N-terminal dipeptides of D(-)-penicillamine as sequestration agents for acetaldehyde

Article abstract of DOI:10.1021/jm9902741

Since acetaldehyde (AcH), a toxic oxidation product of ethanol, may play an etiologic role in the initiation of alcoholic liver disease, we had earlier pioneered the development of β,β-disubstituted-β-mercapto-α-amino acids as AcH-sequestering agents. We now report the synthesis of a series of N-terminal dipeptides of D(-)-penicillamine, prepared from the synthon 3- formyl-2,2,5,5-tetramethylthiazolidine-4S-carboxylic acid (3), a cyclized N- protected derivative of D(-)-penicillamine. These dipeptides were equally or more effective than penicillamine in trapping AcH in a cell-free system. In experiments using a hepatocyte culture system, two of the dipeptides, D- penicillamylglycine (6a) and D-penicillamyl-β-alanine (6d), at 1/20 the molar concentration of ethanol, lowered the concentration of ethanol-derived AcH by 79% and 84%, respectively, at 2 h. The presence of cyanamide (an inhibitor of aldehyde dehydrogenase) in the incubation medium resulted in a 45-fold increase in ethanol-derived AcH; nevertheless, dipeptides 6a and 6c (D-penicillamyl-α-aminoisobutyric acid) were able to reduce this AcH level by approximately one-third.

Full text of DOI:10.1021/jm9902741

J . Med. Chem. 2000, 43, 1029-1033
1029
Brief Articles
N-Ter m in a l Dip ep tid es of D(-)-P en icilla m in e a s Sequ estr a tion Agen ts for
Aceta ld eh yd e
J onathan F. Cohen,† J ames A. Elberling,‡ Eugene G. DeMaster,‡ Renee C. Lin,§ and Herbert T. Nagasawa*,†,‡
Medical Research Laboratories, VA Medical Centers, Minneapolis, Minnesota 55417, and Indianapolis, Indiana 46202,
and Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
Received J une 2, 1999
Since acetaldehyde (AcH), a toxic oxidation product of ethanol, may play an etiologic role in
the initiation of alcoholic liver disease, we had earlier pioneered the development of
â,â-disubstituted-â-mercapto-R-amino acids as AcH-sequestering agents. We now report the
synthesis of a series of N-terminal dipeptides of D(-)-penicillamine, prepared from the synthon
3-formyl-2,2,5,5-tetramethylthiazolidine-4S-carboxylic acid (3), a cyclized N-protected derivative
of D(-)-penicillamine. These dipeptides were equally or more effective than penicillamine in
trapping AcH in a cell-free system. In experiments using a hepatocyte culture system, two of
the dipeptides, D-penicillamylglycine (6a ) and D-penicillamyl-â-alanine (6d ), at 1/20 the molar
concentration of ethanol, lowered the concentration of ethanol-derived AcH by 79% and 84%,
respectively, at 2 h. The presence of cyanamide (an inhibitor of aldehyde dehydrogenase) in
the incubation medium resulted in a 45-fold increase in ethanol-derived AcH; nevertheless,
dipeptides 6a and 6c (D-penicillamyl-R-aminoisobutyric acid) were able to reduce this AcH level
by approximately one-third.
