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The Importance of the Rigidity of the Peptide Backbone for the Inhibitory Properties of BPTI Demonstrated by Semisynthetic Structural Variants.

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give the trimer, the resulting increased electron density on
the PC carbon atom weakens the transannular bond, thereby
facilitating departure of trimer and regeneration of the catalyst 1, in which the transannular interaction is
Strong support for the postulated flexibility of the transannular interaction in 1 (depending upon the phosphorus
substituent) is the stepwise reduction of this distance over a
series of eight compounds from 3.33 8, (which is essentially
the sum of the P and N covalent radii) to 1.967
Experimental Procedure
To a one-necked round-bottomed flask (250 mL, filled with N, and closed with
a septum) containing a solution of 1 (0.1 1 g, 0.50 mmol) in dry benzene (10 mL)
was added by syringe phenyl isocyanate (18.0 g, 99% pure, 150 mmol, Aldrichj.
After the mixture was stirred a t room temperature for 3 min, the white precipitate that had very rapidly formed solidified into a mass within a few seconds.
The solid was cooled to room temperature, dried under vacuum, ground to
powder, stirred with 30 m L of dry benzene for 2 h, filtered in vacuo, washed
again with 15 mL of dry benzene, and finally dried in vacuo to give 17.2 g
(96.6%) of 8 (pure by ' H N M R spectroscopy and TLC (silica gel, hexane:ether = 2:1, or CHCI,. or CHC1,:acetone = 50: 1 as eluents)). M.p. (uncorrected) 279.0-279.5"C (lit. 281 -2XlS'C[lb]);
' H N M R (CDCI,):
6 =7.35-7.51 (m, C,H,); I3CN M R (CD,CN): 6 =128.21. 128.74, 129.16,
133.39,148.46; HRMS ( m / i ) :calcd and found for C,,H,,N,O, 357.11134 (54,
M +); correct C,H.N-analysis.
953; m) R. J. P. Corriu, G. f. Lanneau, V. D. Mehta, Heleroatom Chem.
1991, 2, 46, n) R. Richter, P. Miiller, K. Wagner, Angew. Makromol.
Chem. 1983, f13, 1.
[4] S.-W. Wong, K. Frisch, J. Polym. Sci. Porr A Polym. Chem. 1986,24,2877.
[5] a) M . A. H. Laramay, J. G. Verkade, Z . Anorg. ANg. Chem. 1991,605,163;
b) J. G. Verkade (Iowa State University), US-A 5051 533, 1991 (Chem.
Absrr. 1992, 116, 50379q).
161 W. N. Olmsiead, Z. Margolin, F. G. Bordwell, J. Org. Chem. 1980, 45,
3295. Herein is reported a more reliable value of 32.2 for the pK, of
r-BuOH in DMSO, which thus raises the pK, of 3 to about 30.
[7] J.-S. Tang, M. A. H. Laramay. V. Young. S. Ringrose, R. A. Jacobson,
J. G. Verkade, J. Am. Chem. Soc. 1992. 114, 3129.
[8] S:K. Xi, H. Schmidt, C. Lensink, S. Kim, D. Wintergrass. L. M. Daniels,
R. A. Jacobson. J. G . Verkade, Inorg. Chem. 1990, 29, 2214.
[9] R. G. Arnold, J. A. Nelson, I. J. Verbanc, Chem. Ruv. 1957. 57, 59.
[lo] T. A. Bykova, B. V. Lebedev, E. G . Kipanisova, E. N. Tarasov, T. N.
Frenkel, V. A. Pankratov, S. V. Vinogradova, V. V. Korshak. J. Gen.
Chem. USSR (Engl. Transl.) 1985,2303 (Chem. Absrr. 1984,100,86 1712).
1111 M. I. Bakhitov, N. K. Emal'yanov, A. I. Tikhonova, 1. G. Davletbaev,
Vvsokomol. Soedin. Ser. 5 1983,25,830 (Chem. Absrr. 1984, 100,86 1712).
