69SO
J. Org. Chem. 1995,60,6930-6936
Mechanism-BasedInactivation of Ribonuclease A Jeffkey K. Stowell and Theodore S. Widlanski* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Tatiana G . Kutateladze and Ronald T. Raines Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706-1569 Received April 17,1995@
The first example of a mechanism-based inhibitor of a phosphodiesterase is reported. Although the inactivation brought about by the fluoride 4 is not complete, this compound provides a useful starting point for the synthesis of other more potent inhibitors of ribonuclease A, as well as inhibitors of other nucleases. In addition, an inexpensive method is described for the synthesis of phosphate diesters that cannot be synthesized using standard phosphoramidite methodology. Phosphitylation of the target alcohol with a dialkyl chlorophosphite,followed by activation of the resulting trialkyl phosphite with 12, yields an iodophosphate. The resulting iodophosphate can then be coupled to a second alcohol, phenol, or enolate to give a phosphate triester, which af‘ter subsequent deprotection affords the desired phosphate diester. The novel phosphorylation chemistry presented should greatly facilitate the synthesis of other similar mechanism-based phosphodiesterase inhibitors. Scheme 1 Phosphodiesterases comprise an important group of enzymes whose biological roles include such diverse RO functions as signal transduction,l DNA processing and replication,2phospholipid metab~lism,~ and viral reproRO duction.* Ribonucleases are among the best understood of all the phosphodiesterases. In particular, ribonuclease A (RNase A; EC 3.1.27.5) has been the object of landmark 0, .o work on enzymology; on the folding, stability, and RO OH A 0 0 chemistry of proteins; and on molecular ev~lution.~ RNase A is a small protein (124 amino acid residues; 13.7 RO OH Step I1 + H20 kDa) that catalyzes the two-step hydrolysis of the P-06’ bond of RNA. Scheme 1depicts a catalytic sequence that is widely-accepted and consistent with all known In the enzyme-catalyzed reaction shown in Scheme 1, the side chain of His12 acts as a general base that abstracts a proton from the 2’-hydroxyl of a substrate molecule, thereby facilitating attack on the phosphorus atom.s This attack proceeds in-line to displace a nucleosideeg The side chain of His119 acts as a general acid Both products are then released to solvent. The slow that protonates the 5”-oxygen to assist its departure.8 hydrolysis of the 2’,3’-cyclic phosphate occurs in a separate step that resembles the reverse of transphosphorylation.1° @Abstractpublished in Advance ACS Abstracts, October 1, 1995. (1) Shukla, S. D.; Halenda, S. P. Life Sci. 1991,48, 851-866. Despite our relatively advanced understanding of how (2) Adams, R. L.; Knowler, J. T.; Leader, D. P. The Biochemistry of A works, the design of inhibitors for this enzyme RNase the Nucleic Acids; Chapman and Hall: London, 1986. (3) Gum, M. I.; Hanvood, J. L. In Lipid Biochemistry; Chapman and is still in its infancy. Indeed, there are relatively few Hall: London, 1991. rationally designed phosphodiesterase inhibitors of any (4) (a) Repaske, R.; Hartley, J. W.; Karlick, M. F.; ONeill, R. R.; kind and no published examples of mechanism-based Austin, J. B. J. Virol. 1989, 63, 1460-1464. (b) Schatz, 0.; Cromme, F. V.; Naas, T.; Lindemann, D.; Mous, J.; LeGrice, S. F. J. In Gene phosphodiesterase inhibitors. Regulation and AIDS;Papas, T. S., Ed.; Portfolio: Texas, 1990; pp We recently published several strategies for the design 293-303. and synthesis of mechanism-based phosphatase inacti(5)For reviews, see: (a) Richards, F. M.; Wyckoff, H. W. The Enzymes 1971, IV,647-806. (b) Karpeisky, M. Y.; Yakovlev, G. I. Sou. vators.ll The implementation of one of these strategies Sci. Rev., Sect. D 1981,2, 145-257. (c) Blackburn, P.; Moore, S. The has led to the successful development of phosphotyrosine Enzymes 1982, XV, 317-433. (d) Wlodawer, A. In Biological Macrophosphatase inactivators.12 These approaches are ilmolecules and Assemblies, Vol. II, Nucleic Acids and Interactive Proteins; Jurnak, F. A,, McPherson, A., Eds.; Wiley: New York, 1985; lustrated by the chemical “motifs” shown in Scheme 2.
