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Enzymatic Hydrolysis of Hydrophilic Diethyleneglycol and Polyethyleneglycol Esters of Peptides and Glycopeptides by Lipases.

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could be refined to R :
0.10. Systematic deviations occurred for reflections
with h = 6n. which allowed US to elucidate twin-law involving planes perpendicular to the monoclinic u axis [6]. The resulting effect produce, small hut
significant shifts in the stacking pattern ofthe two-dimensional layers along the
-2.0
-4.0
direction. Further details of the crystal structure investigation may be obtained from the Fachinformationszentrum Karlsruhe. D-76344 Eggenstein-6.0
Leopoldshaien (FRG). o n quoting the depository number CSD-57736. the
names of the authors. and the journal citation.
T
[ 5 ] SHELXTL-PLUS, Version 4.0, Siemcna Industrial Automation. Inc.,
E [eV] -8.0
Madison. WI. USA.
-10.0
[6] Program SFLS (modificationof the program ORFLS): M. Eitel. H. BPrnighausen. Universitit Karlsruhe.
-12.0
[7] A. Simon. H. G . \ o n Schnering. J. Les$-C'oninion Mi,r. 1966. 11. 31.
[XI W. Littke. G . Brauei-. Z. . 4 1 i o r ~Allg.
.
Chem 1963, 325, 122.
-14.0
[9] A. Zalkin, D. E. Sands. Acirr Crj.sru//ngr. 1958, //. 615.
[lo] a-NbBr,. U . Miiller. P. Klingelhdfer. Z. :Vorrrrfor.\ch. B 1983. 38. 559: /$
NbBr,: W. Hdnle. S Furuseth. H. G. von Schnerinp. ;/>id. 1990. 4 j . 952.
[11] U. Miillcr. A < . / C<I ' f ~ i ' . ~ t u / / Srcl.
~ J ~ r .A 1978. 34. 256.
-18.0 I[12] H. A. Skinner. L E Sutton. 7 h i . c Furtickc1 SIC.1940, 36. 668.
[13] .IH. Canterford. R . Colton. Ho/fdi,\ n/r/li, Second mid Tlnvd Row Trrmrrrion
Fig. 2 Total density of states (left) and N b - N b owxlap population (right) for
M c , r i i / . \ . Wiley-Interscience. London. 1968. pp. 154. 161
NbiS21L4.( + ) : bonding N b - N b interactions; ( - ): antibonding N b - N b interac[14] L.Pauling. Thr Yoiirrr of I / W C'lioiiwul Hand. Cornell University Press. Ith;ica.
tions. Dashed line indicates [he Fermi level (hlghest occupied orbital), which occurs
NY. USA, 1960. p. 514.
at ;I small energy gap (0.2 eV) in the DOS that is not completely visible on this scale.
[15] H. Biirnighausen. ,WA1'C'H 1980. 9. 139: U Muller. Inov,yamc S r r u c r u r d
~ ' / i ~ ~ i i i i sWiley.
r r ~ ~ , New York, 1993. p. 235.
[I61 Extended-Htickel calculations using the tight-hinding approximation: M. H.
Whaiigho. R. Hofiniann. R B. Woodward. Proc. K SOC.Lutiduti .4 1979,366.
cule, which indicates a do configuration for these N b atoms. If
23. S iind parameters are from qtandard sources [17. 1x1. two scts of Nh
parameters were obtained by charge iteration. Nb(1-6): c ( 5 5 ) = 1.89.
we compare these energies with those for a Irons-vertex-sharing
H,,(Ss)= - 8.96 eV. i(5p9) = 1.85. ff,,(5p)= - 4.99 eV. ;,(4d) = 4.08 (c, =
chain of NbI, octahedra,[*] which give rise to a broad N b 4d
0.6401). ;,(4d) =1.64 (c, = 0.5516). H,,(4d) = - 9.83 eV: Vb(7). rame <-valband by significant through-bond coupling with the bridging I
ues a s Nb(1-6). H,,(5s) = - 9.85 cV. H,,(5p) = - 5.68 eV. H,,(4d)=
atoms, this band would overlap with the Nb-Nb bonding band
- 11.28 eV.
