced u r e for Deb lock in g t h e C-Ter m in a l
Ald eh yd e. The acetyl tripeptide semicarbazone hydrochloride
(0.33 mmol) 9a -j was dissolved in 5 mL of methanol and 1 mL
of 0.5 M aqueous HCl and chilled to 0 °C. The cold, rapidly
stirring solution was treated with 0.75 µL (1.0 mmol) of 37%
formaldehyde. The mixture was stirred at 0 °C for 4 h. The
product was solidified by the addition of 100 mL of cold THF.
The crude solid was purified via Sephasorb H-P (120 × 5 cm
column) using methanol as the mobile phase. The fractions
demonstrating both positive Sakaguchi and 2,4-DNP tests were
pooled and concentrated under reduced pressure. The resulting
acetyl-tripeptide aldehydes 10a -j were solidified by the addition
of anhydrous ether and were recrystallized from methanol/ether.
Acetyl-L-leu cyl-L-leu cyl-(1-13C)-L-ar gin in al‚HCl (10a): TLC
Rf ) 0.37 (ethanol/water, 6/4, on Whatman MKC18F reversed
Acetyl-L-leu cyl-L-valyl-(1-13C)-L-ar gin in al sem icar bazon e‚
HCl (9b): TLC Rf ) 0.63 (ethanol/water, 6/4, on Whatman
MKC18F reversed phase TLC); HPLC Rv ) 6.56 mL (0.05% TCA
1
in methanol/water, 7/3, v/v, C18, 5 µm); H NMR (DMSO-d6) δ
8.4-8.6 (m, 3H), 7.8-8.0 (m, 3H), 6.9-7.0 (d, J ) 7 Hz, 1H),
5.7-6.0 (m, 3H), 4.6-4.8 (m, 3H), 4.0-4.3 (m, 3H), 3.4-3.6 (t, J
) 7 Hz, 2H), 2.5 (s, 3H), 1.5-1.8 (m, 8H), 0.9-1.1 (d, J ) 7 Hz,
12H). Anal. Calcd for 13CC19H40N9O4Cl: C, 47.57; H, 7.95; N,
24.86. Found: C, 47.79; H, 7.93; N, 24.90.
Acetyl-L-leu cyl-L-isoleu cyl-(1-13C)-L-ar gin in al sem icar ba-
zon e‚HCl (9c): TLC Rf ) 0.65 (ethanol/water, 6/4, on Whatman
MKC18F reversed phase TLC); HPLC Rv ) 6.87 mL (0.05% TCA
in methanol/water, 7/3, v/v, C18, 5 µm); 1H NMR (DMSO-d6) δ
8.3-8.5 (m, 3H), 7.8-8.1 (m, 3H), 6.9-7.0 (d, J ) 7 Hz, 1H),
5.8-6.0 (m, 3H), 4.5-4.7 (m, 3H), 3.9-4.3 (m, 3H), 3.4-3.6 (t, J
) 7 Hz, 2H), 2.4 (s, 3H), 1.5-1.8 (m, 13H), 0.8-1.0 (m, 9H). Anal.
Calcd for 13CC20H42N9O4Cl: C, 48.60; H, 8.12; N, 24.19.
Found: C, 48.82; H, 8.14; N, 24.16.
Acetyl-L-leu cyl-L-p h en yla la n yl-(1-13C)-L-a r gin in a l sem i-
ca r ba zon e‚HCl (9d ): TLC Rf ) 0.72 (ethanol/water, 6/4, on
Whatman MKC18F reversed phase TLC); HPLC Rv ) 5.27 mL
(0.05% TCA in methanol/water, 7/3, v/v, C18, 5 µm); 1H NMR
(DMSO-d6) δ 8.3-8.5 (m, 3H), 7.8-8.1 (m, 3H), 7.1 (s, 5H), 6.9-
7.0 (d, J ) 7 Hz, 1H), 5.7-6.0 (m, 3H), 4.5-4.9 (m, 3H), 3.8-4.2
(m, 3H), 3.1-3.5 (m, 4H), 2.4 (s, 3H), 1.3-1.7 (m, 7H), 0.8-1.0
(d, J ) 7 Hz, 6H). Anal. Calcd for 13CC23H40N9O4Cl: C, 52.11;
H, 7.26; N, 22.71. Found: C, 52.35; H, 7.23; N, 22.67.
