Proteolytic Enzymes: Aspartic and Metallo Peptidases

870 Pages·1995·13.689 MB·English

by  Abelson J.N., Simon M.I., Barrett A.J. (eds.)

Preface
Through the earlier, general volumes on proteolytic enzymes (Vol-
umes ,91 45, and 80), Methods ni Enzymology made available over 002
authoritative articles on these enzymes and their inhibitors. Since the
appearance of the latest of these volumes, however, there have been
many profound advances in this field of study. The biomedical importance
of proteolytic enzymes, suspected for so long, has been established be-
yond reasonable doubt for a number of groups, including the matrix me-
talloproteinases, the viral polyprotein-processing enzymes, and the pro-
hormone-processing peptidases. The more recent, specialized Volumes
222, 223, and 142 have dealt with some of these areas, but others have
remained to be covered.
The resurgence of excitement about proteolytic enzymes has inevita-
bly resulted in an information explosion, but some of the new understand-
ing has also helped us develop novel approaches to the management of the
mass of data. As a result, we can now "see the forest for the trees" a little
more clearly. Like other proteins, the proteolytic enzymes have benefited
from the recent advances in molecular biology, and amino acid sequences
are now available for many hundreds of them. These can be used to group
the enzymes in families of evolutionarily related members. Also, there
has been a major overhaul of the recommended nomenclature for pepti-
dases by the International Union of Biochemistry and Molecular Biology.
In Volume 442 on peptidases of serine and cysteine type and in this
volume on aspartic, metallo, and other peptidases, the chapters on spe-
cific methods, enzymes, and inhibitors are organized within the rational
framework of the new systems for classification and nomenclature.
The peptidases of the aspartic and metallo types dealth with in this
volume depend for their activity on the nucleophilic activity of an ionized
water molecule, unlike the enzymes, described in Volume 244, in which
the nucleophilic character of a serine or cysteine residue is at the heart of
the catalytic mechanism. A wide variety of specificities of peptide bond
hydrolysis is represented in each set of peptidases, together with an
equally wide range of biological functions.
ALAN J. BARRETT
VX
Contributors to Volume 248
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
ANGELA ISATSANA (43), Department of Pa- SEUQCAJ BOUVIER (37), Animal Health De-
thology, The Medical School, St. Luke's partment, Ciba-Geigy Ltd., CH-1566 St.
Hospital, Msida, Malta Aubin, Switzerland
DAVID S. AULD (14), Center for Biochem- YLLOM A. BROWN (32), Department of -DiB
ical and Biophysical Sciences and Medi- chemistry, Strangeways Research Labo-
cine, and Department of Pathology, Har- ratory, Cambridge 1BC 4RN, United
vard Medical School and Brigham and Kingdom
Women's Hospital, Boston, Massachu- MICHAEL BRUNNER (46), Institut fiir Phy-
setts 51120 siologische Chemie, Universitiit Miin-
NRL~IH BARELLI (36), Institat de Pharma- chen, D-80336 Miinchen 2, Germany
cologie Moldculaire et Cellulaire, CNRS, DAVID J. BUTTLE (4), Department of -DiB
dtisrevinU de Nice-Sophia Antipolis, chemistry, Strangeways Research Labo-
F-06560 Valbonne, France ratory, Cambridge CBI 4RN, United
ALAN J. BARRETT (7, 13, 32, 43), Depart- Kingdom
ment of Biochemistry, Strangeways Re- PAUL CANNON (25), Institute of Biochemis-
