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Chemoenzymatic Synthesis of a Modified Pentasaccharide as a Specific Substrate for a Sensitive Assay of -Amylase by Fluorescence Quenching.

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of HPNPP [ZY]. The rate constants were obtained by fitting at least three half-lives
of the reaction according to a first-order kinetics equation. For slower reactions.
rate conslants were calculated from the initial rates. First-order rate constants were
calculated from the slopes of the linear plots of absorbance versus time by converting to concentration units of the phosphate ester (I: = 4500 M - ' em-') and dividing
by the initial phosphate ester concentration. All kinetic experiments were run at
least twice and were reproducible within 5 % error.
Received: November 29, 1994
Revised version: February 21, 1995 [Z 7508 IE]
German version: Angew. Chent. 1995. 107. 1343- 1345
Keywords: enzyme mimics . guanidines . transesterifications
[ I ] Reviews. D. S. Sigman, A. Mazumder. D. M. Perrin. Chem. R P V .1993, 93.
2295-2316; J. R. Morrow. Ariiz. Inorg. Biochem. 1994.9.41-74: J Chin. AK.
Chcvn. R(,$, 1991. 24, 145-152; M . W. Gohel, Angew. Chem. 1994, 106. 12011203; h g ~ w Chem.
.
I n f . Ed. EngI. 1994, 33. 1141-1143.
[ 2 ] J. Smith. K . Arigd, E. V. Anslyn, J. An?. Chrm. Soc. 1993. 115, 362-364.
[3] For recent examples see: J. K. Bashkin, E. I. Frolova. U. Sampath, J. Am.
C h m . So<. 1994. 116. 5981-5982: M. Wall. R . C . Hynes. J. Chin. Angew.
C'hcnr. 1993, /US. 1696-1697; Angcw. Cheni. Inr. Ed. EngI. 1993, 32. 16331635: D. Wahnon, R. C. Hynes. J. Chin, J. Chem. Soc. Chem. Commun. 1994,
1441 1442: B. K. Takasaki. J. Chin, J. Am. C h m . So?. 1993, 115. 9337-9338;
i/m/. 1994. 116, 1121 1122; D. Magda, R. A. Miller, L. Sessler. B. L. Iverson,
ihid 1994, 116. 7439 7440.
[4] C. A. Stein. J. S. Cohen, Cancer Rm. 1988, 48. 2659 -2668.
[ S ] E. Uhlmann. A. Peyman. Chem. R w . 1990. 90, 543--SS4.
[6] J. F. Milligan, M. D. Matteucci. J. C. Martin, J. Med. Chein. 1993. 36, 19231937
[7] F, A. Cotton. E. E. J. Hazen, M. J. Legg. Proc. Null. Awxl. Sci. U S A 1979, 76.
255 I .
[S] D. 1. Weber. A. K. Meeker. A. S. Mildvan. Biochentirrrj. 1991. 30. 6103-6114.
[9] E. H. Serpersu. D. Shortle. A. S. Mildvan. Bioc/ientisti:r 1987. 26. 1289- 1300.
[lo] A. S. Mildvan. E. H. Serpersu. Mer. Ions B i d . Sjsf. 1989. 25. 309-334.
[ i l l R . P. Dixon. S. J. Geib. A. D. Hamilton. J. Am. Cheni. Soc. 1992. 114, 365366
[12] K. Ariga. E. V. Anslyn. J. Org. Chein. 1992. 57, 417-419.
1131 D. M. Kneeland. K. Ariga, V. Lynch. C. Huang, E. V. Anslyn, J. Am. Chum
S ~ 1993,
N
11.5. 10042-10055.
[I41 R. Gross. J. W. Bats. M. W. Gohel. Liehrgs Ann. C h m . 1994, 205-210.
[IS] P. Schiessl. F. P. Schmidtchen. J. Org. Chem. 1994. 59. 509-511.
1161 E P. Schmidtchen, 7i~crahc&on Lett. 1989. 30. 4493-4496.
[17] A. Galan, E. Pueyo. A. Salmeron, J. de Mendoza. Ewuhedron Lett. 1991. 32,
1x17 1x30.