The Importance of the Rigidity of the Peptide
Backbone for the Inhibitory Properties of BPTI
Demonstrated by Semisynthetic Structural
The analogous reaction with p-MeOC,H,NCO carried out over 8 min gave a
98.7% yield of 9 (pure by ' H N M R spectroscopy and TLC (silica gel, same
solvent systems as used for 8)) after the product was washed with benzene
(80 mL) and dried in vacuo at 50°C. M.p. (uncorrected): 261.0-261.5 "C (lit.
By Christian Groeger, Herbert R . Wenzel, and Harald
261-2"C[lb]); 'HNMR(CDC1,): 6 = 3.81 (s,9H,OCH,),6.96(d,4H,CbH4;
'4," = 8.7 Hz), 6.27 (d, 4H, C,H,; 3JH,,= 8.7 Hz); HRMS ( m / z ) : calcd for
C,,H,,N,O,, 447.14304. Found, 447.14358 (50, M ').
Proteinases play an essential role in metabolic processes.
To detect intermediates by "P N M R spectroscopy the reaction was carried out
antagonists, the protein-proteinase inhibitors, block
with 0.037 g (0.17 mmol) of 1 and 0.062 g (0.52 mmol) of PhNCO in 0.7 mL of
the action of proteinases in vivo, and this important control
C,D,, and with 0.057 g (0.26 mmol) of 1 and 0.094 g (0.79 mmol) of pMeOC,H,NCO in 0.7 mL of C,D,. When 0.08 g (0.7 mmol) of PhNCO was
function makes them interesting as potential therapeutics.
added by syringe to a solution of 0.1 g (0.5 mmol) of 1 in ether (10 mL), a
Rational drug design requires detailed knowledge of all
colorless solid precipitated which was dried in vacuo to give 0.14 g of a colorless
of the mode of action of inhibitors; the results of
solid. 'IP NMR (CD,CN): 6 = 31.11. -9.35; MS ( m / z , FAB): 336.1 ( M + H.
investigations into the relationship between structure and
10. 52) and 217.1 ( M + H. 1. 100).
function of naturally occurring inhibitors are the ideal basis
The analogous reaction with P(NMe,), (0.17 g, 1.0 mmol) was carried out in
45 mL of dry benzene with 11.9 g (0.10 mol) of PhNCO. The reaction mixture
for the development of new synthetic proteins with inhibitwas heated at 60-70-C for 5 d at 60-70-C then left a t room temperature for
ory properties.
10 h. whereupon a small amount of white precipitate appeared. The volatiles
Many proteinase inhibitors function as substrate anawere removed under vacuum, the residue was stirred with 5 mL of benzene, and
logues; therefore, besides a low dissociation constant of the
then the suspension was filtered, washed with acetonitrile ( 5 x 2 m t ) , and dried
to give0.48 g(4%jofcyclic dimer. M.p. 182-183 'C (lit. 175"C[lO]); 'H NMR
corresponding enzyme-inhibitor complexes, the resistance to
(CD,CN): 6 =7.4-7.5 (m); HRMS ( m / z ) :calcd for C,,H,,N,O,. 238.07423.
proteolysis (permanence) of the inhibitors against the target
Found, 238.07428 (3.5, M ' ) .
Received: December 30, 1992 [Z5784IE]
German version: Angew. Chem. 1993, 105, 934
[I] a) Z. Bukac, J. Sebenda (Ustav Makromol. Chem.. CSAV Prag), CSA 227247, 1985 (Chem. Absrr. 1986, 105,173224r); b) Chem. Prurn. 1985.
35, 361 (Chem. Abstr. 1984, 103, 123978~);c) J. Horsky, U . Kubanek, J.
Marick. J. Kralicek, ibid. 1982, 32. 599 and (1983, 98, 5459q).
[2] a) H. Ulrich, J. CeN. Plosr. 1981, 17, 31 ; b) P. I. Kordemenas. J. E. Kresta,
Marromolecules 1981, 14, 1434; c) D. K . Hoffmann. J. Cell. Plosr. 1984,
20, 129.