search Laboratory, Cambridge 1BC 4RN, try and Cell Biology, Syntex Discovery
United Kingdom Research, Paid Alto, California 30349
ANDREW B. BECKER (44), Department of NIAMH X. CAWLEY (9), Laboratory of
Molecular Pharmacology, Stanford Uni- Developmental Neurobiology, National
versity School of Medicine, Stanford, Institute of Child Health and Human De-
California 50349 velopment, N1H and Department of
JUDD BERMAN (3), Department of Medici- Biochemistry, Uniformed Services, Uni-
nal Chemistry, Glaxo Inc. Research Insti- versity of the Health Sciences, Bethesda,
tute, Research Triangle Park, North Car- Maryland 29802
olina 90772 CIR~ID~IRF CHECLER (36), lnstitut de Phar-
D. MARK BICKETT (3), Department of Bio- macologie Moleculaire et Cellulaire,
chemistry, Glaxo Inc. Research Institute, CNRS, UniversiN de Nice-Sophia Anti-
Research Triangle Park, North Carolina pulis, F-06560 Valbonne, France
90772 EIR~ILAV CHESNEAU (45), Laboratoire de
HPESOJ G. BIETH (5), INSERM Unitd 392, Biochimie des Signaux R~gulateurs, Cel-
Laboratoire d'Enzymologie, Universitd lulair et Mol~culaires, Unitd de Recher-
Louis Pasteur de Strasbourg, F-67400 ches Associ~e au Centre National de al
Illkirch, France Scientifique, Universitd Pierre et Marie
J6N B. NOSANRAJB (21, 22), The Science In- Curie, 60057 Paris, France
stitute, University of lceland, IS-107 Rey- PAUL COHEN (45), Laboratoire de Biochi-
kjavik, Iceland mie des Signaux Rdgulateurs, Cellulair et
JUDITH S. BOND (20), Department of Bio- Mol#culaires, Unitd de Recherches Asso-
chemistry and Molecular Biology, Col- cide au Centre National de al Scientifi-
lege of Medicine, Pennsylvania State que, Universitd Pierre et Marie Curie,
University, Hershey, Pennsylvania 33071 60057 Paris, France
ix
X CONTRIBUTORS TO VOLUME 248
REHPOTSIRHC A. CONLIN (34), Department MARIE-CLAUDE FOURNII~-ZALUSKI (17),
of Biological Sciences, Mankato State D~partement de Pharmacochimie Mol~-
University, Mankato, Minnesota 20065 culaire et Structurale, Institut National
de al Sante et de la Recherche Medicale,
E~IREIP LOVROC (18), lnstitut National de al
Universit~ Rend Descartes, 75270 Paris
Sant~ et de la Recherche Medicale, Col-
Cedex ,60 France
ldge de France, 50057 Paris, France
YAJ W. Fox (21, 22), Department of Micro-
SAMOHT CRABBE (28), Celltech Therapeu- biology, University of Virginia, Health
tics Ltd., Slough SL1 4EN, United King- Sciences Center, Charlottesville, Virginia
dom 80922
PHILIPPE CRINE (17), D~partement de Bio- ALUSRU GEUSS (51), Boehringer Mannheim
chimie, Facult~ de Mddecine, Universitd GmbH, D-68305 Mannheim, Germany
de Montrdal, Montrdal, Canada H3C 3J7
PAUL GLYNN (23), Medical Research Coun-
PAMELA M. DANDO (32), Department of cil Toxicology Unit, University of Leices-
Biochemistry, Strangeways Research ter, Leicester LE1 9HN, United Kingdom
Laboratory, Cambridge 1BC 4RN, MICHAEL GREEN (3), Department of Medic-
United Kingdom inal Chemistry, Glaxo Inc. Research In-
stitute, Research Triangle Park, North
ELACSAP DAUCH (36), Institut de Pharma-
Carolina 90772
cologie Moleculaire et Cellulaire, CNRS,
Universitd de Nice-Sophia Antipolis, MARIE-LuISE HAGMANN (51), Boehringer
06560-:1 Valbonne, France Mannheim GmbH, D-82372 Penzberg,
Germany
PETER A. DEDDISH (41), Departments of
REHPOTSIRHC J. HANDLEY (4), Department
Pharmacology and Anesthesiology, Uni-
of Biochemistry and Molecular Biology,
versity of Illinois College of Medicine,
Monash University, Clayton Victoria
Chicago, Illinois 21606
,8613 Australia
ANNAIRAM DIOSZEGI (25), Institute of Bio- Louis B. HERSH (16), Department of Bio-