[17] S parameter: M . M. L. Chen. R. Hoffmann. J. Am. C ' h m . Soc. 1976. YK. 1647.
in the Nb,SI, layer. Our calculations strongly suggest, there[IS] I parameter: E. Canadell. 0. Eisenstein. Inor,q. Client. 1983. 22. 2398.
fore, that the monomers form so as to optimize Nb-Nb interac[19] S. Furuseth. W. Hiinle. G . Miller. H. G. von Schnering. 9th Intrrnurionul Contions within the two-dimensional framework, but with little
ferciici, o/ Troniilion Elrnirnts, Ahsrrul /.\, The Royal Society of Chemistry.
charge transfer between the two components. Thus, van der
1988.
[20] B. E. Burcteti, F. A. Cotton. M. B. Hall. R. C . Najjar. Inorg. C'hem. 1982. 2f.
Waals and other London dispersion forces are the dominant
302.
interactions holding these two structural components together.
[21] Twnrlvr A n u / y t u d C ' h r w i i r i r ~ ,Purr 11. &J/. 7. 1ntersclence:Wiley. New York.
Similar calculations on the alternative structure of Nb,SI,
1961.p. 401.
O.O
I
t
t
expected from earlier work on ternary Nb,YX, compounds in
this family['91 reveal that the bicapped Nb, cluster is energetically less favorable than the monocapped alternative.[2o1All of
our attempts to remove the NbI, molecules from Nb,S,I,, have
resulted in the formation of NbI,, I,, and the Nb,SI, trigonal
variant. Thus, a significant structural synergism exists between
these two components, each of which is unstable with respect to
either another structure (Nb,SI,) or towards decomposition
(NbI,), but are stabilized when they "cocondense."
Enzymatic Hydrolysis of Hydrophilic
Diethyleneglycol and Polyethyleneglycol Esters
of Peptides and Glycopeptides by Lipases""
Experinwntal Procedure
Horst Kunz,* Danuta Kowalczyk, Peter Braun, and
Giinther Braum
Elemental N b (foil. A h : washed HF:HNO, and dried under vacuum). S. and I
(sublimed; Alfa) in the molar ratio 7.2: 19 were sealed under Ar in a quart/ ampule
(@ = 10 mm: I = 60 inm) and heated at 1100 K for 2 d. The product forms black.
needle-shaped crystals. Chemical analysis with the Volhard method [21] gave I :
75.0% and S : 2.0%. N b analysir (a, Nb,O,) gave N b : 20.2%. X-ray powder
diffraction using an Enraf- Nonius Guinier camera indicates the phase NbiS,I,, is
accompanied b) a small amount of a hexagonal phase we identify as Nb,Sl, [19].
Magnetic susceptibility measurements show a small Curie- Weiss contribution
(0.018 cm' K m o l ~
' ) from a paramagnetic impurity. Two-probe electrical resistivity
measurements indicated p(300 K ) > lo4 Cl cm.
Received. August 25. 1993 [Z 6316 lE]
German version- Angw . C k m . 1994, 106. 357
[I] a ) Inorgunic A4urer;d.s (Eds. D. W Bruce. D. O'Hare). Wlley. Chichester.
1992: b) D. O'Hare in [l a]. p. 165; c) R. W. McCabe in [la], p. 295.
[2] H. Schiifer. H . G . von Schnering. Angetv. Cheni. 1964, 76, 833.
[3] A. Simon. An,qeti. CAcw?. 1981. Y3. 23; An,qc.w. Clirni. In?. Ed En~Tl. 1981.
20. I
[4] Crystals of Nb,S,I,, were always found as twins: space group P2,:c (No. 14).
Z = 4. 1 =1283.0(1). h = 2209.8(2). c =1465.0(1) pm. p = 94.025(9)
(Siemens P4 diffractometer): Mo,,. 6659 unique reflections. 3366 reflections
with F: 2 3o(F:). The structure was solved with direct methods (SHELXTL
PLUS. [S]) and refined with the program SFLS 161 to R(aniso) = 0.054.