In tr od u ction
the development of AcH sequestration agents.14 Our
structure-activity studies suggested that the ideal AcH-
sequestering agent should have (a) an acidic functional
group to impart H2O solubility and (b) 1,2- or 1,3-
disubstitution with functional groups that can be sulf-
hydryl, amino, or hydroxyl. Of the trifunctional com-
pounds tested that met these requirements, only
â-mercapto-R-amino acids with one or two bulky sub-
stituents at the â-position were effective in sequestering
AcH in vivo. Thus, D(-)-penicillamine (1) and â,â-
tetramethylene-DL-cysteine (2) (Chart 1) were the best
â-mercapto-R-amino acid sequestration agents for AcH
in vivo.13
Ethanol metabolism produces acetaldehyde (AcH), a
toxic oxidation product that may play an etiologic role
in the initiation of alcoholic liver disease (ALD). Of
pertinence in this regard, chronic alcoholics have higher
blood AcH levels compared to nonalcoholics after con-
suming alcoholic beverages1 and are, therefore, more
susceptible to its toxic effects. It is now well-established
that this ethanol-derived AcH binds covalently to cel-
lular proteins, and such AcH-modified proteins can elicit
an immune response manifested by antibody formation
to these AcH-bound epitopes.2 In rodents treated with
ethanol, AcH binds to hemoglobin3 and other plasma
proteins,4 to tubulin,5 and to a specific cystolic liver
protein,6 as well as to liver collagen.7 AcH-protein
conjugates of hemoglobin8,9 have also been detected in
humans after consumption of alcohol. The immunologi-
cal consequences of AcH-protein binding10 and its
possible etiology in ALD11 have been discussed.
L-Cysteine as well as L-cysteinylglycine have been
reported to protect against AcH toxicity in vivo.12
However, we found that while L-cysteine, which readily
condenses with AcH to a cyclic thiazolidine-4-carboxylic
acid at physiological pH, was a good AcH-trapping agent
in vitro, it was totally ineffective in vivo due to its rapid
catabolism.13
Although 1 is used clinically to treat Wilson’s disease
and hereditary cystinuria and to treat rheumatoid
arthritis refractory to conventional therapy, its potential
for renal and hematological toxicity15 limits its useful-
ness as a sequestration agent for AcH. Accordingly,
N-terminal dipeptides of 1 with glycine, â-alanine,
L-valine, and R-aminoisobutyric acid (AIB) were pro-
jected for synthesis as potential AcH-sequestering agents
with possibly attenuated toxicity. These dipeptides were
designed such that one of the C-terminal amino acids
(â-alanine) was of nonprotein origin, while another
(AIB) was a sterically hindered, synthetic R-amino acid.
Since the chiral center of 1 is opposite to that of the
naturally occurring L-cysteine, these dipeptides would
not be expected to be cleaved by endogenous peptidases,
as is the case for L-cysteinylglycine or L-cysteinylva-
line.13
On the basis of the premise that AcH may play an
etiologic role in alcohol-related diseases, we pioneered
† University of Minnesota.
‡ VA Medical Center, MN.
§ VA Medical Center, IN, and Department of Biochemistry/Molecular
Biology, Indiana University School of Medicine, Indianapolis, IN 46223.
10.1021/jm9902741 This article not subject to U.S. Copyright. Published 2000 by the American Chemical Society
Published on Web 02/17/2000

1030 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Brief Articles
Ch a r t 1
results suggest that selected N-terminal D-penicillamyl
dipeptides can sequester ethanol-derived AcH in a
manner comparable to that of D-penicillamine.
Discu ssion
Approximately 30-50% of Orientals lack a functional
low Km hepatic mitochondrial aldehyde dehydrogenase
(AlDH2).19 This enzyme plays a major role in the
metabolic disposition of ethanol-derived AcH to acetate,
this conversion serving as a detoxication mechanism for
AcH. Those individuals lacking AlDH2 drink alcohol
sparingly or not at all because of the consequence of
experiencing a flushing syndrome, manifested by ele-
vated blood AcH, facial flushing, tachycardia, and a
general feeling of malaise reminiscent of the disul-
firam-ethanol reaction,20 on even moderate consump-
tion of alcoholic beverages.21
Resu lts
The synthesis of N-terminal D(-)-penicillamine dipep-
tides required the synthon 3-formyl-2,2,5,5-tetrameth-
ylthiazolidine-4S-carboxylic acid (3; Chart 1), a cyclized
N-protected derivative of 1 patterned after the corre-
sponding L-cysteine derivative,16 for peptide coupling
reactions. The syntheses of D-penicillamylglycine (6a ),
D-penicillamyl-L-valine (6b), and D-penicillamyl-AIB
(6c) are outlined in Scheme 1. Thus, the tert-butyl esters
of glycine (4a ), L-valine (4b), and R-aminoisobutyric acid
(4c) were coupled with the mixed anhydride prepared
from 3 and isobutyl chloroformate in the presence of
Et3N to give the protected dipeptide esters 5. Simulta-
neous deformylation, thiazolidine ring opening, and de-
esterification at the carboxy terminus were readily
accomplished by heating 5 in 1 N HCl to give 6a , 6b,
and 6c as their respective hydrochloride salts. For
D-penicillamyl-â-alanine (6d ), the coupling procedure
was identical to that described in Scheme 1, except that
the commercially available ethyl â-alaninate (4d ) was
used to prepare the N-protected thiazolidine dipeptide
ethyl ester 7. Deblocking as above gave 6d .