[ 3 ] a) Y. Taguchi, I. Shubuya. M. Yasumoto. T. Tsuchiya, K. Yonemoto, Bull.
Chem. SOC.Jpn. 1990, 63, 3486; b) Y Taguchi, I. Shibuya (Agency of
Industrial Sciences and Technology), HP-A 03 109382.1991 (Chem. Abstr.
1991, 115, 208022;); c) J. Mizuya, T. Yokozawa, T. Endo, J. Po1j.m. SCI
Purr A Polym. Chem. 1991.29.1545; d) T. Endo, J. Suike (Arokawa Chemical Industries, Ltd.), JP-A 01 226878, 1989 (Chem. Absrr. 1990, 112,
7 7 2 3 6 ~ ) ;e) K. Ashide (BP Chemicals Ltd.). EP-A 169708, 1986 (Chem
Ahsrr. 1987, 107. 134825;); f ) F. R. Gubaidullin, M. I. Bakhitov, L. Sh.
Zainutdinova, F. L. Kligman, R. G. Miftakhova (Kazan Chemical-Technological Institute), SU-A 1555328, 1990 (Chem. Abstr. 1990, 113,
7 8 4 2 5 ~ )g)
; D. Kermis, H. P Mueller (Bayer AG), DE-A 3 543925. 1987
(Chem. Absrr. 1987, 107, 176705a); h) W. Broda, E. V. Dehmlow, H:J.
Schulz, Isr. J. Chem. 1985. 26, 222; i) T. Endo, Y Nambu (Asahi. Denka
Kogyo, K . K.), EP-A 447074, 1991 (Chem. Abstr. 1992, 116, 41 486v);
j) H. J. Fabris. E. M. Mexey, H. Uelzmann (General Tire and Rubber
Co.), US-A 3980594, 1976 (Chem. Absrr. 1976, 85, 193324~);k ) E. A.
Barsa, C. Conn (Upjohn Co.), US-A 4540781, 1985 (Chem. Ahsrr. 1985,
104, 89741d); I)I. Wakishima, Z. Kijima, Bull. Chem. Sor. Jpn. 1975, 48,
Verlagsgesellschaji mhH. W-6940 Weinheim, 1993
enzymes is essential.
In this paper we want to demonstrate with three semisynthetic "backbone" variants of the trypsin-kallikrein inhibitor BPTI (BPTI = basic pancreatic trypsin inhibitor = Kunitz trypsin inhibitor) from bovine organs, that
a strong permanent trypsin inhibitor can be converted into a
trypsin substrate by minor changes in the peptide backbone.
In contrast to the specificity-determining amino acid
PI = L Y S ' ~ , [ ' the
P region of BPTI, (Pl= AlaL6,
P2 = Arg17, Fig. I), can be replaced by a variety of residues
without significantly reducing the anti-tryptic activity of the
inhibitor.@ 31 Even the complete removal of the side chains
in this region is tolerated. Thus, the dissociation constant of
the [Gly16,Gly'7] BPTI-trypsin complex is 2 x l o - " M, only
one order of magnitude higher than that of the native complex.
Since the influence of the side chains in this region is apparently small, it seemed instructive for further investiga[*] Prof. Dr. H Tsrhesche, Dip].-Chem. C. Groeger, Dr. H. R. Wenzel
Lehrstuhl fur Biochemie der Universitat
Fakultit fur Chemie. D-W-4800 Bielefeld 1 (FRG)
Telefax: Int. code + (521)106-6146
This work was supported by the Deutsche Forschungsgemeinschaft (Ts
8/24-2). We thank Werner Beck, Universitat Tubingen, for recording the
ion-spray mass spectra.