[ I X ] B. Dietrich. T. M. Fyles, J. M. Lehn, L. G. Pease, D. L. Fyles. J. C h m . So<.
C'hcw7. C ~ n r ~ n ~
1978,
i i ~ ~21.
. 934-936.
[19] V. Juhian. R. P. Dixon. A. D. Hamilton, J. Am. Chem. Soc. 1992, 114. 11201121.
[ZO] M. W. Giihel. J. W. Bats. G . Durner. Angew. Chrm. 1992, 104, 217-218;
. A I I ~ ~ ~Clrrni.
I I ~ . lnt. Ed Engl. 1992. 31. 207-209.
[ r l ] R. Gross, Ci. Durner. M. W. Gobel. Liehigs Ann. Cheni. 1994, 49-58.
[22] D. M. Brown. D. A. Usher. J. C h m . Soc. 1965. 6558-6564.
[23] For :I discussion on using activated esters see: F. M. Menger. M. Ladika, 1 An?.
C / J ~ ~So<
I I I . 1987. I U Y . 3145-3146.
[24] C. R Rasmuasen. F. J. J. Villani. L. E. Weaner. B. E. Reynolds, A. R. Hood,
L. R. Hecker, S. 0. Nortey. A. Hanslin. M. J. Costanzo, E. T. Powell, A. J.
Molinari. Si.nrhi~si$1988. 456-459.
[25] C , R . Rasmussen. F. J. J. Villani. B. E. Reynolds. J. N. Plampin. A. R . Hood,
1 . R. Hecker. S. 0 . Nortey. A. Hanslin, M. J. Costanzo. K. M. J. Howse, A. J.
M 01 i nori. S 1.17rhesis 1988. 460 465.
[26] M. S. Bernntowicz. Y. Wu, G. K. Matsueda. J. Orgy Chem. 1992. 57. 2497~
-
202.
[27] L. S. Flatt. V. Lynch. E. V. Anslyn. Tefruhedron Lerr. 1992. 33, 2785-2788.
[ZX] F. Chu. L. S. Flatt. E. V. Anslyn, J. Am. Chem. Soc. 1994. 116, 4194-4204.
1291 [18]Crown-6 (1 equiv) and water (8 x lo-' M) were added to the stock solution
of the barium salt of HPNPP in CH,CN to increase solubility.
Chemoenzymatic Synthesis of a Modified
Pentasaccharide as a Specific Substrate
for a Sensitive Assay of a-Amylase
by Fluorescence Quenching**
Nathalie Payre, Sylvain Cottaz, and Hugues Driguez*
Most living species such as microorganisms. higher plants,
and animals generate energy by the metabolism of starch. The
enzymes involved in the degradation of starch include a-amylase
(1,4-a-~-glucanglucanohydrolase) [EC 3.2.1.11, an endoglycanase, and exoglucanases such as glucoamylase [EC 3.2.1.31
and b-amylase [EC 3.2.1.2].[']
The detection and the determination of a-amylase activity are
not only necessary for catalysis studies but also important for
technological and medical applications, for example for monitoring fermentation processes and for diagnosing diseases like
parotitis (mumps) and acute pancreatitis. The assay of amylases
usually requires specific substrates to distinguish between the
endo- and exoglucanases, which both act on the a-(1 -+ 4)-glycosidic bond in D-G]c-or-(l -+ 4)-D-Gk units. A survey of the literature from the mid-1980s shows the considerable amount of
work that has been directed toward this goal.[21
The specific substrates can be divided into two groups: substrates with a single chromophore or fluorophore, which are
used in most of the commercially available diagnostic kits, and
substrates with an additional fluorescence-quenching group.