Acetyl-L-p h en yla la n yl-L-leu cyl-(1-13C)-L-a r gin in a l sem i-
ca r ba zon e‚HCl (9e): TLC Rf ) 0.70 (ethanol/water, 6/4, on
Whatman MKC18F reversed phase TLC); HPLC Rv ) 6.01 mL
(0.05% TCA in methanol/water, 7/3, v/v, C18, 5 µm); 1H NMR
(DMSO-d6) δ 8.3-8.6 (m, 3H), 7.9-8.1 (m, 3H), 7.0 (s, 5H), 6.8-
6.9 (d, J ) 7 Hz, 1H), 5.8-6.0 (m, 3H), 4.5-4.9 (m, 3H), 3.8-4.2
(m, 3H), 3.1-3.5 (m, 4H), 2.5 (s, 3H), 1.4-1.7 (m, 7H), 0.8-1.0
(d, J ) 7 Hz, 6H). Anal. Calcd for 13CC23H40N9O4Cl: C, 52.11;
H, 7.26; N, 22.71. Found: C, 52.33; H, 7.24; N, 22.69.
Acet yl-L-leu cyl-L-a la n yl-(1-13C)-L-a r gin in a l sem ica r b a -
zon e‚HCl (9f): TLC Rf ) 0.79 (ethanol/water, 6/4, on Whatman
MKC18F reversed phase TLC); HPLC Rv ) 3.73 mL (0.05% TCA
in methanol/water, 7/3, v/v, C18, 5 µm); 1H NMR (DMSO-d6) δ
8.4-8.6 (m, 3H), 7.9-8.2 (m, 3H), 6.9-7.0 (d, J ) 7 Hz, 1H),
5.7-6.0 (m, 3H), 4.6-4.9 (m, 3H), 3.7-4.2 (m, 3H), 3.0-3.3 (t, J
) 7 Hz, 2H), 2.5 (s, 3H), 2.0-2.2 (d, J ) 7 Hz, 3H), 1.2-1.6 (m,
7H), 0.9-1.1 (d, J ) 7 Hz, 6H). Anal. Calcd for 13CC17H36N9O4-
Cl: C, 45.35; H, 7.58; N, 26.32. Found: C, 45.54; H, 7.56; N,
26.28.
phase TLC); HPLC Rv ) 5.64 mL (0.07% TCA in ethanol/water,
1
7/3, v/v, C18, 5 µm); [R]25 ) -28.4° (c 1.0, methanol); H NMR
D
(DMSO-d6) δ 10.0 (s, 0.4H, CHO), 7.6-7.9 (m, 3H), 6.0 (m, 0.6H,
carbinol amine), 5.4 (m, 2H), 4.6-4.7 (m, 1H), 4.1-4.3 (m, 3H),
3.0-3.3 (t, J ) 7 Hz, 2H), 2.5 (s, 3H), 1.3-1.7 (m, 10H), 0.9-1.1
(d, J ) 7 Hz, 12H). Anal. Calcd for 13CC19H39N6O4Cl: C, 51.98;
H, 8.47; N, 18.11. Found: C, 52.08; H, 8.44; N, 18.06.
Acetyl-L-leu cyl-L-va lyl-(1-13C)-L-a r gin in a l‚HCl (10b): TLC
Rf ) 0.65 (ethanol/water, 6/4, on Whatman MKC18F reversed
phase TLC); HPLC Rv ) 5.27 mL (0.07% TCA in ethanol/water,
1
7/3, v/v, C18, 5 µm); [R]25 ) -35.0° (c 1.0, methanol); H NMR
D
(DMSO-d6) δ 10.1 (s, 0.4H, CHO), 7.5-7.9 (m, 3H), 5.9 (m, 0.6H,
carbinol amine), 5.4 (m, 2H), 4.6-4.7 (m, 1H), 4.1-4.4 (m, 3H),
3.0-3.3 (t, J ) 7 Hz, 2H), 2.5 (s, 3H), 1.5-1.8 (m, 8H), 0.9-1.1
(d, J ) 7 Hz, 12H). Anal. Calcd for 13CC18H37N6O4Cl: C, 50.94;
H, 8.29; N, 18.68. Found: C, 51.02; H, 8.37; N, 18.61.
Acetyl-L-leu cyl-L-isoleu cyl-(1-13C)-L-a r gin in a l‚HCl (10c):
TLC Rf ) 0.27 (ethanol/water, 6/4, on Whatman MKC18
F
reversed phase TLC); HPLC Rv ) 5.39 mL (0.07% TCA in
ethanol/water, 7/3, v/v, C18, 5 µm); [R]25 ) -29.6° (c 1.0,
D
1
methanol); H NMR (DMSO-d6) δ 10.1 (s, 0.4H, HCO), 7.3-7.7
(m, 3H), 6.1 (m, 0.6H, carbinol amine), 5.6 (m, 2H), 4.6-4.7 (m,
1H), 4.2-4.3 (m, 3H), 3.0-3.2 (t, J ) 7 Hz, 2H), 2.6 (s, 3H), 1.1-
13
1.5 (m, 10H), 0.8-1.0 (d, J ) 7 Hz, 12H). Anal. Calcd for
-
CC19H39N6O4Cl: C, 51.98; H, 8.47; N, 18.11. Found: C, 52.17;
H, 8.52; N, 18.04.