It is noteworthy that in the coupling of the tert-butyl
ester of the sterically hindered AIB with the mixed
anhydride of the equally sterically hindered 3, the major
product isolated was the N-carbethoxylated amino ester
8 (Chart 1), where the amino group of the amino ester
had attacked the sterically less-hindered carbonyl group
of the mixed anhydride. Thus, the yield of the desired
product 5c was commensurately low for this reaction.
The sequestration of AcH by the four N-terminal
dipeptides of 1 in a cell-free system is shown in Figure
1. It can be seen that these dipeptides were equally or
more effective than 1 itself (positive control) in trapping
AcH. As predicted, methionine (Met), threonine (Thr),
and serine (Ser) were totally without AcH-sequestering
activity. In agreement with our previous results, L-
homocysteine (Hcy) was comparable to 1 in sequestering
AcH in vitro; however, Hcy was shown not to be an
effective AcH-trapping agent in vivo.13
By genotyping individuals for AlDH2, an indication
of AcH exposure after ethanol consumption can be
deduced.22 The observation that a higher risk factor for
esophogeal cancers exists among heavy drinkers with
a mutant AlDH2 allele underscores the importance of
AcH as a possible etiological factor in this disease.23
Individuals with this heritable, mutant AlDH2 allele
who refrain from drinking are generally protected from
alcoholic liver disease as well as alcohol-induced cancers,
whereas excessive drinking by subjects with this mutant
allele places them at risk of developing not only eso-
phogeal cancer but also cirrhosis of the liver. If total
abstinence from alcohol cannot be voluntarily sustained,
we envision the potential use of AcH-sequestering
agents to protect chronic alcoholics, as well as those
individuals with a nonfunctional AlDH2, from acetal-
dehydemia and its adverse pathologic consequences.
Other uses of these aldehyde-sequestering agents can
also be envisioned. For example, methanol is metabo-
lized in humans to formaldehyde and then to formic
acid, and while metabolic acidosis elicited by the latter
is a major problem, blindness induced by methanol is
believed to be due to retinal destruction by formalde-
hyde.24 Sequestration of methanol-derived formalde-
hyde25 should, therefore, provide protection against
methanol-induced blindness. Aldehyde sequestration
agents may also be applicable (a) for the treatment of
accidental overexposure in the workplace to toxic alde-
hydes, such as formaldehyde and glutaraldehyde, or (b)
to counteract the toxic effects of AcH when subjects
being treated for alcoholism with disulfiram, an inhibi-
tor of AlDH, accidentally ingest alcohol.
In experiments using a hepatocyte culture system,17
D-penicillamylglycine (6a ) and D-penicillamyl-â-alanine
(6d ), at 1/20 the molar concentration of ethanol (20 mM),
lowered the concentration of ethanol-derived AcH by
79% and 84%, respectively, at 2 h (Figure 2A). We are
unable to explain the anomalous result with compound
Exp er im en ta l Section
1H NMR spectra were recorded at ambient temperature on
either a GE-300 or a Bruker AC-200 NMR spectrometer.