The chromogenic substrates are usually nitrophenylmaltooligosaccharides bearing blocking groups at the nonreducing
end to prevent action of exoenzymes. The nitrophenyl group
and the blocking substituent are introduced by demanding multiple-step reactions and/or low-yielding procedures, either by
chemical synthesis from maltodextrins with an appropriate
degree of polymeri~ation,'~]
or by chemoenzymatic transformations of modified linear or cyclic d e ~ t r i n s . 'Since
~ ] the enzymatic
hydrolysis of these substrates never occurs a t the reducing unit,
a-glucosidase or P-glucosidase and glucoamylase have to be
added during the assay to liberate nitrophenol from the residual
nitrophenylmaltooligosaccharides. Not only are these drawbacks costly but complications may also arise from the multitude of possible side reactions.
These problems may be overcome by the use of specific substrates that display intramolecular fluorescence quenching, in
which the variation of fluorescence between a fluorophore/
quencher pair on the same molecule can be monitored by using
the intramolecular resonance energy transfer effect.[51 This
strategy is analogous to that developed for the specific assay of
proteases.[61and for glycanases this was first achieved by Omishi
and Ikenaka, who prepared a disubstituted maltopentaoside by
a complex chemoenzymatic synthesis.''] In a very recent paper,
Bock et al. described the preparation of acylated inaltotriosides
which appeared to be pH sensitive and poor substrates for
a-amyIases.[81
The merit of substrates that display intramolecular fluorescence quenching is confirmed here by the specific detection of
x-amylase activities with micromolar concentrations of the sub[*] Dr. H. Driguez, N . Payre. Dr. S. Cottaz
Centre de Recherches sur les Macromolecules Vkgetales (CERMAV-CNRS)
BP 53 X, F-38041 Grenohle cedex 9 (France)
Telefax : Int. code +76547203
e-mail : hdriguez(n cermav.grenet.fr
[**I This work was supported by the Centre National de la Recherche Scientifique
(France) and by the European Biotech Program (1994-1996). We thank Prof.
P. Jardon (U. J. F. Grenohle) for providing his help in recording the fluorescence spectra.
Anpan.. Chm7. In/. Ed. EngI. 1995, 34, N o . I 1
'd3 VCH Vrr/ug,~gesellschafimhH. 0-69451 Wemherm, 1995
0570-0833,'9511I 1 1-123'9 $ 1 U . U U f .25:0
1239
COMMUNICATIONS
emission
(Amm = 490 nm)
synthetic considerations. Our strategy requires one fluorophore
with an amino function that can form an amino linkages with
the oligosaccharide, and the other fluorophore with an aliphatic
hydroxyl group that can form an ether linkage. These two linkages are much more stable than an ester bond during the enzymatic
Compound 1 was prepared chemoenzymatically (Scheme 2);
this approach is very valuable for the synthesis of regiospecifically modified oligosaccharides.[lal The key steps of the synthesis of 1 are the a-amylase-catalyzed coupling of a modified s(maltosyl fluoride donor 7 with an indolylethylmaltotrioside 10,
and the oxidation of pentasaccharide 11 by galactose oxidase
[EC 1.I .3.9], such that EDANS can be introduced regiospecifically at the nonreducing end of the oligosaccharide. a-D-Galactosyl-(I + 4)-Cr-D-glUCOSyl fluoride 7 and indolylethylmaltotrioside 10 were obtained by conventional chemical
modifications from known hexa-0-acetyl maltose 2["] and maltotriosyl bromide 8[12](Table l).
The glycosylation of 10 with 7 was achieved by using conditions originally described by Kobayashi et al. for the enzymatic
intramolecular energy
transfer
H
1
Scheme 1. Fluorescence quenching and intramolecular energy transfer in 1. After
irradiation of 1 at 290 nm, fluorescence at 490 nm was observed.
strate indolylethyl- and 5-(2-aminoethylarnino)-l -naphthalenesulfonate-substituted maltopentaoside 1 (Scheme 1).
The design of 1 was based on several observations: maltopentaose is the best substrate for our model enzyme, porcine pancreatic a-amylase (PPA) ,['] and the emission band of excited
3-(2-hydroxyethyl)indole (indolylethanol) (L,,, = 345 nm) and
the absorption band of 5-(2-aminoethylamino)-I -naphthalenesulfonate (EDANS) (n,,, = 340 nm) show excellent overlap.