-
1
pp 395-439. (e) Eftink, M. R.; Biltonen, R. L. In Hydrolytic Enzymes; Neuberger, A., Brocklehurst, K., Eds.; Elsevier: New York, 1987; pp 333-375. (0 Beintema, J . J.; Schiiller, C.; Irie, M.; Carsana, A. Prog. Biophys. Molec. Biol. 1988, 51, 165-192. (6) For a mechanism, see: Findlay, D.; Herries, D. G.; Mathias, A. P.; Rabin, B. R.; Ross, C. A. Nature 1961, 190, 781-784. (7)For other proposed mechanisms, see: (a) Witzel, H. Progr. Nucleic Acid Res. 1963,2, 221-258. (b) Hammes, G. G. Adu. Protein Chem. 1968, 23, 1-57. (c) Wang, J . H. Science 1968, 161, 328-334. (d) Anslyn, E.; Breslow, R. J.Am. Chem. SOC. 1989,111,4473-4482. (8)Thompson, J . E.; Raines, R. T. J. Am. Chem. SOC. 1994, 116, 5467-5468.
0022-326319511960-6930$09.00/0
(9) (a) Usher, D. A.; Erenrich, E. S.; Eckstein, F. Proc. Nat. Acad. Sci. U.S.A. 1972, 69, 115-118. (b) Usher, D. A.; Richardson, D. I.; Eckstein, F. Nature 1970, 228, 663-665. (10) (a) Thompson, J . E.; Venegas, F. D.; Raines, R. T. Biochemistry 1994, 33, 7408-7414. (b) Par& X.; NoguBs, M. V.; de Llorens, R.; Cuchillo, C. M. Essays Biochem. 1991,26, 89-103. (11) (a)Myers, J. IC;Widlanski, T. S. Science 1993,262,1451-1453. (b) Stowell, J. IC;Widlanski, T. S. J.Am. Chem. SOC. 1994,116, 789790. (c) Myers, J . K.; Cohen, J . D.; Widlanski, T. S. J.Am. Chem. SOC., in press.
0 1995 American Chemical Society
J. Org. Chem., Vol.60,No.21, 1995 6931
Mechanism-Based Inactivation of Ribonuclease A
Scheme 2 Motif A
Motif B
Scheme 3 Motif
:wra
C
0. ,O OH
Pi
HL B r Quinone methide
Acid halide or ketene
a-bromo aldehyde
An integral design consideration for these inhibitory motifs was that they be applicable to the development of phosphodiesterase or phosphotriesterase inhibitors simply by incorporating the appropriate chemical functionalities into suitable substrates. In this paper, we describe a chemical strategy for the introduction of these inhibitory motifs into substrates for RNase A. We then examine the inactivation of the enzyme brought about by use of inhibitory motif A.
