Enzymatic synthesis of carbon-fluorine bonds [8]

Full text of DOI:10.1021/ja005855q

4350
J. Am. Chem. Soc. 2001, 123, 4350-4351
Enzymatic Synthesis of Carbon-Fluorine Bonds
2,4-dinitrophenyl â-glycoside substrate in the presence of fluoride
2 M KF, pH 6, 25 °C), substantial glycosidic bond activity is
(
12
†
†
‡
restored (Table 1). Because fluoride is acting in the place of
the missing catalytic nucleophile, the product of the glycosidic
bond cleaving reaction for Abg and Man2A nucleophile mutants
David L. Zechel, Stephen P. Reid, Oyekanmi Nashiru,
†
‡
†
Christoph Mayer, Dominik Stoll, David L. Jakeman,
‡
,†
R. Antony J. Warren, and Stephen G. Withers*
13
is the corresponding R-glycosyl fluoride (Scheme 1, first step)
1
19
14a
Protein Engineering Network of Centres of Excellence and
Departments of Chemistry and Microbiology
UniVersity of British Columbia
as confirmed by TLC, H NMR, and F NMR. In the case of
Abg E358G, the turnover for carbon-fluorine bond formation
-1 15
exceeds 2 s . However, because these nucleophile mutants, or
VancouVer, B.C., Canada V6T 1Z1
10,16
“
glycosynthases”,
also act as catalysts for oligosaccharide
synthesis, the R-glycosyl fluoride product in turn acts as a glycosyl
donor in a subsequent transglycosylation reaction with a second
molecule 2,4-dinitrophenyl â-glycoside, thus forming new gly-
cosidic bonds (Scheme 1, second step).1 The wild-type enzymes
did not catalyze the formation of R- or â-glycosyl fluorides, as
ReceiVed December 6, 2000
Halogenated secondary metabolites are ubiquitous in Nature.
Over 3000 natural products containing chlorine, bromine, or iodine
have been characterized to date, and scores are added to the list
4a
1
19
each year. There is, however, a notable scarcity of fluorinated
indicated by F NMR, presumably due to electrostatic or steric
2
17
natural products, of which only 13 are known. This may in part
effects.
stem from the limited bioavailability of fluoride, which exists
primarily in insoluble mineral forms.3 Equally elusive is an
enzymatic mechanism for fluorination, as no enzyme with
The Abg E358G and E358A mutants can also catalyze
nucleophilic halogenation of 2,4-dinitrophenyl â-glucoside with
chloride and bromide (2 M). Although the corresponding R-glu-
,4
2
14b
fluorinating activity has yet been isolated. A key aspect of the
cosyl halides are too unstable to isolate directly, transglyco-
fluorination mechanism will be the nature of the fluorinating
species: electrophilic, radical, or nucleophilic. The electrophilic
halogenation mechanism is well-known for the haloperoxidases,5
and a radical chlorination mechanism is thought to be involved
sylation products resulting from transfer to a second equivalent
of substrate were observed by TLC and ESI-MS. A comparison
of kcat/K
M
values for E358G and E358A (Table 1) indicates an
-
-
-
order of halide reactivity (F > Cl > Br ) opposite to that
expected in aqueous solution. Although this may be the result of
steric constraints in the Abg active site, this order of halide
nucleophilicity has also been observed in organic solvents and
the gas phase.18 Desolvation of the halide may well occur in the
6
in the biosynthesis of barbamide. However, the chemical
+
7
oxidation of fluoride to “F ” (E° ) 2.87 V), the strongest
oxidizing agent known, is infeasible and therefore these mech-
anisms are unlikely. The nucleophilic halogenation mechanism
is rare, having only been demonstrated for the methylation of
active site of Abg. The reactivity order may also reflect a
-
-
-
8
18,19
Br , Cl , and I by S-adenosylmethionine methyl transferases.
stabilizing “synergism”
in the halogenation transition state
A nucleophilic mechanism also appears to be used by FAD/
9
(12) Evolved halide specificity in a wild-type enzyme can be expected to
produce equivalent catalysis at “physiological” concentrations of fluoride.
NADH dependent halogenases in the biosynthesis of pyrrolnitrin,
and is thought to be involved in the biosynthesis of fluoroacetate.4
Given the scarcity of data on nucleophilic halogenation by
enzymes, particularly with fluoride, it is significant that specific
active site mutants of two retaining glycosidases, Agrobacterium
sp. â-glucosidase (Abg) and Cellulomonas fimi â-mannosidase
(13) This is likely a concerted single-displacement mechanism since
glycosyl oxocarbenium ion intermediates do not exist in the presence of
2
2,26
anions.