Despite the relatively short lifetime of excited indolylethanol
(2 ns), the efficiency of the fluorescence quenching is satisfactor ~ . [The
~ ] EDANS/indolylethanol pair was also chosen based on
Table 1. Physical and spectroscopic data for I , 3-6, 9, and 11. NBA
zyl alcohol.
=
3-nitroben-
3: M.p. 197-199'C; [ar];' = +50 (c = 0.5 in CHCI,); ' H N M R (300 MHz, CDCl,): 6 = 5.80 (d. 'J(H-l ',H-2') = 8.04 Hz. 1 H; H-1 I ) , 5.36 (d, 'J(H-I',H2,) = 4.02 Hz, 1 H ; H-l'), 3.00 (s, 6 H ; 2 x CH,SO,), 2.00 (m, IXH: 6 x CH,CO);
"CNMR (75.4 MHz. CDCI,): 6 = 95.50 (C-1'). 91.00 (C-l'), 38.50 and 37.50
(2xCH,SO,); C,,H,,O,,S,
(750.5): calcd. 41.61, H 5.10, S 8.54;
found: C 41.46. H 5.08, S 8.48.
= +62 (c = 0.49 in CHCL,); ' H N M R
(300 MHz, CDCI,): 6 = 8.05-7.50 (m. 1 0 H ; Ph), 5.83 (dd, 3J(H32.H-42)= 3.2 Hz, 'J(H-4,,H-5') < 1 Hz, 1 H: H-4'). 5.73 (d, 'J(Hl'?H-2') = 8.1 Hz. 1 H ; H - l ' ) , 5.55 (d, 'J(H-I',H-Z') = 3.7 Hz, 1 H :
H-1'). 5.40 (dd. 1 H ; H-3,). 5.30 (t, 'J(H-2',H-3') = 'J(H-3I.H4') = 9.5 Hz. 1 H: H-3'). 5.23 (dd, 3J(H-2'.H-32) =7.3 Hz, 1 H ; H22), 4.95 (dd, 1 H ; H-2'), 4.53 (dd, 'J(H-6a1.H-6b') =12.3 Hz, ,J(H5',H-6b1) = 2.5 Hz, 1 H ; H-6b'). 4.48 (dd, 'J(H-6a2.H-6b2) =
9.3 Hz, 1 H ; H-6b'). 4.42 (ddd, 1 H; H-5'). 4.26 (dd, 'J(H-S',H6a2) = 4.0 Hz. 1 H : H-6a'). 4.20 (dd. 3J(H-5i,H-6a') = 4.6 Hz. 1 H:
H-6al). 4.05 (t. 3J(H-4',H-5') = 9.5 Hz, 1 H ; H-4'). 3.80 (ddd. 1 H ;
(802.7): cdlcd: C
H-5'). 2.07-1.93 (in, 18H; CH,CO): C,,H,,O,,
56236. H 5.27; found: C 56.65, H 5.25.
4: M.p. 165-166T: [a];'
2. R = H
4, R' = OAC; R' = H ; R3=Ac; R4 = BZ
R=S4CH3
"3,
5. R' = H ; R2 = F ; R3=Ac; R4 =Bz
dC
A c 0 4 O A r
6 , R' = H ; R' = F ; R 3 , R4 =Ac
7, R' = H ; R2 = F; R 3 , R4 = H
5 : M.p. 113-114'C; [a];' = +91 (1. = 0.5 in CHCI,); "CNMR
,
(75.4MH2, CDCI,): 6=170.62, 169.93, 169.76, 169.56 ( ~ x C O acetates), 165.52, 165.26 (2 x C 0 , benzoates), 103.4 (d, ' J ( C I',F) = 229 Hz; C-l'), 96.0 (C-12), 20.66, 20.45, 20.39, 20.29, 20.18
( 5 x CH,CO); C,,H,,FO,, (762.7): calcd: C 56.69. H 5.15, F 2.49;
found: C 56.39, H 5.10. F 2.50.