R,(aniso) = 0.058. The initial structural model found by direct iuethods [5]
Dedicated to ProfeJJor Reinhnrd W Hoffrnann
on the orcarion of his 60th birthdup
The esters of primary alcohols were used as protective groups
for carboxyl groups quite early in peptide synthesis."] However,
alkaline hydrolysis of these esters bears a high risk of racemization. In glycopeptide synthesis['' with 0-glycosyl-serine and
-threonine derivatives the [I elimination of the carbohydrate
constitutes an additional problem.[''
New perspectives open up with enzymatic methods that are
carried out in neutral media,[41as is shown by the selective hydrol[*I Prof.
Dr. H. KunL. DipLIng. D. KoMalczyk. DiplLChem. P. Braun.
Dr. G . Branm
lnstitut fur Orgdnische Chemie der Universitit
J.-J.-Becher-Weg 18-20
D-55099 Mainr (FRG)
Telefax: Int. code + (6131)39-4786
[**I This work W B S supported by the Deutsche Forschungsgemeinschaft and the
Fondr derchemisuhen Industrie. We thank the Amano Pharmaceutical Co. for
donating eni.ymca.
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ysis of the heptyl esters of amino acids and peptides by l i p a ~ e s . ' ~ ]
In peptide synthesis lipases have the advantage that they d o not
attack the peptide linkage. A limitation is the substrate selectivity
of the enzymes; for example, the heptyl esters of hydrophobic
dipeptides such as -Val-Phe- are attacked by lipases either very
slowly or not at
Similarly the heptyl esters of peptides with
C-terminal p r ~ I i n e . [ ~0-azidogalactosyl-threonine.
"]
o r 0-galact o ~ a m i n y l - s e r i n e are
[ ~ ~not
~ hydrolyzed by either lipase M (Amano) from Mucor jmanicus or lipase N (Amano) from Rhizopus
nivcus. This lack of reactivity can be explained not only by the
fact that the substrate structure is not suited to the active center
of the enzyme. but also by simple physical factors such as the
poor solubility and water-repelling properties of the heptyl ester
in water. Heptylr5Iand similar alkyl esters were originally chosen for the reaction with lipases, since their hydrophobic properties simulate those of the natural substrates for these enzymes.r61
Many researchers have found that lipases incorporate the lipid
boundary surface during the catalytic process.['] Hence, it was
generally accepted that compounds containing a polar region
and a hydrophobic region provided suitable substrate properties for lipase-catalyzed hydrolysis.
During glycopeptide synthesis, however, we have observed that
2-morpholinoethyl esters (MoEt ester) of peptides. which d o not
contain any especially hydrophobic regions. are cleanly hydrolyzed by lipases.['] This surprising result can be interpreted by
postulating that complexation of the amphiphilic morpholino
ester, for example with N a + , causes the exposure of a hydrophobic surface which functions as a recognition region for the
interaction with the lipase (Scheme 1).
H
b
H-Xaa-OH
+
TsOH * H-Xaa
benzene. A
B: EDCfHOBt,
CH,CIz
Iiwe N
or CE
- 04 0 + o M e
wateriacetone 10: I
pH 71 37°C
The MEE esters are stable under the cleavage conditions for
most N-terminal protective groups. For example the Boc group
in Sa can be removed quantitatively with HCl/diethyl ether without the MEE ester bond in 7 being attacked.
\
H.7H\
quantitatively, but the products still contained traces of 2 after
drying at 80 "C under high vacuum and washing with diethyl
ether. The impure products were therefore converted into the
protected dipeptide esters S by treatment with N-protected
amino acids under the usual peptide condensation conditions.
The enzymatic hydrolysis of the dipeptide MEE esters 5 was
carried out in water/acetone ( 1 O : l ) at 37°C; the reaction mixture was maintained at pH 7 with 0.2 M sodium phosphate buffer
solution. Lipase N (Amano) from Rhizopus nivcus proved to be
suitable. Only in the case of alanyl-proline MEE ester Sd was it
necessary to use lipase CE (Amano) from Hurnicolo lanuginosri
(Scheme 2 and Table 1 ) .