14) (a) TLC analysis of a reaction of Man2A E519S, 2,4-dinitrophenyl
â-mannoside, and 1 M KF indicated the formation of R-mannosyl fluoride as
(
1
19
the major product. H and F NMR spectra of the isolated, per-O-acetylated
1
product agreed with that of an authentic sample: H NMR (200 MHz, CDCl
3
)
(
Man2A), can catalyze the formation of carbon-fluorine bonds
δ 5.55 (dd, 1 H, J ) 48.3, 1.7 Hz, H-1), 5.40-5.30 (m, 3 H, H-2,3,4), 4.29
10
with nucleophilic fluoride. Replacement of the catalytic glutamate
nucleophile in Abg (E358) or Man2A (E519) with alanine,
glycine, or serine renders the usual double displacement mech-
anism11 inoperable and virtually no glycosidic bond cleaving
activity is detectable. However, when assayed with the appropriate
19
(
dd, 1 H, J ) 12.7, 5.4 Hz, H-6ax), 4.14 (m, 2 H, H-5, H-6eq); F NMR
(188 MHz, CDCl , referenced to CF CO H) δ -62.5 (d, J ) 49.2 Hz).
3
3
2
R-Glucosyl fluoride could not be isolated from a preparative reaction with
Abg E358S, 2,4-dinitrophenyl â-glucoside, and 2 M KF due to the more potent
glycosynthase activity of this mutant, leading to its rapid consumption via
transglycosylation. The rate constant for turnover of R-glucosyl fluoride with
Abg E358S exceeds 1 s- (unpublished). Di- and trisaccharide products were
the major products detected by TLC and ESI-MS. However, a low concentra-
1
*
Address correspondence to this author. Phone: (604) 822-3402. Fax:
(
604) 822-2847. E-mail: withers@chem.ubc.ca.
tion of R-glucosyl fluoride produced by the reaction with Abg E358G was
†
19
Department of Chemistry
Department of Microbiology
confirmed by F NMR (282 MHz, referenced to CF
3
CO
2
H): δ -74.3 (dd, J
‡
) 55.4, 24.9 Hz,). Moreover, the analogous reaction with 2,4-dinitrophenyl
â-galactoside and Abg E358G resulted in the formation of R-galactosyl fluoride
as the major product. R-Galactosyl fluoride cannot donate to a second
â-galactoside substrate due to the absence of a suitably positioned (equatorial)
(
(
(
1) Gribble, G. W. Chem. Soc. ReV. 1999, 28, 335-346.
2) O’Hagan, D.; Harper, D. B. J. Fluorine Chem. 1999, 100, 127-133.
3) Cotton, F. A.; Wilkinson, G. AdVanced Inorganic Chemistry, 5th ed.;
1
6 1
19
John Wiley and Sons: New York, 1988.
4-hydroxyl. H and F NMR spectra of the isolated, per-O-acetylated product
1
(
4) Harper, D. B.; O’Hagan, D. Nat. Prod. Rep. 1994, 123-133.
3
agreed with that of an authentic sample. H NMR (200 MHz, CDCl ) δ 5.78
(
5) (a) Butler, A. Coord. Chem. ReV. 1999, 187, 17-35. (b) Littlechild, J.
(dd, 1 H, J ) 2.4, 53 Hz, H-1), 5.50 (dd, 1 H, J ) 2.9, 1 Hz, H-4), 5.34 (dd,
1 H, J ) 3.2, 10.7 Hz, H-3), 5.16 (ddd, 1 H, J ) 2.9, 10.7, 23.2 Hz, H-2),
Curr. Opin. Chem. Biol. 1999, 3, 28-34. (c) Hofmann, B.; T o¨ lzer, S.; Pelletier,
I.; Altenbuchner, J.; van P e´ e, K.-H.; Hecht, H. J. J. Mol. Biol. 1998, 279,
89-900. (d) van P e´ e, K.-H. Annu. ReV. Microbiol. 1996, 50, 375-99.
1
9
4.38 (m, 1 H, H-5), 4.11 (m, 2 H, 2 × H-6); F NMR (188 MHz, CDCl
3
,
8
referenced to CF CO H) δ -75.1 (dd, J ) 53.4, 22.9 Hz). (b) During the
3 2
(
6) (a) Hartung, J. Angew. Chem., Int. Ed. 1999, 38, 1209-11. (b) Sitachitta,
reaction of Abg E358G with 2,4-dinitrophenyl â-galactoside and 2 M NaCl
1
N.; Rossi, J.; Roberts, M. A.; Gerwick, W. H.; Fletcher, M. D.; Willis, C. L.
(pD 5.6, 300 K) a small resonance was observed by H NMR that may
J. Am. Chem. Soc. 1998, 120, 7131-32.
correspond to R-galactosyl chloride: δ 5.45 (d, J ) 3.1 Hz, H-1, referenced
(
7) CRC Handbook of Chemistry and Physics, 71st ed.; CRC Press: Boston,
to HDO). The instability of this compound precluded synthesis of a standard.