chemistry and Cell Biology, Syntex Dis- chemistry, College of Medicine, Univer-
covery Research, Palo Alto, California sity of Kentucky, Lexington, Kentucky
30349 63504
VINCENT DIVE (36), D~partment d'lng~- LINDA HOWARD (23), Department of Cell
nierie et d'Etude des Prot~ines, C.E.N. de Biology and Lombardi Cancer Center,
Saclay, Laboratoire de Structure des Pro- Georgetown University School of Medi-
tdines en Solution, 91191 GiflYvette, cine, Washington 20007
France GRAZIA ISAYA (33), Department of Genet-
ics, Yale University School of Medicine,
TREBOR ETGES (37), Department of Bio-
New Haven, Connecticut 01560
chemistry, University of Puerto Rico, San
Juan, Puerto Rico 63900 KARL E. KADLER (49, 50), Department of
Biological Sciences, Research Division of
STEPHAN FISCHER (51), Boehringer Mann-
Biochemistry, University of Manchester,
heim GmbH, D-82372 Penzberg, Ger-
Manchester M13 9PT, United Kingdom
many
TAKASHI KAGEYAMA (8), Department of
YRREIHT FOULON (45), Laboratoire de Bio- Cellular and Molecular Biology, Primate
chimie des Signaux R~gulateurs, Cellulair Research Institute, Kyoto University, In-
et Mol~culaires, Unit~ de Recherches As- uyama, Aichi 484, Japan
soci~e au Centre National de la Scientifi- KESITNARF KESUOLAK (33), Department of
que, Universit~ Pierre et Marie Curie, Genetics, Yale University School of Medi-
60057 Paris, France cine, New Haven, Connecticut 01560
CONTRIBUTORS TO VOLUME 248 xi
CHIH-MIN KAM (1), School of Chemistry ALAN MELLORS (47), Department of Chem-
and Biochemistry, Georgia Institute of istry and Biochemistry, Guelph-Waterloo
Technology, Atlanta, Georgia 23303 Centre for Graduate Work ni Chemistry,
University of Guelph, Guelph, Ontario,
EERAT KESSLER (48), Maurice and Gabriela
Canada NIG 1W2
Goldschleger Eye Research Institute,
Sackler Faculty of Medicine, Tel-Aviv CHARLES G. MILLER (34), Department of
University, Tel-Hashomer 52621, Israel Microbiology, University of Illinois at -rU
C. GRAHAM KNIGHT (2, 6, 32), Department bana-Champaign, Urbana, Illinois 10816
of Cell Adhesion and Signalling, Strange- EUQINOR~IV MONNET (35), 1NRA Centre De
ways Research Laboratory, Cambridge Recherches De Jouy-en-Josas, Station De
1BC 4RN, United Kingdom Recherches Laitidres, Domaine de
GEORG-B URKHARD KRESSE (51), Boehringer Vilvert, 78352 Jouy-en-Josas, Cedex,
Mannheim GmbH, D-82372 Penzberg, France
Germany CESARE MONTECUCCO (39), Centro CNR
CHINGWEN LI (16), Department of Bio- Biomembrane and Dipartimento di
chemistry, College of Medicine, Univer- Scienze Biomediche, Universita di Pa-
sity of Kentucky, Lexington, Kentucky dora, 00153-57 Padova, Italy
63504 KAZUYUKI MORIHARA (15)~ Institute of Ap-
SAMANTHA J. LIGHTFOOT (49), Department plied Life Sciences, Graduate School,
of Biological Sciences, Research Division University of East Asia, Yamaguchi ,157
of Biochemistry, University of Manches- Japan
ter, Manchester M13 9PT, United King- GILLIAN MURPHY (28, 30), Department of
dom Cell and Molecular Biology, Strangeways
XINLI LIN (11), Protein Studies Program, Research Laboratory, Cambridge 1BC
Oklahoma Medical Research Founda- 4RN, United Kingdom
tion, Oklahoma City, Oklahoma 40137 HIDEAKI NAGASE (27), Department of Bio-
REGGIE Y.C. LO (47), Department of -orciM chemistry and Molecular Biology, Uni-
biology, University of Guelph, Guelph, versity of Kansas Medical Center, Kan-
Ontario, Canada N1G 1W2 sas City, Kansas 06166
Y. PENG LOH (9), Section on Cellular Neu- WALTER NEUPERT (46), Institut fiir Phy-