Chemical shifts are reported as δ values (ppm). Mass spectra
(EI or FAB) were obtained on a Kratos MS 25 or Finnegan
LCQ mass spectrometer. For TLC analyses, Analtech silica
gel GF plates were used. The solvent systems used for TLC
were as indicated. The plates were visualized by spraying with
ninhydrin or ceric sulfate solution and heating. Column
chromatography was carried out using columns packed with
Kieselgel 60 (230-400 mesh) silica gel (EM Science). A rotating
evaporator was used to remove solvents in vacuo.
6c. The presence of cyanamide (an inhibitor of AlDH18
)
in the incubation medium resulted in a 45-fold increase
in ethanol-derived AcH (Figure 2B, control without
dipeptide), and while 6a and 6c were able to reduce this
concentration by 33% and 37%, repectively, it is clear
that considerably higher dipeptide concentrations would
be required under the latter conditions for effective AcH
sequestration. Additional in vivo work in rodents is
necessary to assess the degrees of efficacy, or toxicity,
if any, of these compounds. Nevertheless, the present
3-F or m yl-2,2,5,5-t et r a m et h ylt h ia zolid in e-4S-ca r b ox-
ylic Acid (3). A suspension of 59.7 g (400 mmol) of D(-)-
penicillamine in 1200 mL of acetone was heated under reflux
with mechanical stirring for 2 h. The solids were allowed to

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1031
boxylic acid. This was used directly for formylation as de-
scribed for the racemic compound.26 Compound 3, obtained in
69% yield, had a mp of 180-181 °C (lit.26 mp 179-180 °C).
N-(3-F or m yl-2,2,5,5-t et r a m et h ylt h ia zolid in yl-4S-ca r -
bon yl)-â-a la n in e Eth yl Ester (7). To a solution of 3 (2.17 g,
10.0 mmol) in 50 mL of dry CH2Cl2 was added dry Et3N (2.80
mL, 2.03 g, 20.1 mmol), and the solution was stirred under
N2 with ice bath cooling. To this solution were added isobutyl
chloroformate (1.30 mL, 1.37 g, 10.0 mmol) and an additional
25 mL of CH2Cl2. The clear solution was stirred ca. 1 h, and
â-alanine ethyl ester hydrochloride (1.56 g, 10.1 mmol) was
added. The reaction mixture was allowed to warm to room
temperature overnight and was then washed successively with
1 N HCl, 5% NaHCO3, and water (50 mL of each). The solution
was dried and the solvent evaporated to dryness in vacuo to
give 2.89 g of viscous, colorless oil. This was purified by flash
chromatography using EtOAc:hexane (1:4, 2:3, and 3:2) as
eluent to give 2.12 g (67% yield) of 7 as a colorless oil. 1H NMR
(CDCl3): δ 1.28 (t, J ) 7.1 Hz, 3H, CH3CH2), 1.42, 1.44 (s,
1:6, 3H, CH3CCH), 1.64, 1.69 (s, 7:1, 3H, CH3CCH), 1.83, 1.94
(s, 1:5, 3H, CH3CN), 1.97, 1.99 (s, 5:1, 3H, CH3CN), 2.57 (m,
2H, CH2CO2), 3.6 (m, 2H, CH2N), 4.17 (q, J ) 7.1 Hz, 2H, CH2-
CH3), 4.34, 4.55 (s, 1:10, 1H, CH), 6.6 (br s, 1H, NH), 8.31,
8.37 (s, 1:10, 1H, HCO). Mass Spectrum (EI): m/ z (rel
intensity) 316 (M+•, 7), 200 (32), 172 (100), 144 (80). Anal.
(C13H24N2O4S) C,H,N.
F igu r e 1. Sequestration of AcH by N-terminal dipeptides of
D(-)-penicillamine and selected amino acids in a cell-free in
vitro system. See Experimental Section for details.