6: M.p. 117'C: [a];'= +I09 ( c = 0 . 5 in CHCI,); I3CNMR
(75.4 MHz, CDCI,): 6 = 170.64-169.53 (7 x CO. acetates), 103.4 (d,
'J(C-1', F) = 229Hz: C-l'), 95.9 (C-1'). 20.61-20.21 (7 x CH,CO):
C,,H,,FO,, (638.6): calcd: C 48.91, H 5.52. F 2.98: found: C 48.77,
H 5.52, F 2.91.
HO
7 + 10-
h
9 : [z];~= +58.5 ( c = 0.9 in CHCI,); ',CNMR (75.4 MHz, CDCI,):
H
6 =170.33-169.19 ( 1 0 x C 0 , acetates). 135.94, 122.04, 121.66,
119.06,118.43, 112.12, 110.88 (indole), 99.97 (C-1'). 95.46 (C-l', C1 3 ) . 62.84, 62.15, 61.19(C-6'. C-6,, C-6,). 25.32 (OCH,CH,), 20.61
20.19 (10 x CH,CO); FAB-MS (NBA + KCI, positive-ion rnode):'m/
z : 1067 [ M ' ] : C,,H,,NO,, (1068.0) = calcd: C 53.98. H 5.76, N 1.31;
found: C 53.90, H 6.04, N 3.45.
11: [a];' = +99 (t.= 0.54 in H,O); ' H N M R (500MHz. D,O).
6 =7.63-7.08 (m. 5 H ; indole), 5.27. 5.25, 5.24, 5.22 (each d, 4 H ;
H-12, H-13, H-14, H-1'). 4.26 (d, 3J(H-l',H-21) = 8.04Hz. 1 H : HI ' ) ; FAB-MS (NBA + KCI, positive-ion mode): mi;: 972 [ M ' ] .
1: [a]:' = +87 ( c = 0.42 in DMSO); 'H NMR (500 MHz, D,O):
S=8.20-6.75(rn,llH;Harom.),5.20(d,3J(H-12,H-22)=3.83Hz.
1 H ; H-12), 5.16 (d, 'J(H-13,H-2') = 3.83 Hz. I H ; H-1'). 5.05 (d.
-
-
H
11, R = C H # I
Scheme 2. Preparation of pentasaccharide 1. a) MeSO,CI, pyridine, 0 ° C to room temperature.
94%: b) sodium benzoate, hexamethylphosphoric triamide, 110 'C, 52 %: c) HF/pyridine, 0 "C,
86%; d) NdOMe/MeOH, then Ac,O, pyridine, 90%; e) NaOMe/MeOH. quant.; f) 3-(2-hydroxyethyl)indole, Hg(CN),. HgBr,. toluene/MeNO,, 44%; g) NaOMeiMeOH, quant.; h) ar-amylase
(Aspergillus oryzue). MeOH/phosphate buffer. 59% ; i) galactose oxidase (Duct.p/iumdendroidrs),
catalase (bovine liver), phosphate buffer. 37°C: j) EDANS, MeOH, reflux, then NaBH,CN, i)-J)
54% overall.
1240
c
VCH krlugsgesellschuft mhH. 0-69451 Weinheim. 199s
3J(H-14.H-24) = 3.97 Hz,
1H ;
H-14),
4.94
(d,
3J(H-lS,H-
2') = 3.28 Hz. 1 H: H-l'), 4.73 (d, J = 2.25 Hz, 1 H: H-4'), 4.10 (d,
3J(H-li,H-21)=7.95 Hz, 1 H; H-1'); FAB-MS (positive-ion mode):
m / z : 1246.364, calcd. for C,,H,,N,NaO,,S
[ M H,O Nd']:
1246.371.
OS70-0833:95!lllI-i240 $10.00+ .2S!0
~
+
Angew. Chrm. Ini. Ed. Engl. 1995, 34, No. if
COMMUNlCATlONS
polymerization of r-maltosyl fluoride with a-amylase as cataKeywords: a-amylase assay
chemoenzymatic syntheses
enzymes . fluorescence . oligosaccharides
l y ~ t . ~Under
' ~ ] these conditions I 1 was obtained in 78% yield
(determined by HPLC, 59 YOisolated yield). The epimerization
a) D. French in Eiochemistry nf Curhohjdrufrs, Serie one, V d . 5 (Ed. W. J.
at the 4'-position in the formation of 4 is beneficial for two
Whelm). Butterworths. London, 1975, p. 267; b) P. J. Reilly. Food Sci. R,chreasons. Firstly, 7 is accepted by the a-amylase as a donor, but
nn/. 1985. 14, 101 -142.
neither 7 nor 11 are acceptors because of the axial 4-OH at the
See for example: a) K.Omishi, T. Ikenaka. J Biochem. 1985.97.977-982: b) S.