3
1
990-1991.
(15) The activity of a mutant glutamate dehydrogenase was enhanced 10 -
(
(
8) Wuosmaa, A. M.; Hager, L. P. Science 1990, 249, 160-162.
9) (a) van P e´ e, K.-H.; Keller, S.; Wage, T.; Wynands, I.; Schnerr, H.;
fold by fluoride fulfilling an electrostatic role (Hayden, B. M.; Dean, J. L. E.;
Martin, S. R.; Engel, P. C. Biochem. J. 1999, 340, 555-560).
(16) Mayer, C.; Zechel, D. L.; Reid, S. P.; Warren, R. A. J.; Withers, S.
G. FEBS Lett. 2000, 466, 40-44.
(17) The precision of chemical rescue in glycosidase mutants is well
documented (Ly, H. D.; Withers, S. G. Annu. ReV. Biochem. 1999, 68, 487-
522).
(18) (a) Olmstead, W. N.; Brauman, J. I. J. Am. Chem. Soc. 1977, 99,
4219-4228. (b) Parker, A. J. J. Chem. Soc. 1961, 1328-1337.
(19) Pearson, R. G.; Songstad, J. J. Org. Chem. 1967, 32, 2899-2900.
Zehner, S. Biol. Chem. 2000, 381, 1-5. (b) Keller, S.; Wage, T.; Hohaus, K.;
H o¨ lzer, M.; Eichhorn, E.; van P e´ e, K.-H. Angew. Chem., Int. Ed. 2000, 39,
2
300-2302.
(
10) We first noted fluoride dependent glycosidic bond cleaving activity
with Man2A nucleophile mutants (Nashiru, O.; Zechel, D. L.; Stoll, D.;
Mohammadzadeh, T.; Warren, R. A. J.; Withers, S. G. Angew. Chem., Int.
Ed. 2001, 40, 417-419).
(11) Zechel, D. L.; Withers, S. G. Acc. Chem. Res. 2000, 33, 11-18.
1
0.1021/ja005855q CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/13/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 18, 2001 4351
Table 1. Kinetic Parameters for Abg and Man2A Nucleophile
Mutants Catalyzing Nucleophilic Halogenation of 2,4-Dinitrophenyl
â-Glucoside and 2,4-Dinitrophenyl â-Mannoside, Respectively
Scheme 2. Man2A E429A Catalyzing Nucleophilic
Fluorination of the Mannosyl-Enzyme Covalent Intermediate,
Forming â-D-Mannosyl Fluoride. (DNP ) 2,5-dinitrophenyl)
k
cat
K
(mM)
M
kcat/K
M
min 1)a
-
(min ‚mM-1)
-1
(
Abg wt
5040
3600
0.0214
0.024
235 000
150 000
+
2 M KF
Abg E358A
<0.001
21.5
1.00
0.08
131
44.8
11.2
+
2 M KF
2 M NaCl
0.131
0.112
164
8.93
+
Abg E358G
+
+
+
+
2 M KF
0.157
0.086
0.144
834
521
77.8
2 M NaCl
2 M NaBr
2 M KI
hydrogen bonds known, with hydrogen bond energies of 30 kcal/
mol realized with methanol or ethanol as partners in the gas
phase.25 Moreover, the departure of fluoride during the solvolysis
b
n/a
Abg E358S
2 M KF
<0.001
28.9
+
0.044
657
26
of R-glucosyl fluoride is acid catalyzed, even when trifluoro-
ethanol (pK
a
) 12.4) acts as the proton donor.2
2,27
Likewise, a
Man2A wt
2 M KF
Man2A E519A
2 M KF
Man2A E519S
2 M KF
27 000
7 900
<0.008
20.7
0.0015
14.0
0.6
0.22
45 000
36 000
+
hydrogen bond formed with the departing fluoride ion in the
glycosynthase transition state (Scheme 1, second step) may also
explain the ∼25-fold greater glycosynthase activity of Man2A
+
0.131
0.036
158
389
10,16
and Abg serine mutants relative to the alanine mutants.
+
The generality of enzymatic nucleophilic fluorination is further
demonstrated by the ability of the general acid-base mutant of
Man2A (E429A) to synthesize â-mannosyl fluoride. This mutant
readily forms the covalent intermediate with the reactive substrate
a
cat, K
M
apply to glycoside at 2 M halide, pH 6.0, 25 °C. b No
k
activity.