robiology, Laboratory of Developmental siologische Chemie, Universit~it Miin-
Neurobiology, National Institute of Child chen, D-80336 Miinchen ,2 Germany
Health and Human Development, and FLORENCE NOBLE (17), D~partement ed
Department of Biochemistry, Uniformed Pharmacochimie Molgcalaire et Strac-
Services, University of the Health Sci- turale, Institut National de al Sante et ed
ences, Bethesda, Maryland 29802 al Recherche Medicale, Centre, National
HIROSHI MAEDA (24), Department of Micro- de al Recherche Scientifique, Universitg
biology, Kumamoto University Medical Rend Descartes, 75270 Paris Cedex ,60
School, Kumamoto 860, Japan France
GERARD M. MCGEEHAN (3), Department of ADRIAN R. PIEROTTI (45), Department of
Biochemistry, Glaxo Inc. Research Labo- Biological Sciences, Glasgow Caledonian
ratories, Research Triangle Park, North University, Glasgow GL4 OBA, Scotland
Carolina 90772 ANDREW G. PLAUT (38), Department of
NORMAN McKIE (32), Department of Bio- Medicine, Gastroenterology Division,
chemistry, Strangeways Research Labo- Tufts University School of Medicine and
ratory, Cambridge 1BC 4RN, United New England Medical Center, Boston,
Kingdom Massachusetts 11120
xii CONTRIBUTORS TO VOLUME 248
SEMAJ C. POWERS (1), School of Chemistry WALTER REKCOTS (19), Zoologisches lnsti-
and Biochemistry, Georgia Institute of tut der Universitiit Heidelberg, Physiolo-
Technology, Atlanta, Georgia 23303 gie, Im Neuenheimer FeN, D-69120 Hei-
ANNIK PRAT (45), Laboratoire de Biochimie delberg, Germany
des Signaux Rdgulateurs, Cellulair et Mo- KENJI IHSAHAKAT (10), Department of Bio-
Idculaires, Unitd de Recherches Associde physics and Biochemistry, Faculty of Sci-
au Centre National de al Scientifique, ence, The University of Tokyo, Tokyo ,311
Universitd Pierre et Marie Curie, 60057 Japan
Paris, France FULONG TAN (41), Departments of Pharma-
NEIL D. RAWLINCS (7, 13, 32), Department cology and Anesthesiology, University of
of Biochemistry, Strangeways Research Illinois College of Medicine, Chicago, Illi-
Laboratory, Cambridge 1BC 4RN, nois 21606
United Kingdom NADROJ TANG (11), Oklahoma Medical Re-
DRANREB P. ROQUES (17), Ddpartement de search Foundation, Protein Studies Pro-
Pharmacochimie Moldculaire et Struc- gram, Oklahoma City, Oklahoma 40137
turale, Institut National de la Santd et de DLARAH EHCSEHCST (26), Biochemistry De-
la Recherche Medicale, Centre National partment, University Bielefeld, ,51633-D
de la Recherche Scientifique, Unioersitd Bielefeld Germany
Rend Descartes, 75270 Paris Cedex ,60
HAROLD E. VAN WART (25), Institute of
France
Biochemistry and Cell Biology, Syntex
DRAHCIR A. ROTH (44), Department of Mo- Discovery Research, PaiD Alto, Califor-
lecular Pharmacology, Stanford Univer- nia 30349
sity School of Medicine, Stanford, Cali-
BRUNO VINCENT (36), Institut de Pharma-
fornia 50349
cologie Moldculaire et Cellulaire, CNRS,
NANHSIRK NARAKNAS (12), Department of Universitd de Nice-Sophia Antipolis,
Microbiology and Immunology, Uni- F-06560 Valbonne, France
formed Services, University of the Health JEAN PIERRE VINCENT (36), Institut de
Sciences, Bethesda, Maryland 41802 Pharmacologie Moldculaire et Cellulaire,
GIAMPIETRO SCHIAVO (39), Centro CNR CNRS, Universitd de Nice-Sophia Anti-
Biomembrane and Dipartimento di polls, F-06560 Valbonne, France
Scienze Biomediche, Universitd di Pa-
ROD B. WATSON (49), Department of Bio-
dora, 00153-57 Padova, Italy logical Sciences, Research Division of
LACSAP SCHNEIDER (37), Department of Biochemistry, University of Manchester,