Acet yl-L-leu cyl-â-a la n yl-(1-13C)-L-a r gin in a l sem ica r b a -
zon e‚HCl (9g): TLC Rf ) 0.81 (ethanol/water, 6/4, on Whatman
MKC18F reversed phase TLC); HPLC Rv ) 3.93 mL (0.05% TCA
in methanol/water, 7/3, v/v, C18, 5 µm); 1H NMR (DMSO-d6) δ
8.4-8.7 (m, 3H), 7.9-8.2 (m, 3H), 6.8-6.9 (d, J ) 7 Hz, 1H),
5.7-6.0 (m, 3H), 4.6-4.9 (m, 3H), 3.7-4.1 (m, 3H), 2.9-3.4 (m,
6H), 2.4 (s, 3H), 2.0-2.2 (d, J ) 7 Hz, 3H), 1.2-1.6 (m, 7H),
Acet yl-L-leu cyl-L-p h en yla la n yl-(1-13C)-L-a r gin in a l‚H Cl
(10d ): TLC Rf ) 0.56 (ethanol/water, 6/4, on Whatman MKC18
F
reversed phase TLC); HPLC Rv ) 6.79 mL (0.07% TCA in
ethanol/water, 7/3, v/v, C18, 5 µm); [R[25 ) -31.3° (c 1.0,
D
1
methanol); H NMR (DMSO-d6) δ 7.6-7.9 (m, 3H), 7.1 (s, 5H),
6.1 (m, 1H, carbinol amine), 5.7 (m, 2H), 4.7-4.9 (m, 1H), 4.1-
4.3 (m, 3H), 3.0-3.3 (m, 4H), 2.4 (s, 3H), 1.3-1.7 (m, 7H), 0.9-

Notes
J . Org. Chem., Vol. 63, No. 16, 1998 5655
1.1 (d, J ) 7 Hz, 6H). Anal. Calcd for 13CC22H37N6O4Cl: C,
55.67; H, 7.49; N, 16.87. Found: C, 55.73; H, 7.64; N, 16.76.
Acet yl-L-p h en yla la n yl-L-leu cyl-(1-13C)-L-a r gin in a l‚H Cl
HCl (10h ): TLC Rf ) 0.46 (methanol/water, 7/3, on Whatman
MKC18F reversed phase TLC); HPLC Rv ) 6.89 mL (0.07% TCA
in ethanol/water, 5/5, v/v, C18, 5 µm); [R]25 ) -33.5° (c 1.0,
D
1
(10e): TLC Rf ) 0.63 (ethanol/water, 6/4, on Whatman MKC18
F
methanol); H NMR (DMSO-d6) δ 10.3 (s, 0.4H, CHO), 7.7-7.8
reversed phase TLC); HPLC Rv ) 6.94 mL (0.07% TCA in
(m, 3H), 7.0-7.5 (m, 7H), 5.9 (m, 0.6H, carbinol amine), 5.7 (m,
2H), 4.9-5.0 (m, 1H), 4.1-4.4 (m, 3H), 3.0-3.3 (m, 4H), 2.4 (s,
3H), 1.3-1.6 (m, 7H), 1.0-1.1 (d, J ) 7 Hz, 6H). Anal. Calcd
for 13CC22H36N6O4Cl2: C, 52.07; H, 6.81; N, 15.78. Found: C,
52.19; H, 6.89; N, 15.65.
ethanol/water, 7/3, v/v, C18, 5 µm); [R]25 ) -41.4° (c 1.0,
D
methanol); 1H NMR (DMSO-d6) δ 9.9 (s, 0.3H, CHO), 7.6-7.8
(m, 3H), 7.1 (s, 5H), 6.0 (m, 0.7H, carbinol amine), 5.6 (m, 2H),
4.9-5.0 (m, 1H), 4.2-4.4 (m, 3H), 3.0-3.2 (m, 4H), 2.3 (s, 3H),
1.3-1.6 (m, 7H), 1.0-1.1 (d, J ) 7 Hz, 6H). Anal. Calcd for
13CC22H37N6O4Cl: C, 55.67; H, 7.49; N, 16.87. Found: C, 55.76;
H, 7.59; N, 16.75.