0570-0833/93j0606-0898 3 10.00+ ,2510
Angew Chem. Inr. Ed. Engl. 1993, 32, No. 6
H-Ile' '...
pH 4.75
C4H8, CO,
...~ y s ' ~ - ~ ; ~ y ' ~ ~ [ ~ ~ ] C'...l y " - ~ l e '
Fig. 1. A projection of the tertiary structure of the BPTI-trypsin complex 161
(section). residues Tyr", His4', and Phe4' belong to trypsin. The P strand
including side chains and the specificity-determining amino acid Lys" as well
as the N terminus and &-aminogroups of BPTI are shown in boldface. The
dotted lines represent hydrogen bonds.
tions into structure-function relationships to replace positions P', and P2 with a series of peptide analogues derived
from glycylglycine (Fig. 2). The introduction of the struc-
Fig. 2. Peptide analogues derived from glycylglycine. The box indicates the
changed amide bond.
turally related building blocks 5-aminovalerianic acid (Ava),
N-aminoethylglycine (Aeg), and 5-aminolevulinic acid (Ale),
into the Pregion of the inhibitor results in a complete or
partial replacement of the Pl-P2 amide bond with methylene
groups. To introduce the building blocks, the Pl-Pl (Lys"AlaI6) bond in the reactive site of the inhibitor is cleaved
with trypsin, and the amino acids Ala16 and Arg" are removed with aminopeptidase K. This results in the formation
of the inactive fragment de(Alal6-Arg' 7)-[seco-l 5/
18]BPTI.*41The new N terminus at P3 = Ile18 can now be
acylated by using the N-hydroxysuccinimide ester of BocAva, (Boc = butoxycarbonyl), (Boc),-Aeg, and Z-Ale
(Scheme 1).
Although the inhibitor fragment possesses a total of six
potential sites for acylation (two N termini as well as E-amino
groups of four lysine residues, Fig. I), the reaction can be
carried out with a high degree of selectivity by controlling the
pH value. For example, at a pH of 4.75, up to 25 % of the
product is monoacylated at I1eL8.After cleavage of the Boc
or Z protecting group, the open LysI5, Gly16Y[XX]Gly17
bond is closed by using trypsin.['I
Anxeu C'hrm. Inr. E d Eng/. 1993, 32. No. 6
$3 VCH
Scheme 1. The exchange of AlaI6 and Arg" by peptide analogues, (exemplified by a Boc-protected activated ester), starting from de(Ala"-Arg")-[sero15/18]BPTI (XX = CH,CH,, CH,NH, COCH,). TFA = Trifluoroaceticacid.
Compared with [Gly16-Gly'7]BPTI, [Ava16- "IBPTI and
[Aeg'6-'7]BPTI have proved to be much weaker and, furthermore, temporary trypsin inhibitors; that is, they are degraded by the enzyme. The dissociation constants are about
2 x lo-' and 7 x 1 0 - 9 ~respectively.
In contrast, the inhibitor [Ale16- 17]BPTIwith a dissociation constant of 5 x 1 0 - l ' ~is a strong and permanent inhibitor of trypsin.
A look at the subsite interactions[61in the native BPTItrypsin complex (Fig. 1) shows that the keto function of the
Pl-P2amide bond is involved in neither intramolecular hydrogen bonds in BPTI nor intermolecular hydrogen bonds
between BPTI and the enzyme. The NH group alone serves
as donor for a hydrogen bond to the CO unit of Phe41 in
trypsin and thus should contribute to the stability of the
complex. The experiment shows, however, that
[AegI6- 17]BPTI has a slightly higher dissociation constant
than [Ava'6-'7]BPTI.
The good inhibitory properties and especially the resistance of the Ale derivative to breakdown by trypsin must be
due to the restricted flexibilty of the bonding loop, because
the keto function fixes four atoms in the same plane; the Ale
derivative thus possesses some of the rigidity of [Gly16GlyL7]BPTI.A rigid binding region of the inhibitor means
easier docking and a lower entropy loss[71during complexation by the enzyme, which also leads to a higher affinity for
the enzyme. At the same time the rigidity of the binding
region explains the extremely slow proteolysis of the Pl-P;
peptide bond.[*]
In contrast, a flexible bonding loop, which additionally
forms a hydrogen bond to the enzyme in the complexed state
(Aeg derivative), leads to a much stronger reduction in degree of freedom than in a reaction with a loop with the same
degree of flexibility that cannot form this bond (Ava derivative). Only the combined effects of backbone rigidity and
hydrogen bond formation-as in [Gly16-Gly17]BPTI-result in the net energy gain for the complex formation, as
shown by the lower dissociation constant of 2 x 1 0 - l 2 in
contrast to the Ale derivative.