H
HCV EtzO
5a
-
HCI. H-Val-Phe-OMEE
'I
Llolit rater+Na-
quant.
MEE eater+Na
Scheme 1
By generalizing this interpretation we came to the conclusion
that diethyleneglycol esters as well as oligo- and polyethyleneglycol esters of amino acids and peptides ought to be enzymatically
cleavable protective groups.['I To test this hypothesis, the hydrotosylates of esters 3 (2-[2-methoxyethoxy]ethyl ester; MEE) were
prepared from amino acids 1 and diethyleneglycol monomethyl
ether 2 by azeotropic esterification. Esters 3 were formed almost
In accord with this stability and the favorable solubility of the
amino acid and peptide MEE esters. peptide syntheses with these
components can be conducted without complications (cf. the
total yields of 5 for the two-step sequence in Table 1). In lipasecatalyzed hydrolyses the MEE esters have proved to be superior
to all the other esters hitherto inve~tigated.'~.
5. Thus, the completely selective and practically quantitative hydrolysis of the
MEE ester 5 a was achieved with lipase N (Amano). whereas the
corresponding heptyl esterL5'did not react with any of the lipases investigated. It should be emphasized that even the proline-
Fable 1. Synthesis of amino acid MEE esters 3 from 1 and 2. conversion o f 3 with protected amino acids 4 l o provide M E € esters, and the hydrolysis of 4 with lipases to
give 6.
PG
Xaii
Boc
Val
Ser
Ala
Ala
BOC
Tcoc [el
Z
Xaa
Phe
Phe
Ser
Pro
Method [a]
A
A
A
B
5 [b]
5a
5b
5c
5d
Yield
[%I
71
64
74
50
[c]
[TI;'
Lipase
(i,
6
1, CHCI,)
23.3
N
- 6.6
N
- 3.4
-53.1
N
CE
6a
6b
6c
6d
Yield
[dl
["/.I
97
94
92
31
[.I?
(c
Id1
1. MeOH)
14.3
15.4
- 1 .0
- 33.1
-
[a] For A.B \ee Scheme 2. [b] Characteristic ' H N M R signals of the MEE group. 6 = 4.2 (m. 1 H . COOCH,). 3.7-3.4 (m. 6H. OCH,). 3.3 (s, 3H. OCH,). [ c ] Total yleld
for two steps. [d] The dicyclohexylammonium salt was isolated: its elemental analysis and 200 MH7 ' H N M R spectra are consistent with the proposed structure.
[el Tcoc
=
2.2.2-trichloroethoxycarbonyl,
terminated MEE ester 5 d was hydrolyzed not with lipase N but
with lipase CE (Amano). 5 d is the first proline ester for which
a lipase hydrolysis has been found.
To test the MEE esters in glycopeptide syntheses, the O-glycosyl amino acid esters 8 were prepared from serine and threonine
MEE esters by the introduction of Aloc,"" Frnoc,["' or Z protective groups by known literature methods. and subsequent
glycosylation with 2-azido-2-deoxy-3.4,h-tri-O-acetyl-?-l,-alactopyranosyl bromider"] or ethyl-2-azido-2-deoxy-3,4.6-tri-Oacetyl-/~-o-l-thiogalactoside.~'
31 Compounds 8 were converted
10 X = N J
11 x = m c
into their galactosamine conjugates (T,-antigen) 9 with thioacetic
acid. The MEE esters once again have more favorable properties
than the corresponding heptyl and bromoethyl estersf4. in lipase-catalyzed hydrolyses (Table 2).
with TOTU['hlwas smoothly and selectively hydrolyzed to 13.1171
The reaction was quantitative according to thin-layer chromatography. All other protective groups and the glycosidic bond remained untouched.
Encouraged by these results we have prepared the Fmoc-peptide polyethyleneglycol esters (PEG esters) 14 by the method of
Mutter and Bayer"*] and hydrolyzed them with lipase N. The
loading of the polyethyleneglycol in conjugate 14 was verified by
calibration with Fmoc-Phe-OH. According to the first results.
the highly hydrophilic peptide PEG esters were hydrolyzed by
lipases in practically neutral media. The base-sensitive Fmoc
group in 15 remained intact in the polymer-free peptide.