D-P en icilla m yl-â-a la n in e Hyd r och lor id e (6d ). A stirred
solution of 7 (1.00 g, 3.16 mmol) in 40 mL of dioxane:2 N HCl
(1:1) was heated under N2 in a water batch at 85-90 °C. An
aliquot sampled after 1.75 h for NMR analysis indicated about
8% starting material remaining. After 3.75 h, the reaction
mixture was evaporated to dryness in vacuo, water was added,
and the mixture was evaporated to dryness again. NMR
analysis indicated about 3% of the formyl group was still
uncleaved, although the ester moiety was hydrolyzed. The
product was reheated in 15 mL of 1 N HCl for an additional
3.25 h under N2 when NMR analysis indicated the reaction
was complete. The solution was lyophilized to give 687 mg
(79% yield) of 6d as a hygroscopic solid, mp 71-75 °C. 1H NMR
(D2O): δ 1.32 (s, 3H, CH3), 1.38 (s, 3H, CH3), 2.52 (t, J ) 6.4
Hz, 2H, CH2CO2H), 3.31 (td, J ) 6.4 Hz, J ) 14.0 Hz, 1H,
CHNCO), 3.50 (td, J ) 6.4 Hz, J ) 14.0 Hz, 1H, CHNCO),
3.78 (s, 1H, CHCS). Mass Spectrum (FAB): m/ z 221 (MH+).
Anal. (C8H16N2O3S‚HCl‚H2O) C,H,N.
N-(3-F or m yl-2,2,5,5-t et r a m et h ylt h ia zolid in yl-4S-ca r -
bon yl)glycin e ter t-Bu tyl Ester (5a ). To an ice-cooled solu-
tion of 3 (5.75 g, 26.5 mmol) and Et3N (7.50 mL, 5.45 g, 53.8
mmol) in CH2Cl2 (80 mL) was added isobutyl chloroformate
(3.43 mL, 3.61 g, 26.5 mmol). The milky suspension was stirred
for 1 h, and glycine tert-butyl ester hydrochloride (4a ; 4.44 g,
26.5 mmol) was added. The reaction mixture was stirred with
continued cooling for 30 min, then allowed to warm to room
temperature overnight, and washed successively with 60-mL
portions of 1 N HCl, 5% NaHCO3, and H2O. After drying over
Na2SO4, the solution was evaporated to dryness in vacuo to
give 8.38 g (97% yield) of 5a as an amber oil which slowly
crystallized. This product was dissolved in ether (100 mL) and
diluted with hexane (100 mL), and the solution was evaporated
to ca. 80 mL and cooled to give 5.19 g (60% yield) of 5a as
colorless needles, mp 112.5-113.5 °C. 1H NMR (CDCl3): δ
1.49, 1.50 (s, 3:1, 9H, CH3CO), 1.67, 1.72 (s, 7:1, 6H, CH3CCH),
1.96, 1.98, 2.02 (s, 3:3:1, 6H, CH3CN), 3.99 (m, 2H, CH2), 4.4,
4.66 (s, 1:5, 1H, CHCON), 6.5 (br s, 1H, NH), 8.37, 8.41 (s,
1:6, 1H, HCO). Mass Spectrum (EI): m/ z (rel intensity) 330
F igu r e 2. A. Effect of N-terminal D(-)-penicillamine dipep-
tides on AcH levels at 2 h in rat hepatocytes incubated with
20 mM ethanol and 1 mM dipeptide; N ) 4. AcH was
undetectable in controls without ethanol (not shown). B. Same
as in A, except that cyanamide (50 µM), an inhibitor of AlDH,
was added to the system.
settle, and the supernatant solution was decanted through a
filter. The residual solids were heated again in 400 mL of fresh
acetone until complete solution resulted (5-10 min). The hot
solution was poured through the same filter. This process
dissolved most of the solids previously held on the filter.
Cooling the combined filtrates resulted in the precipitation of
a mass of solids which were collected. Concentration (by
distillation of the acetone) of the mother liquor followed by
cooling gave two additional crops of product giving a total of
68.4 g (90% yield) of 2,2,5,5-tetramethylthiazolidine-4S-car-
Sch em e 1

1032 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Brief Articles
(M+•, 35), 257 (100), 200 (15), 172 (100), 144 (50). Anal.