Biochemistry, University of Dundee, Manchester M13 9PT, United Kingdom
Dundee 1DD 4HN, Scotland SECNARF KCORBNELLIW (30), Department
ATSUSHI SERIZAWA (32), Sapporo Research of Biochemistry, Queen Mary and West-
Laboratory, Snow Brand Milk Products field College, University of London, Lon-
Co., Ltd., Sapporo 065, Japan don 1E 4NS, United Kingdom
RANDAL A. SKIDGEL (40, 41), Department TRAcY A. WILLIAMS (18), Institut National
of Pharmacology and Anesthesiology, de la Santd et de la Recherche Medicale,
University of lllinois College of Medicine, Colldge de France, 75005 Paris, France
Chicago, Illinois 21606 JEFFREY S. WISEMAN (3), Department of
FLORENT SOUBRIER (18), Institut National Biochemistry, Glaxo Inc. Research Insti-
de al Santd et de al Recherche Medicale, tute, Research Triangle Park, North Car-
Colldge de France, 50057 Paris, France olina 90772
VALENTIN M. STEPANOV (42), Protein J. FREDERICK ,RENSSEOW JR. (29, 31), De-
Chemistry Laboratory, Institute of Micro- partment of Biochemistry and Molecular
bial Genetics, Moscow ,545311 Biology, University of Miami, School of
Russia Medicine, Miami, Florida 10133
CONTRIBUTORS TO VOLUME 248 xiii
LLESSUR L. WOLZ (20), Department of Bio- YRNEH C. Wu (12), Department of Microbi-
chemistry and Molecular Biology, Col- ology and Immunology, Uniformed Ser-
lege of Medicine, Pennsylvania State vices, University of the Health Sciences,
University, Hershey, Pennsylvania 33071 Bethesda, Maryland 41802
ANDREW WRIGHT (38), Department of Mo- TREBOR ZWILLING (19), Zoologisches Insti-
lecular Biology and Microbiology, Tufts tut der Universitiit Heidelberg, Physiolo-
University School of Medicine, Boston, gie lm Neuenheimer Feld, D-69120 Hei-
Massachusetts 11120 delberg, Germany
1 EDITPEP RETSEOIHT SETARTSBUS 3
1 Peptide Thioester Substrates for Serine Peptidases
and Metalloendopeptidases
By JAMES C. POWERS and CHIH-MIN KAM
Introduction
Synthetic peptide substrates are widely used in biochemical and
physiological studies of proteolytic enzymes. Synthetic peptide substrates
can be used to detect enzyme during isolation, to assay enzyme activity,
to determine enzyme concentrations, to investigate enzyme specificity,
and to determine inhibitor potency. The three most commonly used
synthetic substrates are peptide 4-nitroanilides, peptide thioesters, and
peptide derivatives of 7-amino-4-methylcoumarin. Amino acid and peptide
thioesters are sensitive substrates for serine peptidases and metalloendo-
peptidases because the substrates have high kcat/Km values for enzymatic
hydrolysis rates and low background hydrolysis rates, and the thiol-
leaving group can be easily detected at low concentrations. Cleavage of
the thioester bond can be monitored continuously by reaction with a
thiol reagent such as 4,4'-dithiodipyridine or 5,5'-dithiobis (2-nitrobenzoic
acid) contained in the assay mixture to produce a chromogenic com-
pound. 2"1 Alternately, the thiol detection reagent can be used after the
reaction has been concluded. Synthetic peptide thioester substrates have
been used for substrate mapping of elastases, chymotrypsin-like enzymes,
coagulation enzymes, and complement proteins, and they are also useful
for detecting various new serine peptidase activities in cell extracts such
as lymphocyte and natural killer cell granules. Several synthetic peptide
thioesters have been used to monitor enzyme activities of various metal-
loendopeptidases such as collagenases, stromelysin, gelatinase, and ther-
molysin.