The structural variants of the peptide backbone of BPTI
described here show that complementarity in the sense of a
Verlagsge.sellschaji mhH, W-6940 Weinheim, 1993
0570-0833/93/0606-0899 $ 10.00i .25/0
perfect fit between enzyme and inhibitor is not as important
as the rigidity of the peptide structure in the binding region.
This rigidity is an absolute requirement for the proper function of the inhibitor as a permanent inhibitor.
Experimental Procedure
Ava'HCI was produced by acid hydrolysis of 2-piperidone. Aeg was synthesized from 1.2-diaminoethane and chloroacetic acid according to [9]. Ale.HCI
was purchased from Merck. The addition of Boc or Z protecting groups and the
esterification with hydroxysuccinimide were carried out according to standard
procedures [lo]. All derivatives and intermediates were characterized by
'H N M R spectroscopy and mass spectrometry.
A general procedure for the synthesis of BPTI derivatives: De(Ala"-Arg")[seco-1511XIBPTI (25 mg) (preparation see refs.12.31) was dissolved in bidistilled
water ( 5 mL) and treated with a 30-fold molar excess of activated ester. dissolved in 1 mL of dioxane or dimethyl sulfoxide. The pH was maintained at 4.75
by addition of NaOH. After 4-5 h the solution was gel filtrated on Sephadex
G-25, and the monoacylated product was separated by ion-exchange chromatography on C M Sepharose F F at pH 8.6 (NaCI gradient). The corresponding fractions were desalted on Sephadex (3-25, and the protecting groups were
cleaved with trifluoroacetic acid (15 min for Boc: 10 h for Z). The acid was
removed under vacuum, and the residue taken up with the equivalent amount
of bovine trypsin in Tris buffer pH 8. After 10 min the pH was lowered to 1.7
with dilute HCI, and the solution was subjected to gel filtration on Sephadex
G-50 at pH 1.7. Finally. the inhibitor-containing fractions were repurified on
CM Sepharose F F by ion-exchange chromatography, desalted on Sephadex
G-25, and lyophilized. Yield: 0.5-2.5 mg, 2-10%. Ion-spray mass spectra of
the synthesized inhibitor variants gave the expected molecular weights. The
identity of the Aeg derivative was also confirmed by complete sequencing with
the Edman degradation method. [Glylb-Gly' 'IBPTI was synthesized as described in [ 3 ] .
Received: January 18, 1993 [258381E]
German version: Angew. Chem. 1993, /08, 948
[i]H. Tschesche, Angew. C k m . 1974.86, 23 -40: Angeir. Chem. In!. Ed. Engi
1974, 13, 10-28.
[2] C . Groeger, H. R. Wenzel, H. Tschesche. J. Prorein Chem. 1991, 10, 245251
131 C. Groeger, H. R. Wenzel. H. Tschesche, J. Prorern Chrm. 1991, 10. 527
141 H. Jering, H. Tschesche, Eur. .f Biochem. 1976. 6/, 453-463.
[S] U. Quast, J. Engel, E. Steffen, H. Tschesche, Bzoc'hemi.sfry 1978, 17, 16751682.
(61 R. Huber, D. Kukla, W. Bode. P. Schwdger, K. Bartels, J. Deisenhofer, W.
Steigemann, 1 Mol. B i d . 1974. 89, 73-101.
[7] R. J. Read, M. N . G . James in Proreinuse Inhibrrors (Eds.: A J. Barrett, G .
Salvesen). Elsevier, Amsterdam, 1986. p. 323.
[8] W. R. Finkenstadt, M. A. Hamid, J. A. Mattis. J. Schrode. R. W. Sealock.