(C15H26N2O4S) C,H,N.
37 °C was quantified by headspace GC analysis.13 DTT was
included in the reaction mixture to maintain the thiols in their
reduced state.
D-P en icilla m ylglycin e Hyd r och lor id e (6a ). 5a (931 mg,
2.82 mmol) was hydrolyzed over 10 h using the same procedure
for 6d to give 587 mg of 6a (77% yield) as a hygroscopic solid,
mp 67-71 °C. 1H NMR (D2O): d 1.36 (s, 3H, CH3), 1.44 (s,
3H, CH3), 3.92 (s, 1H, CHCS) 3.92 (d, J ) 17.8 Hz, 1H,
CHNCO), 3.97 (d, J ) 17.8 Hz, 1H, CHNCO). Mass Spectrum
(FAB): m/z 207 (MH+). Anal. (C7H14N2O3S.HCl.1.5H2O)
C,H,N.
N-(3-F or m yl-2,2,5,5-t et r a m et h ylt h ia zolid in yl-4S-ca r -
bon yl)va lin e ter t-Bu tyl Ester (5b). 5b was prepared as for
5a using 2.17 g (10.0 mmol) of 3 in 30 mL of CH2Cl2, Et3N
(3.0 mL, 2.78 g, 22.0 mmol), isobutyl chloroformate (1.30 mL,
1.37 g, 10.0 mmol), and 4b (2.10 g, 10.0 mmol). After workup,
the reaction mixture was evaporated to ca. 10 mL, diluted with
hexane (20 mL), and evaporated to ca. 10 mL when a mass of
colorless needles precipitated, mp 145.5-147 °C. A second crop
of crystals was obtained by evaporation of the filtrate to ca. 1
mL and dilution with isopentane (5 mL). The total yield of 5b
was 2.11 g (57%). 1H NMR (CDCl3): δ 0.94, 0.98 (d, 1:1, J )
6.8 Hz, J ) 6.8 Hz, 6H, CH3CH), 1.50, 1.51 (s, 2:1, 9H, CH3-
CO), 1.64, 1.67, 1.73 (s, 8:4:1, 6H, CH3CCH), 1.97, 2.02 (s, 4:1,
6H, CH3CN), 2.20 (m, 1H, CHCH3), 4.4 (m, 1H, CHCO2), 4.66
(s, 1H, CHCON), 6.6 (br d, 1H, NH), 8.36, 8.43 (s, 2:7, 1H,
HCO). Mass Spectrum (EI): m/ z (rel intensity) 372 (M+•, 65),
316 (100), 200 (100), 172 (100), 144 (100). Anal. (C18H32N2O4S)
C,H,N.
D-P en icilla m yl-L-va lin e Hyd r och lor id e (6b). 5b (1.07 g,
2.87 mmol) was hydrolyzed for 6 h using the same procedure
for 6d to give, after lyophilization, 780 mg of 6b (96% yield)
as a fluffy solid, mp 263-265.5 °C dec. 1H NMR (D2O): δ 0.84,
0.86 (d, 1:1, J ) 6.8 Hz, J ) 6.8 Hz, 6H, CH3CH), 1.36 (s, 3H,
CH3CS), 1.43 (s, 3H, CH3 CS), 2.10 (m, 1H, CHCH3), 3.95 (s,
1H, CHCON), 4.15, 4.16 (d, 1:1, J ) 5.7 Hz, J ) 5.7 Hz, 1H,
CHCO2). Mass Spectrum (FAB): m/ z 249 (MH+). Anal.
(C10H20N2O3S‚HCl) C,H,N.