The results obtained in Iipase-catalyzed hydrolysis of polar,
solubilizing MEE and PEG esters open up new possibilities in
both peptide and glycopeptide syntheses as well a s syntheses on
polymer-bound substrates. The lipases have no protease activity
and do not attack peptide linkages. The MEE esters confer wettability and solubility in water and so ensure that the esters of
hydrophobic peptide sequences will be hydrolyzable. Some of
these polar peptide esters. for example 14a and 14b. form clear
Fmoc- V d -
Phe- OPEG
lipasc N
pH 7 0 I 17°C
Fmoc- Val-
Table 2. Hydrolyses of 0-glycosyl amino acid M E € esters 8 and 9 catalyed by
lipase M (Arnano) from MUCOF
j f i w i i i c u s to give 10 and 11.
lipase N
Fmoc- Phe- Ala - V d -
14b
8b
Aloc
FmoL
8c
Z
Ya
Aloc
9b
Z
8a
sei
Ser
Thr
Ser
Thr
IOa
10 b
IOC
Ila
Ilb
89
64
5
17
18
132 8
101 5
-
113 3 [b]
94.4
[a] The products IOa, IOb, and 11 b were characterized by their correct eleineiital
analyses and 400 MHz ' H N M R spectra consistent with the proposed structures.
[b] (( 1. CHCI,).
Phe - OH
15a 53 %
14a
OPEG
pH 7.0 / 37°C
Fmoc- Phe- Ala - Val- OH
15b 77 70
solutions in water. Their hydrolysis (see above) brings into question the general validity of the postulate that lipases are only
effective on heterogeneous surFaces.[''] The exposed hydrophobic surface resulting from complexation (see Scheme 1) is obviously sufficient for the formation of the enzyme-substrate complexes with lipases. Since the hydrolyses take place in neutral
media. sensitive structural elements of the deblocked products.
such as protective groups and glycosidic bonds, remain intact.
The differently protected azidogalactosyl-serine MEE esters
8 a and 8 b were hydrolyzed smoothly with lipase M to give 10a
and I0 b. respectively. The corresponding threonine MEE ester
8c reacted too slowly for preparative purposes. The acetamidogalactosyl-serine and -threonine MEE esters 9 a and 9b, whose
heptyl and bromoethyl ester analogues showed no reactivity
with lipases, were hydrolyzed sufficiently fast to be useful. This
reaction was not optimized further, since in the meantime i t had
been shown that glycosyl amino acid MEE esters can be effectively deblocked at the C-terminus with the protease papain." 5 1
For glycopeptides whose saccharide side chains are not C-terminally located, and for which deblocking with papain is restricted because of its protease activity, the lipase-catalyzed cleavage of the MEE ester is quite efficient (Scheme 3). The T,,-antigen
glycopeptide ester 12 prepared from I 1 a and 7 by condensation
12 67 %
13 72 %
Scheme 3. TOTU = 2 4 (ethox~carbonylcyanomethylene)arnino]-l
.1 3.3-tetramethyluronium tetrafluoroboraLe[I 61.
Gencral method hi-thc hydrolysis o f M E € esters: A ~olutionof lipase N or C E
(200 rng) i n 0 . 2 .iqueou\
~
sodium phosphate buffer (2 mL;pH 7) was stirred with
phenylmethylsulfonyl lluoride (PMSF: 3.5 mg) for I h a t 0 C to inhibit residual
protease activity. After the addition of more sodium phosphate buffer solution
( I S inL). the mixture was shaken at 37 C for another 1 h. (With lipase M this
preparatory treatmenr 15 not necessark.) A solution of peptide ester 5 (0.5 mmol) iii
acetone (2 mL) wiis added dropwise to 20 rnL ol' Llir lipase solution prepared 'IS
above The mixture was well shaken for 24 h a t 37 C.The solution was saturated
with NaCl a n d exrracted five times with ethyl acetate. The combined organic phase\
were dried over MgSO, and concentrated. The N-protected peptide 6 was obtained
by llash chrormtograph) (petroleum ether. ethyl acetate-ethyl acetate). dissolved
iii diethyl ether. ; i n d concerted to its dicgclohes).l;imnionium salt The precipit.rled
s i l t was pure accordin5 to its elementill analysis a n d 200 M H r ' H N M R speclrir.