Satomura, T. Iwata. Y. Sakata, Curhohydr. Rrs. 1988, 176. 107-115: c)S.
nonreducing end unit. Therefore only one disaccharide is added
Satomura. K. Omishi, T. Ikenaka, ihid. 1988, 180. 137- 146; d ) E. 0. Higele,
and 11 is obtained in good yield. Secondly, the nonreducing
M. Kratzer, E. Schaich. E. Rauscher, Chn. Chem. (Win.r/iw-Sulm?N C ) 1989,
galactosyl residue at the C-6 position in I1 can be oxidized
35, 188-189; e) K. Ogawa. H. Matsui, T. Usui, Eio.sci. Bmtechnol. Eiochem.
regiospecifically by the galactose oxidase/catalase system to give
1992. 56. 1933-1936; f ) K . Ogawa. Y. Taki. H . Ishida, Y. Yamagata. S.
Satomura, M. Kiso, A. Hasegawa, ihid. 1993.57. 821 -828; g) S. Tokutake. T.
aldehyde 12.1'4JIn the final step the EDANS group can be
Oguma. K. Tobe. K. Kotani. K . Saito. N. Yamaji. Curhohrdr. Res. 1993. 238.
introduced by reductive amination, giving 1 in 54% isolated
193-213; h) K. Ozawa, Y Yamagata, S. Satomura. H. Ishida. M. Kiso, A.
yield (Table 1 ) .
Hasegawa. ihid. 1994. 262. 137-145.
The intramolecular fluorescence quenching in 1 was demonSee refs. [2c.d, f-h].
See refs. [2a. b, e, g].
strated in preliminary experiments at concentrations of 10 p ~ .
R. H. Fairclough. C. R. Cantor. Mrfhods Enrjmoi. 1978. 4X, 347-379.
When 1 was excited at 290 nm, the EDANS fluorescence emita ) A. Yaron. A. Carmel, E. katchdkki-Katzir. A n d . Biochern. 1979. 95. 228ted at 490 nm was 12 times more intense than that of free
235; b) E. D. Matayoshi, G . T. Wang. G. A. Krafft. J. Erickson, Science 1990.
EDANS. We also detected a fivefold decrease in the intensity of
247. 954 958.
K. Omishi, T. Ikenaka. .l
5inchem. 1986, 99. 291 -294.
the indolylethyl emission at 345 nm. At this concentration we
V. Ferro. M. Meldal. K. B0ck.J. Chem. Suc. Perkin Truris 1 1994. 2169-2176.
can assume that variation of fluorescence intensity due to colliC. Seigner. E. Prodanov, G. Marchis-Mouren, Biochim. Biophps. Aclu 1987,
sional quenching is negligible. In fact a 2% increase in fluoresY13. 200-209.
cence intensity at 490 nm was observed for solutions of free
a) D. G . Drueckhainmer. W. J. Hennen, R. L. Pedersen, C. F. BdrbaS I l l . C. M.
Gautheron. T.Krack, C.-H. Wong. Sjnfhesi.c 1990. 499 525. and references
EDANS with indolylethylpentasaccharide11 when the EDANS
therein; b) S. Cottaz, C . Apparu, H. Driguez. J Chem. Sor. Perkin Truns. 1
concentration was increased from 10 to 20pM.
1991, 2235 2241 : c) C . Simiand, S. Cottaz, C. Bosso. H . Driguez. Eiochiinie
When a 5 p~ solution of 1 was incubated with PPA, the inten1992. 74. 75-79; d ) B. Evers. P. Miscbnick. J. Thiem. C'rrrhoh.~dr.Rex 1994,
sity of the fluorescence at 490 nm decreased rapidly. After 6 min
252. 335-341.