1 .D .R Grassetti dna .J .F Murray, Jr., Arch. .mehcoiB .syhpoiB 119, 14 .)7691(
.D .A Farmer dna .J H. Hageman, .J Biol. .mehC 250, 6637 .)5791(
Copyright © 1995 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 248 All rights of reproduction in any form reserved.
4 SDOHTEM 11
Synthetic Methods
Materials. CDI, 3 DCC, HOBt, and benzyl mercaptan can be obtained
from Aldrich Chemical Company, Inc. (Milwaukee, WI). All Boc amino
acids can be obtained from Chemical Dynamics Corp. (South Plainfield,
NJ), Bachem Bioscience, Inc. (Philadelphia, PA), and numerous other
sources.
Boc-Ala-Ala-AA-SBzl. Boc-AA-SBzl derivatives are prepared by cou-
pling Boc-AA-OH and benzyl mercaptan using the DCC/HOBt method.
Boc-AA-SBzl can then be deblocked with HC1 in dioxane or ethyl acetate
to give HC1. AA-SBzl which is further coupled with Boc-Ala-Ala-OH to
give the final product Boc-Ala-Ala-AA-SBzl. 5,4 When the amino acid AA
is glutamic acid or aspartic acid, the side-chain carboxyl group is protected
with a tert-butyl group, which along with the Boc group can be removed
by trifluoroacetic acid or HC1 in ethyl acetate. 5
Boc-Ala-Ala-Nva-SBzl. To prepare Boc-Ala-Ala-Nva-SBzl, 4 Boc-Nva-
OH (2.17 g, 10 mmol) is dissolved in dry THF (10 ml), CDI (1.62 g, 10
mmol) is added, and the reaction is stirred at 0 ° for 45 min. Benzyl mercaptan
(1.16 ml, 10 mmol) is added, and the reaction mixture is allowed to warm
to 25 ° overnight. The solvent is removed under reduced pressure, ethyl
acetate (20 ml) is added, and the organic solution is washed with 10% (w/v)
citric acid, 4% (w/v) NaHCO3, and saturated aqueous NaC1. The ethyl
acetate solution is dried over MgSO4, then filtered, and the solvent is
removed. Boc-Nva-SBzl is solidified with hexane or petroleum ether (80%
yield), produces one spot on thin-layer chromatography (TLC)
CHC13 : methanol (9 : 1, v/v), and is used for subsequent reaction without
further purification.