Received: August 23. 1993
Revised version: October 26. 1993 [Z6308 I € ]
German kersion: 4 n ~ e i r ' .Clieni. 1994, /Oh. 393
[ l ] a ) T. Curtiris. F Goehel. J. Prukr. Ch~vn[ 2 / 1888. 37. 150: b) E. Fischer. Brr
Dix / I . C/iiwi. Ge\. 1906. 39. 2893.
[2] H. titin-/. Pure .App/. Cliwi. 1993. (55, 1223: ,411rihinrii\ i i n d , A n / i w u l ( ' o n p~ii~id
-\C l i m i i ( ~ i S
d ~ n i / i ~ .mid
\ i . ~Modificoriom (Ed.;.: t i . Krohn, H. tiirst. H.
Maas). VCH. Weiiiheini. 1993, p 251
[3] R. D. Marchall, A. Neuberger, A d r . C d d i d ~C/wiii.
.
Bn)~/i~~
1970,
i n . 35. 407
[4] A. Reidel. H. Waldrnann, JT Priikr. Chcni. 1993. 335. 109.
[S] a) P. Hraun. H. Waldrnann. W. Vogt. H.Kunz. Liehiy h i i , Clicm. 1991. 165:
h) P. Brarm. H. Waldinanii. H.K u n i . Srnlerr 1992. 39.
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J. B West. C.-H Wong. 7 t / r a h f r / r m Lctl. 1987. 28. 1629.
C -S. Chen. C.. J Sih. Angcii.. Chrrn. 1989. 101. 711; A r i : ~Chrrii. I n r . Ed.
078i. 1989. 28. 695. and references thcrein.
G B r a u m . P. Braun. D. Kowalcryk, H. Kunr. Terruhedron Lerr. 1993. 34. 31 11
H. K u n / . G . Braum.P. Braun.(Hoechst AG).DE-AP413312') 7(7Oct. 1991).
H . K u n r . C . Unverzagt. Arigeii. Chern. 1984. 96. 426: ,4ri,qew. C/im7. In[. Ed.
Engi. 1984. 33. 436.
1.A C'avpiiio, G. Y. Han. J. Ani. C ~ P I H
SOC.
. 1970, YZ, 5748.
H P n u l w i , J.-P. Holck. Carhoiijdr. Rrs. 1982, 109. 89.
H. P:iulseii. M. Rauw,ald. U. Weichert. Liebig\ Ann. C/iwi. 1988. 75.
T. R o s x . J. M. Lico. D. T. W. Chu. J. Org. Clirni. 1988. 53. 1580.
H . K u n r . P. Braun. unpublished.
G. Breipohl. W. Kbiiiy (Hoechst AG). EP-A 0460446. A1 (23 May 1990).
13. [x]h2 = f 7 1 . 3 ( ~ . = 1 . 2 . CHCI,): R, = 0.33 (ethyl acetate'methanol
2 . 1 ) : ' H N M R (200 MHr. CDCI,. TMS): 8 = 5.33--5.24 (m. 2 H . H-4,
). 5.20 (dd. 1 H, Jgc,m
= 1.0 Hz. J,,, = 10.4 Hz. - C H = C H *',, 1,
5.04 (dd. 1 H, J , =11.5 Hz, J,,+ = 2 9 Hr, H-3). 4.80 (m. 1 H. r-CH. Phe).
4.72 ( d . 1 t1. .J, = 3.2 Hz. H-1). 0.82 (2d. 6 H , CH,. Val): FAB-MS
( 3 - N O B A ) . I ) / : : . 763 2 (M-H)- (calc. 763.2).
a ) M. Mutter. E. Bayer. .4ri,q,w. C/irni. 1974. 86, 101: Angew. C/IP?II.
In/. Ed
0 1 g . i .1974. 13. 88. b) V. N. R. Pillai. M. Mutter. E. Bayer. 1. Gatfield, J. Org.