Scheme 1. Abg E358S Catalyzing Nucleophilic Fluorination
of 2,4-Dinitrophenyl â-Glucoside and Subsequent Transfer of
R-Glucosyl Fluoride to a Second Equivalent of Substrate (R)
2,5-dinitrophenyl â-mannoside (Scheme 2). Due to the absence
of base catalysis in the second step, deglycosylation is slowed
and the covalent intermediate accumulates, as evidenced by very
-
1
low kcat and K
M
values (kcat ) 12 min , K < 1 µM). However,
M
fluoride (1 M KF) can attack the anomeric carbon of the covalent
intermediate, resulting in faster turnover of the enzyme (kcat
)
-
1
8
7 min ) and a corresponding rise in K
M
(7.7 µM), as well as
the formation of â-mannosyl fluoride. The â-mannosyl fluoride
product could not be isolated and is not expected to accumulate
2
8
as it is also a good substrate for Man2A. Nevertheless, while
monitoring the reaction directly by 19F NMR spectroscopy a
resonance corresponding to a low steady-state concentration of
â-mannosyl fluoride was observed.29
The catalysis of carbon-fluorine bond formation by mutants
of two unrelated glycosidases, each using a nucleophilic fluorina-
tion mechanism, demonstrates the feasibility of such a mechanism
between hard, weakly polarizable attacking and leaving groups
20
(i.e. fluoride and phenolate oxygen). Synergism may also
30
occurring in Nature. Indeed, it is particularly interesting that
account for similar reversals in halide reactivity that have been
2
one of the known fluorinated natural products, nucleocidin, is
21
observed for the reaction of methyl glycosides and R-glucosyl
itself a glycosyl fluoride and may well be formed by an analogous
mechanism. As suggested by this work, catalytic features of
nucleophilic fluorination may involve hydrogen bonding and
desolvation of the anion. This work also raises the possibility of
engineering other enzymes to catalyze nucleophilic fluorination
of medicinally significant compounds.
fluoride22 with halides in aqueous solution.
Also noteworthy from the kcat/K
M
values is the fluorination
activity of the Man2A and Abg serine mutants (Table 1). This
activity is greater than or comparable to the fluorination activity
of the glycine and alanine mutants, despite the extra bulk of the
serine side chain. It is probable that this difference arises from a
hydrogen bond to the serine hydroxyl group. Abg E358G may
also benefit from a hydrogen bond between fluoride and Tyr 298,
a residue that interacts with the E358 oxygen in the wild-type
Acknowledgment. We thank the Natural Sciences and Engineering
Research Council of Canada (NSERC) and the Protein Engineering
Network of Centres of Excellence (PENCE) for financial support.
23
enzyme. Although fluorine is, at best, a poor hydrogen bond
JA005855Q
acceptor,24 desolvated fluoride can form some of the strongest
(25) Hibbert, F.; Emsley, J. AdV. Phys. Org. Chem. 1990, 26, 255-379.
(
26) Banait, N. S.; Jencks, W. P. J. Am. Chem. Soc. 1991, 113, 7958-61.
(
20) This is consistent with the halide reactivity order observed for the
(27) Sinnott, M. L.; Jencks, W. P. J. Am. Chem. Soc. 1980, 102, 2026-
-
-
-
S-adenosylmethionine methyl transferase reaction (I . Br > Cl ), which
32.
8
involves a “soft” leaving group, sulfur. Fluoride is not a substrate. However,
halide specificity may have evolved in these enzymes.
(28) Stoll, D.; He, S.; Withers, S. G.; Warren, R. A. J. Biochem. J. 2000,
351, 833-8.
(
(
(
21) Bennet, A. J.; Sinnott, M. L. J. Am. Chem. Soc. 1986, 108, 7287-94.
22) Banait, N. S.; Jencks, W. P. J. Am. Chem. Soc. 1991, 113, 7951-58.
23) (a) Gebler, J. C.; Trimbur, D. E.; Warren, R. A. J.; Aebersold, R.;
(29) Man2A E429A (4 µM) was reacted with 2,5-dinitrophenyl â-manno-
side (23 mM) in 1 M KF, pH 7, at 300 K. After 20 min an apparent doublet
1
9
was observed by F NMR (282 MHz, referenced to CFCl ): δ -146.7 (d, J
3
Namchuk, M.; Withers, S. G. Biochemistry 1995, 34, 14547-53. (b)
) 49 Hz). This agreed with an authentic sample of â-mannosyl fluoride.
(30) In fact, there is strong evidence that haloperoxidases may not be
involved in the biosynthesis of most halometabolites and that nucleophilic
Hakulinen, N.; Paavilainen, S.; Korpela, T.; Rouvinen, J. J. Struct. Biol. 2000,
1
29, 69-79.
24) Dunitz, J. D.; Taylor, R. Chem. Eur. J. 1997, 3, 89-98.
5
d,9a
(
halogenation may be Nature’s preferred biosynthetic mechanism.