Boc-Nva-SBzl (0.97 g, 3 mmol) is treated with 10 equivalents of 2.2 N
HC1 in dioxane and allowed to stir at 25 ° for 45 min. The solvent is then
removed by evaporation, and ether is added to solidify the product. The
resulting Nva-SBzl. HCI is dried in vacuo and used for subsequent steps
3 AA, Amino acid residue; Abz, ;lyoznebonima-2 AMC, ;niramuoclyhtem-4-onima-7 Boc, tert-
;lynobracyxolytub Bu-i, isobutyl; Bzl, benzyl; CDI, ;elozadimiidlynobrac-'1,1 DCC, N,N'-
;edimiidobraclyxeholcycid DMF, ;edimamroflyhtemid EDC, -)onimalyhtemid(-3-3-lyhte-1
propylcarbodiimide hydrochloride; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul-
fonic acid; HOBt, N-hydroxybenzotriazole; MES, cinoflusenahte)onilohprom-N(-2 ;dica
.uM N-morpholinocarbonyl; Nba, 4-nitrobenzylamine; NNap-OCH3, -hpan-3-yxohtem-1
thylamine; ,)1C(IzBS ;IC-4-4H6C2HCS Suc, succinyl; THF, tetrahydrofuran; Tricine,
;enicylglyhtem)lyhtemyxordyh(sirt-N Z, .lynobracyxolyzneb
4 j. oW Harper, .R .R Cook, .J Roberts, .B .J ,nilhgualcM dna .J .C Powers, Biochemistry ,32
5992 .)4891(
s .S Odake, C.-M. Kam, .L Narasimhan, .M Poe, .J T. Blake, .O Krahenbuhl, .J Tschopp,
dna .J .C Powers, Biochemistry ,03 7122 .)1991(
1 PEPTIDE THIOESTER SUBSTRATES 5
without further purification. Nva-SBzl-HCI (0.78 g, 3 mmol), Boc-Ala-
Ala-OH (0.78 g, 3.0 mmol), HOBt (0.61 g, 4.5 mmol), and triethylamine
(0.42 ml, 3.0 mmol) are dissolved in 10 ml of DMF, and the solution is
cooled to - 10 ° in an ice-water-salt bath. DCC (0.68 g, 3.3 mmol) is added,
and the reaction mixture is stirred at - 10 ° for 2 hr and at 25 ° overnight.
Dicyclohexylurea is removed by filtration, DMF is removed by evaporation,
and the residue is dissolved in ethyl acetate and washed as described above.
The final product is recrystallized from CHC13-petroleum ether with cool-
ing, mp 140°-141°; TLC Rf of 0.61 CHCI3 : methanol (9 : ,1 v/v). Analysis
calculated for C23H35N3OsS: C, 59.38; H, 7.58; N, 9.02. Found: C, 59.58; H,
7.62; N, 9.14.
Boc-Ala-Ala-Asp-SBzl. To prepare Boc-Ala-Ala-Asp-SBzl, 5 to a THF
solution (60 ml) of Boc-Asp(O-tert-Bu)-OH (11.6 g, 40 mmol) is added
HOBt hydrate (3.1 g, 20 mmol), benzyl mercaptan (5.2 ml, 44 mmol), and
DCC (9.9 g, 48 mmol) in 51 ml of THF at -5 .° After stirring at 0 ° for 21
hr and at room temperature for 24 hr, the reaction mixture is filtered and
the solvent removed by evaporation. Ethyl acetate is added to the residue,
and the solution is washed successively with 1 N HCI, 10% (w/v) Na2CO3,
and a saturated NaC1 solution. The organic layer is dried over MgSO4, and
the solvent is removed by evaporation. The crude product is purified by
silica gel chromatography using ethyl acetate :n-hexane (1:10, v/v) as an
eluant to give Boc-Asp(O-tert-Bu)-SBzl (13.5 g, 85% yield) as a pale yellow
oil. An ethyl acetate solution (70 ml) saturated with HC1 is added to Boc-
Asp(O-tert-Bu)-SBzl (4.0 g, 8.8 mmol) at 0 ,° and the solution is stirred at
25 ° for 2.5 hr. The solvent is removed, and ethyl acetate is added to the
residue to give a white precipitate of H-Asp-SBzl. HC1 which is filtered
and dried in vacuo (2.1 g, 87% yield).
To a THF solution (20 ml) of Boc-Ala-Ala-OH (0.52 g, 2.0 mmol) is
added successively N-methylmorpholine (0.22 ml, 2.0 mmol) and isobutyl
chloroformate (0.26 ml, 2.0 mmol) at -15 .° After stirring for 2 min and
addition of a cold THF solution (3 ml) of triethylamine (0.56 ml, 4 mmol),
the mixture is added to a DMF solution (1 ml) of H-Asp-SBzl • HC1 (0.55
g, 2 mmol) at -15 .° After stirring for 1 hr, the reaction mixture is quenched
by the addition of 1 N HC1 (4 ml) and is then concentrated in vacuo. An
ethyl acetate solution of the residue is washed with water and dried over
MgSO4. The solvent is removed by evaporation, and the crude product is
purified by silica gel chromatography using CHC13:methanol (50:1, v/v)
as an eluant and solidified with n-hexane to give the final product (0.67 g,
70% yield) as a white powder; mp 72°-79°; TLC eR of 0.52 (CHC13 : metha-
nol : CH3COOH, 80 : 10 : 5, v/v/v). Analysis calculated for S3NTO13H22C "
0.25H20-0.25C6H14: C, 55.61; H, 6.95; N, 8.28. Found: C, 55.71; H, 7.00;
N, 7.94.