Acetyl-L-leu cyl-L-p-n itr oph en ylalan yl-(1-13C)-L-ar gin in al‚
HCl (10i): TLC Rf ) 0.66 (methanol/water, 7/3, on Whatman
MKC18F reversed phase TLC); HPLC Rv ) 5.34 mL (0.07% TCA
Acetyl-L-leu cyl-L-alan yl-(1-13C)-L-ar gin in al‚HCl (10f): TLC
Rf ) 0.74 (ethanol/water, 6/4, on Whatman MKC18F reversed
phase TLC); HPLC Rv ) 4.44 mL (0.07% TCA in ethanol/water,
in ethanol/water, 5/5, v/v, C18, 5 µm); [R]25 ) -39.4° (c 1.0,
D
1
methanol); H NMR (DMSO-d6) δ 10.2 (s, 0.3H, CHO), 7.7-7.9
(m, 3H), 7.2-7.6 (m, 7H), 6.0 (m, 0.7H, carbinol amine), 5.7 (m,
2H), 4.9-5.0 (m, 1H), 4.0-4.4 (m, 3H), 3.0-3.4 (m, 4H), 2.5 (s,
3H), 1.3-1.6 (m, 7H), 1.0-1.1 (d, J ) 7 Hz, 6H). Anal. Calcd
for 13CC22H36N7O6Cl: C, 51.06; H, 6.68; N, 18.06. Found: C,
51.21; H, 6.73; N, 18.19.
7/3, v/v, C18, 5 µm); [R]25 ) -22.5° (c 1.0, methanol); H NMR
1
D
(DMSO-d6) δ 10.0 (s, 0.2H, CHO), 7.7-7.9 (m, 3H), 6.0 (m, 0.8H,
carbinol amine), 5.4 (m, 2H), 4.5-4.7 (m, 1H), 4.1-4.3 (m, 3H),
3.0-3.3 (t, J ) 7 Hz, 2H), 2.5 (s, 3H), 2.0-2.2 (d, J ) 7 Hz, 3H),
1.3-1.7 (m, 7H), 0.9-1.1 (d, J ) 7 Hz, 6H). Anal. Calcd for
13CC16H33N6O4Cl: C, 48.63; H, 7.88; N, 19.92. Found: C, 48.69;
H, 7.94; N, 19.83.
Acet yl-L-leu cyl-L-cycloh exyla la n yl-(1-13C)-L-a r gin in a l‚
HCl (10j): TLC Rf ) 0.27 (ethanol/water, 6/4, on Whtaman
MKC18F reversed phase TLC); HPLC Rv ) 8.14 mL (0.07% TCA
Acetyl-L-leu cyl-â-alan yl-(1-13C)-L-ar gin in al‚HCl (10g): TLC
Rf ) 0.79 (ethanol/water, 6/4, on Whatman MKC18F reversed
in ethanol/water, 7/3, v/v, C18, 5 µm); [R]25 ) -28.3° (c 1.0,
D
1
phase TLC); HPLC Rv ) 4.54 mL (0.07% TCA in ethanol/water,
methanol); H NMR (DMSO-d6) δ 10.1 (s, 0.8H, CHO), 7.7-7.9
1
7/3, v/v, C18, 5 µm); [R]25 ) -17.9° (c 1.0, methanol); H NMR
D
(m, 3H), 6.0 (m, 0.2H, carbinol amine), 5.4 (m, 2H), 4.5-4.6 (m,
1H), 4.0-4.3 (m, 3H), 3.0-3.3 (t, J ) 7 Hz, 2H), 2.5 (s, 3H), 2.0-
2.3 (m, 2H), 1.3-1.7 (m, 12H), 0.8-1.2 (m, 12H). Anal. Calcd
for 13CC22H43N6O4Cl: C, 55.01; H, 8.51; N, 16.67. Found: C,
55.24; H, 8.59; N, 16.54.
(DMSO-d6) δ 10.1 (s, 0.1H, CHO), 7.7-7.9 (m, 3H), 6.0 (m, 0.9H,
carbinol amine), 5.3 (m, 2H), 4.6-4.7 (m, 1H), 3.6-4.1 (m, 2H),
2.9-3.5 (m, 6H), 2.5 (s, 3H), 1.2-1.7 (m, 7H), 0.9-1.1 (d, J ) 7
Hz, 6H). Anal. Calcd for 13CC16H33N6O4Cl: C, 48.63; H, 7.88;
N, 19.92. Found: C, 48.71; H, 7.93; N, 19.81.
Acetyl-L-leu cyl-L-p-ch lor oph en ylalan yl-(1-13C)-L-ar gin in al‚
J O980070C