D. Wang, M. Laskowski in Buyer Symposium V. Proteinuse Inhibitors
(Eds.: H. Fritz, H. Tschesche. L. J. Greene. E. Truscheit), Springer, Berlin,
1974, S. 389-411.
[9] E. P. Heimer, H. E. Gallo-Torres. A. M. Felix. M. A. Ahmad, T. J. Lambros. F. Scheidl, J. MeIenhofer, Int. 1 Peplide Protein Rex 1984, 23, 203211.
[lo] M. Bodanszky. A. Bodanszky. The Pructrre o/Peplide Synrhesu. 1st ed.,
Springer. Berlin, 1984. p. 12. 20, 125.
The design and synthesis of receptor molecules for the
selective complexation of ions has attracted much attention
during the past two decades.['] However, the number of host
molecules for anions is very low compared to the number for
cations. The reported host molecules contain either positive[*] Prof. Dr. Ir. D. N. Reinhoudt, Dr. s. Valiyaveettil, Dr. J. F J. Engbersen.
Dr. W. Verboom
Laboratory of Organic Chemistry
University of Twente
P.O. Box 217. NL-7500 AE Enschede (The Netherlands)
W-6940 Weinheim,1993
3 R=CH,Cl
R = (CH,),CH,
R = 4-MeOC6H,
Willem Verboom, and David N . Reinhoudt*
G VCH VeriuRc~esell.schaf~
Synthesis and Complexation Studies of Neutral
Anion Receptors
By Suresh Valiyaveettil, Johan I? J. Engbersen,
ly charged sitesc2]or Lewis acid metal centers13] to accomplish anion binding.[41
In nature, the selective ion flow to and from the cell is
regulated by ion-binding proteins that act as ion carriers and
channels across the cell membrane. Quiocho et a1.['] have
shown that the high specificity of phosphate transport by
proteins is determined by extensive hydrogen bonding in the
binding site. The phosphate binding site is formed by two
similarly folded globular protein domains and is located in a
cleft approximately 8 8, inside from the protein surface.
Phosphate binding involves the formation of twelve hydrogen bonds, five from the main chain and seven from the
side-chain residues.[5"]
In our attempts to mimic nature in the recognition of
anions by the formation of multiple hydrogen bonds in
three-dimensional arrangements, we have designed a new
series of ligands with hydrogen-bond donor as well as acceptor sites. This strategy was based on the known structures of
phosphate binding sites of proteins. In this communication
we demonstrate that surprisingly strong anion binding can
be achieved with relatively simple, neutral host molecules in
which the H-bond donor and H-bond acceptor sites are
present in a tetrahedral arrangement and interact with complementary groups on the anions. Since these receptor molecules are uncharged they are of particular interest as
ionophores in membrane transport and for the introduction
of anion selectivity in potentiometric membrane sensors.
The host molecules 1,2, and 3-8 were synthesized starting
from diethylenetriamine and tris(aminoethyl)amine, respectively, by reaction with the appropriate acid chlorides in the
presence of triethylamine as base.16] Compounds 1-8 were
isolated in 70-90
yield after recrystallization from
methanol, and were characterized by 'H and 13CNMR
spectroscopy, mass spectrometry, and elemental analysis.
8 R
In 'H N M R titration experiments of the Iigands with
Bu,N+A- (A- = H,PO;, HSO;, Cl-) in chloroform, the
N H signal of the ligands shifted Ah = 1.5-2.0 to lower field
until a host-guest ratio of 1 :1 was reached. N o shift was
observed in the N M R signal when the concentration of the
anion was increased; however, in the titration with H,PO,
the NH signal shifted until a host-guest ratio of 1:2 was
attained. These stoichiometries were confirmed by the characteristic 6(31P) values for all host-guest complexes in acetonitrile upon addition of two equivalents of Bu,N+H,PO,.
For example, the signal for ligand 8 showed a shift of
A6 = 0.345 with one equivalent of H,PO; and a further shift
B 10.00f ,2810
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structure, properties, inhibitors, demonstrated, bpti, semisynthetic, variant, importance, backbone, rigidity, peptide
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