6 SDOHTEM 11
Lysine- or Arginine-Containing Thioesters. Z-Lys-SBzl is prepared by
coupling Z-Lys-OH with benzyl mercaptan using the DCC method. 6 Simple
N-blocked arginine thioesters are synthesized by coupling an N-blocked
arginine derivative with a thiol using either the DCC-HOBt or DCC meth-
ods. 7 Di- and tripeptide thiol esters are then prepared by deblocking the
Boc-Arg-SR with HC1 in dioxane followed by coupling with the appropriate
peptide acid using the pentachlorophenyl active ester method. The arginine
side chain is protonated with HC1 during the synthesis of arginine thioesters;
therefore, it is not necessary to protect the guanidino group with other re-
agents.
Z-Arg-SBzl. HCl. To prepare Z-Arg-SBzl. HC1, 8 Z-Arg-OH-HC1
(3.45 g, 10 mmol) is dissolved in DMF, HOBt (1.35 g, 1.0 mmol) is added,
and the solution is cooled to 0 .° Benzyl mercaptan (1.24 g, 10 mmol) and
DCC (2.16 g, 10.5 mmol) are then added. The reaction mixture is stirred
overnight at 0 °, followed by removal of the dicyclohexylurea by filtration
and the solvent by evaporation. The crude product is purified by flash
column chromatography on silica gel (32-64/xm) using 15% methanol in
CHCI3 as an eluant. The product is obtained as a white foam after trituration
with petroleum ether and drying in vacuo (20% yield); TLC Rf of 0.64
CHC13 : methanol : CH3COOH (10 : 3 : 1, v/v/v). Analysis calculated for
"lC304NTzH12C 0.5H20: C, 54.83; H, 6.14; N, 12.18. Found: C, 54.76, H,
6.10; N, 12.40.
Thioester Substrates for Metalloendopeptidases. Thioester substrates for
metalloendopeptidases typically contain a thioester bond in the interior of
a peptide sequence and require additional synthetic steps. The synthesis
usually involves coupling of an N-terminal peptide fragment with the thiol-
containing fragment using standard peptide condensation methods to
form the thioester bond. For example, CH3CO-Pro-Leu-Gly-SLeu-Leu -
Gly-OCzH5 is prepared from CH3CO-Pro-Leu-GIy-OH and
HSCHCHzCH(CH3)z-CO-Leu-GIy-OC2H5 using the DCC-HOBt
method. The required HSCHCH2CH(CH3)z-CO-Leu-Gly-OCzH5 is syn-
thesized by coupling L-a-mercaptoisocaproic acid and Leu-Gly-OCzH5 us-
ing EDC-HOBt. 9
Boc-Abz-Gly-Pro-Leu-SCHzCO-Pro-Nba. Boc-Abz-Gly-Pro-OH and
Leu-SCH2CO-Pro-Nba • HC1 are synthesized by standard peptide coupling
methods. Boc-Abz-Gly-Pro-OH (0.78 g, 2 mmol) and Leu-SCH2CO-Pro-
Nba. HC1 (0.94 g, 2 mmol) are then coupled using the mixed anhydride
6 .G .D Green dna .E ,wahS Anal Biochem. ,39 322 .)9791(
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yrtsimehcoiB ,02 6917 .)1891(
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9 .H ,netragnieW .R Martin, dna .J Feder, yrtsimehcoiB ,42 0376 .)5891(