K . Takeo. K. Shinmitsu. Curhohjdr. Rrs. 1984. 133, 135 145.
the substrate was completely hydrolyzed (Fig. 1). At this stage,
K. Takeo. K. Mine, T. Kuge. Curhohjdr. Re,. 1976, 48. 197 208.
S. Kobayashi, J. Shimada, K. Kashiwa, S.-I. Koda. Mrri.roino/rcuk.\ 1992, 25.
3237-3241,
a ) G. Avigad, D . Amaral. C. Asensio. B. L. Horecker. J B i d . Chrm. 1962.237.
2736 2743; b) R. A. Schlegel, C. M. Gerbeck, R. Montgomery, CurholiJdr.
Res. 1968, 7, 193-199.
For the glucoamylase used (Sigma) low hydrolytic acti%itywas detected but
attributed to contamination by r-amylase (sigma).
a-amylase activity was previously determined with the Sigma Amylase Diagnostic Kit by using nitrophenyl 4.6-ethylidenemaltoheptaoside as substrate.
0
2
t/min
4
6
Fig. 1 . Progress of the PPA-catalyzed hydrolysis of 1 monitored by steady-state
fluorescence based on the relative fluorescence intensity I,,, at 490nm. A 5 p ~
. 7) was incubated with PPA (Sigma,
solution ol I i l l phosphate buffer ( 0 . 0 2 ~ pH
1 0 0 U L . ' ) a t 3 0 C[16].
HPLC analysis of the reaction mixture confirmed the complete
conversion of I into two shorter oligosaccharides (EDANSgalactosyl-x-(1 -+ 4)-maltose and indolylethylmaltoside; the retention times were compared to those of authentic standards
prepared independently).
When the same experiment was conducted with different concentrations of r-amylase, a linear relationship was found for the
initial rate of hydrolysis of 1 and the concentration of the enzyme. In contrast. even under optimal conditions. a-galactosidase, [&amylase, x-glucosidase, and glucoamylase were inactive." '1
Compound 1 is thus the first specific, sensitive, and readily
available substrate for the in vitro assay of human salivary and
pancreatic x-amylases (HSA and HPA). Further studies on 1
and the preparation of new substrates with improved performances are under way.
The Synthesis of Tris(perfluoroalky1)phosphanes""
Joel J. Kampa, John W. Nail, and Richard J. Lagow*
Many papers and review articles have dealt with the synthesis,
characterization, and reactions of highly fluorinated phosphanes.".'] Much attention has been drawn to the fact that
fluorinated phosphanes form complexes with transition metals.
Phosphorus trifluoride (PF,), the first member of the fluorophosphane family, has been shown to be an analogue of carbon
monoxide. In its reactions with transition metals, PF, closely
resembles CO electronically. Other highly fluorinated phosphorus(i1i) compounds similar to PF, exhibited similar reaction
chemistry. Recent work in our laboratory has shown that
P(CF,), forms complexes with numerous transition metals.13J
Tris(trifluoromethyl)phosphane, P(CF,), ,[41 has been known
for years; however, its preparation from trifluoromethyl iodide
[*I
+
[**I
Received: December 28, 1994 [27591 IE]
German version. Angebv. Chem. 1995, 107, 1361-1 364
Angot.. Chrm. 1ni. Ed. Lngl. 1995. 34, No. 1 i
;i-'j
Prof. R. J. Lagow. Dr. J. J Kampa. Dr. J. W. Nail
Department of Chemistry, University of Texas at Austin
Austin, TX 78712 (USA)
Telefax: Int. code (512)471-8648
This research was supported by the Air Force Office of Scientific Research
(F49620-92-0104) and the US. Department of Energy (DE-FG0591ER12119).
VCH Verlujisge.sell.icliuftmhH. 0-69451 WeOihrim, 1995
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synthesis, quenching, chemoenzymatic, fluorescence, amylases, pentasaccharide, specific, modified, substrate, sensitive, assays
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