C/lC?77 1980. 45, 5364.
H. L . Brockinann in Lipu.\c.s(Eds.: B. Borgstrom. H 1.Brockmann). Elsevier,
Anistertl;im. 1984. p. 3.
Progress toward an Antibody Glycosidase""
J a e h o o n Yu. Linda C. Hsieh, Lynn Kochersperger,
Shirlee Yonkovich, J a m e s C. S t e p h a n s ,
M a r k A. Gallop,* and Peter G. Schultz"
The development of catalysts for sequence-specific oligosaccharide cleavage would have a significant impact on the study of
carbohydrate structure and function."] Catalytic antibodies
may be particularly well-suited for this task. since antibody
specificity may be programmed through hapten design. Mechanistic studies of enzymatic oligosaccharide hydrolysis suggest
that a catalytic antibody combining site would need to stabilize
the delocalized positive charge and/or the half-chair conformation of the high-energy oxocarbenium ion formed by acid-catalyzed leaving group expulsion [Eq. (a)].[" As part of a program
for generating glycosidase antibodies, we have elicited monoclonal antibodies against several transition state analogues for
the hydrolysis of model acetal and glycoside substrates (1-4).
The design of hapten 5 is based on known cyclic amine glycosidase inhibitors such as nojirimycin and ca~tanosperrnine.['~
The
positively charged ammonium ion is expected to induce functional groups in the antibody combining site that stabilize the chargeProf. P G Schultr. L. C. Hsieh. 1. C . Srephans
Department o1'Chemistry
University of California
1
" 1
+
ROH
delocalized transition state in the hydrolysis of substrate 1 and/or
assist in the acid-catalyzed expulsion of the leaving group. The
5-bromoindolyl leaving group, which i s rapidly oxidized in air to
form bromoindigo, provides a simple, direct assay of cell culture
supernatants for catalytic activity, greatly facilitating the screening process during hybridoma p r ~ d u c t i o n . ' ~In] addition, this
group serves as a common recognition element between substrate and hapten to ensure substrate binding. Hapten 6 is based
on the potent amidine-containing glycosidase inhibitors which
are thought to mimic both the half-chair conformation and the
positive charge of the high-energy oxocarbenium ion.[51The less
activated benzylic leaving group in substrate 2 ensures protonation of the amidine group in the corresponding hapten and
provides the common recognition element. For comparison,
antibodies were also raised against the D-galactal derivative 7, a
neutral analogue of the half-chair oxocarbenium ion intermediate formed in the hydrolysis of substrate 3.I6I
2 n = 1, R = NH(CS)NHEt
4 n=0,R=N02
HO
-
-
HO'
3
4
NOz
0
bB
0
and
reiicc Berkeley L'ihoratory
Berkeleq. CA 94720 (USA)
Tclet'a\: Int code + (510)642-8369
Lab\
Di-. M A Gallop. Dr. J. Yu."' Dr. L. Kochersperger, S. Yonkovich
Aff)m,ix Research Institute
4001 Mirand'i Avenue. Palo Alto. CA 94304 (USA)
Present address
Department of Chemistry,
Korea lristittite o f Technology
Seoul (Korea)
This work w a s supported by the Office ofNaval Research (Grant No. N0001491-J-I 130) and the Assistant Secretary for Conservation and Renewable Energy, Ad\anced Industrial Concepts Division of the U.S. Department of Energy
(Contract No. DE-AC03-76SF00098). L C. H. is supported by a National
Sciencc Foundation Graduate Fellowship. We thank Holly C. Wessling for her
:issiCtiinee.
The first step in the synthesis of hapten 5 was formylation of
5-bromoindole with POCl, in dimethylformamide (DMF), followed by protection of the indole nitrogen with dimethyl pyrocarbonate. The formyl group was then converted to the corresponding primary bromide by sequential treatment with NaBH,
and PPhJBr,, and the resulting bromide was used to alkylate
2-hydroxymethylpiperidine. Derivatization of the primary hy-
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polyethyleneglycol, enzymatic, diethyleneglycol, esters, lipase, glycopeptides, hydrophilic, peptide, hydrolysis
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