N-(3-F or m yl-2,2,5,5-t et r a m et h ylt h ia zolid in yl-4S-ca r -
bon yl)-r-a m in oisobu tyr ic Acid ter t-Bu tyl Ester (5c). 5c
was prepared as for 5a using 913 mg (4.20 mmol) of 3 in 50
mL of CH2Cl2, Et3N (1.2 mL, 0.87 g, 8.6 mmol), isobutyl
chloroformate (0.55 mL, 0.58 g, 4.2 mmol), and 4c‚HCl‚0.2H2O
(811 mg, 4.07 mmol). After workup, the CH2Cl2 solution was
evaporated to dryness, and the residue was purified by flash
chromatography using EtOAc:hexane (1:4) as eluent to give
647 mg (61% yield) of a colorless oil as the byproduct 8. 1H
NMR (CDCl3): δ 0.90 (d, J ) 6.7 Hz, 6H, CH3CH), 1.44 (s,
9H, CH3CO), 1.49 (s, 6H, CH3CN), 1.88 (m, 1H, CHCH3), 3.80
(b) In h ep a tocyte system s: Hepatocytes from male wistar
rats were isolated and cultured under conditions previously
described.17 On culture day 2, ethanol and the test dipeptides
were added to the culture medium to final concentrations of
20 and 1.0 mM, respectively. After 2 h, 1.0 mL of medium was
removed and added to a semicarbazide/sodium azide mixture
and frozen until assayed for AcH by gas chromatography.17
AcH was measured before the additions and 2 h after incuba-
tion, and the values were compared against ethanol control
without dipeptides. In experiments with cyanamide (to inhibit
AIDH), the cyanamide was added with the ethanol to give a
final concentration in the culture medium of 50 µM.
Ack n ow led gm en t. This work was supported by
General Medical Research funds from the Department
of Veterans Affairs. We thank Sagar Kawle for technical
assistance in the synthesis of some intermediates and
Thomas P. Krick of the Mass Spectrometry Laboratory,
Department of Biochemistry, for the mass spectral
analyses. The Mass Spectrometry Laboratory is main-
tained by the Minnesota Agricultural Experiment Sta-
tion.
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Further elution with the same solvent mixture (2:3) gave
407 mg (28% yield) of 5c as a colorless solid. Recrystallization
from EtOAc:hexane (1:10) gave an analytical sample, mp
149.5-152 °C. 1H NMR (CDCl3): δ 1.45, 1.48 (s, 5:1, 9H, CH3-
CO), 1.54, 1.55, 1.56, 1.62, 1.66 (s, 2:6:6:5:1, 12H, CH3CCH
and CH3CCO2), 1.91, 1.95, 1.97 (s, 3:3:1, 6H, CH3CN), 4.24,
4.49 (s, 2:7, 1H, CHCON), 6.74 (br s, 1H, NH), 8.37, 8.41 (s,
1:6, 1H, HCO). Mass Spectrum (EI): m/ z (rel intensity) 358
(M+•, 2), 200 (50), 172 (87), 144 (52). Anal. (C17H30N2O4S)
C,H,N.
D-P en icilla m yl-r-a m in oisobu tyr ic Acid Hyd r och lor id e
(6c). 5c (192 mg, 0.536 mmol) was hydrolyzed for 5 h using
the same procedure for 6d (except the water bath temperature
was 65-70 °C) to give, after lyophilization, 150 mg of 6c (100%
1
yield) as a colorless solid, mp 145-148 °C. H NMR (D2O): δ
1.37, 1.39, 1.448, 1.452 (s, 4:6:3:3, 12H, CH3CS and CH3CCO2),
3.79, 3.80 (s, 1:1, 1H, CHCS). Mass Spectrum (FAB): m/ z 235
(MH+). Anal. (C9H18N2O3S‚HCl) C,H,N.
Sequ estr a tion of AcH. (a ) In a cell-fr ee system : AcH
(40 nmol) was incubated without (control) and with the
synthetic dipeptide or the amino acid (400 nmol) in a reaction
mixture containing 4.0 mM dithiothreitol (DTT) and 40 mM
potassium phosphate buffer (pH 7.4) in a total volume of 1.2
mL. The AcH remaining after a 30-min incubation period at

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