Results and Discussion Inhibitor Design. RNase A binds the bases of adjacent RNA residues in three enzymic subsites: B1, B2, and B3.13 Catalysis by RNase A results in the cleavage of the P-Or bond specifically on the 3'-side of pyrimidine nucleotides that are bound in the B1 subsite.14 Indeed, the B1 subsite appears to bind14J5only residues having a pyrimidine base. Although the B2 subsite has a preference for residues having an adenine base and the B3 subsite has a preference for residues having a purine base, the B2 and B3 subsites can accommodate all residues. Indeed, the enzyme readily catalyzes the transphosphorylation of other phosphodiesters, such as the uridine 3'-phosphate diester with p-nitrophenol.8 A number of important phosphodiesterases such as phospholipases D and C show a similar proclivity to RNase A in that enzyme specificity is conferred largely by the alcohol that remains phosphorylated.16 In these cases, this type of selectivity permits the design of potential mechanism-based enzyme inactivators based on the motifs shown in Scheme 2. Starting with the appropriate phosphate ester, simply appending one of the latent reactive functionalities shown in Scheme 2 gives rise to a potential mechanism-based enzyme inactivator. Using this principle, we envisioned the structures of potential mechanism-based ribonuclease inactivators (46)shown below (Scheme 3). Inhibitor 4 would induce the enzyme-catalyzed liberation of a quinone methide. Inhibitor 5 gives rise to an acylating agent, and inhibitor 6 would yield an a-bromo aldehyde, which is a reactive alkylating agent. The three motifs shown below repre(12) Wang, Q.; Dechert, U.; Jirik, F.; Withers, S. G . Biochem. Biophys. Res. Comm. 1994,200,577-583. Taylor, W. P.; Myers, J. K.; Widlanski, T. S.; Zhang, Z.-Y.; Dixon, J . E. J. Am. Chem. SOC., submitted. (13) Parks, X.; NoguBs, M. V.; de Llorens, R.; Cuchillo, C. M. Essays Biochem. 1991,26, 89-103. (14) delcardayrk, S. B.; Raines, R. T. Biochemistry 1994,33,60316037. (15) (a) McPherson, A.; Brayer, G.; Cascio, D.; Williams, R. Science 1986,232,765-768. (b) Aguilar, C. F.; Thomas, P. J.; Mills, A.; Moss, D. S.; Palmer, R. A. J. Mol. Biol. 1992,224, 265-267. (16)(a) Hendrickson, E. K.; Johnson, J. L.; Hendrickson, H. S. BioMed. Chem. Lett. 1991, 1, 615-618. (b) Heller, M. Adu. Lipid Res. 1978, 16, 267-326.
qF
RNase
I--\
&iB
r
1
RNase
Br "*
0
1
RNase
0
H
L H
B
r
sent just a few of the many possible ways in which our strategy could be used to generate the structures of potential enzyme inactivators. Inhibitor Synthesis. Although there are a variety of useful methods available for the synthesis of nucleoside phosphodiesters, it was clear that the synthesis of enol phosphate diesters such as 5 and 6 would not be possible using standard phosphoramidite chemistry.l7 Moreover, the expense of the reagents used for phosphoramidite chemistry prompted us to examine the possibility of developing other methods suitable for the synthesis of phosphodiesters. Following up on earlier reports on the oxidative activation of phosphite triesters,18we recently developed a protocol for the synthesis of phosphate esters from trialkyl phosphites, 12, pyridine, and the target alcohol (eq l).19
As shown below (eq 21, this methodology can easily be used for the synthesis of phosphodiesters simply by starting with a dialkyl chlorophosphite. Phosphitylation CI-P(OR)z
R'OH
R'O-P(OR)z
1. 12 2. R"OH, Py
PR
R'O-Y=O OR,,
(2)
of the target alcohol gives a trialkyl phosphite that can be oxidized in situ with iodine. The resulting activated ester (the phosphoryl iodide is probably formed initially and then reacts with chloride ion to give the chlorophosphate) can then be treated with a second alcohol, phenol, or enolate to give the requisite triester. For the synthesis of complex phosphodiesters, the chemistry outlined in eq 2 provides a useful and inexpensive alternative to phosphoramidite-type syntheses. We have applied this methodology to the synthesis of two initial target compounds as shown below (Schemes 4 and 5). Starting with doubly-protecteduridine (71, treatment with diethyl chlorophosphite gives the trialkylphosphite, which was not isolated. In situ oxidation of this material, followed by the addition of p-hydroxybenzaldehyde (for the synthesis of compound 41, led to the desired triester 8 (53.8% from 7 ) in reasonable overall yield. Compound 8 is formed as a mixture of two diastereomers owing to the newly created chiral center at phosphorus. Since final deblocking of the phosphate triester destroys this chirality, the mixture was not separated but carried on (17) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993,49,10441-10488. (18) Wantanabe, Y.; Inada, E.; Jinno, M.; Ozaki, S. Tetrahedron Lett. 1993,34,497-500. (19) Stowell, J. K.; Widlanski, T. S. Tetrahedron Lett. 1995, 36, 1825-1826.
Stowell et al. 100
80 n
s 60
W
CHo
1.NaBH4 2. DAST 3. TMSBr
TBSO
80 pM
40
110 pM
2o
I
I
I
0
10
20
-D- 200pM
-+ 220vM
01
I
Time (min)
TBsoum 1
RIBUO, ,,O OEt@p\oll
RlBU =
Figure 1. Loss of enzymatic activity of RNase A during incubation with different concentrations of 4.
Scheme 6
I OTBS
in the synthesis. Inhibitor 4 was then obtained by reduction of 8 with sodium borohydride (94.9%),followed by fluorination with (diethy1amino)sulfur trifluoride (78.6%)and deblocking with TMSBr and fluoride ion as shown (72.8%). Although our most immediate concerns centered on the enzymology of the benzylic fluoride 4, we were interested in ascertaining whether the phosphorylation chemistry we had developed would be suitable for construction of the enol phosphate diesters ultimately required for the synthesis of compounds 5 and 6. A second question we wished to address concerned the possibility of regioselective dehydrobrominationof the dibromide derived from enol phosphate 9 (Scheme 5). Recently, we had reported that good regioselectivity in the elimination of halides from 1,2-dihalo phosphates could be obtained by taking advantage of complex-inducedproximity effects.llb In the current case, we were worried that the highly oxygenated nature of the nucleoside might preclude the use of such effects to guide the elimination. Enol phosphate 9 was synthesized in a manner analogous to that described for triester 8. Phosphitylation of 7 followed by oxidation and trapping of the activated phosphate ester with the enolate of acetaldehyde gave the expected enol phosphate 9 (67.4%from 7, mixture of two diastereomers) in reasonable overall yield. No attempt was made to separate these diastereomers. Bromination of 9 gave the dibromide 10 as an inseparable mixture of all four diastereomers. Gratifymgly,we found that treatment of these dibromides with LiHMDS in toluene gave the 1-bromoenol phosphate 11 (56.8%from 9, mixture of two diastereomers) as the sole product, indicating that complete regioselective control of the dehydrobromination is possible. Although the phosphitylatiodoxidatiodphosphorylation sequence shown in Schemes 4 and 5 provides a relatively facile method to construct phosphate esters with three different substituents (e.g., vinyl phosphate 9), an intrinsic drawback to this methodology is that the last coupling reaction proceeds via a phosphorochloridate, an acid halide that often couples poorly to alcohols. In contrast, we have found that phosphoryl iodides give
3. 12
TBsowra
RlBU =
I OT6S
superior yields in coupling reactions with a variety of nucleophile^.^^ When the reaction protocol shown in Schemes 4 and 5 is used, a phosphoryl iodide is generated as an obligate intermediate. Under the assumption that generating and using this iodide in the absence of chloride ion would improve the yield of phosphate esters, the initial phosphitylation reaction was run in ether. The pyridine hydrochloride that formed was filtered off and the reaction mixture evaporated to remove excess pyridine. The resulting phosphite was then dissolved in dichloromethane or tetrahydrofuran (the iodophosphate is unstable in tetrahydrofuran and must be used immediately) and the oxidatiodphosphorylation run as previously described. This simple change in procedure resulted in substantially better reaction yields. Aldehyde 8 was obtained in 72.7%yield (vs 53.8%for the procedure run in the presence of chloride ion), and vinyl phosphate 9 was obtained in 74.2%yield (vs 67.4%for the procedure run in the presence of chloride ion). In addition, the nitrophenyl ester 12,an intermediate in the synthesis of a ribonuclease substrate, was obtained in 79.3%yield (Scheme 6). Inactivationof Ribonuclease A. Compound 4 is an irreversible inactivator of RNase A. The kinetics of inactivation appear to be multiphasic (Figure 1). A n initial phase appears to reach completion within 60 s, while other phases reach completion on the time scale of min. The rate and extent of inactivation does not increase when the concentration of 4 is greater than approximately 200 pM. These data are consistent with the steady-state kinetic parameters for the enzymatic cleavage of the uridine 3'-phosphate diester with p nitrophenol, a substrate that resembles 4. For that
J. Org. Chem., Vol. 60, No. 21, 1995 6933
Mechanism-Based Inactivation of Ribonuclease A substrate, kcat= 1 9 s-l a n d K , = 330 P M . ~Enzyme that had been inactivated by 4 is not reactivated by prolonged dialysis, suggesting that the enzyme is indeed modified covalently. The inactivation of RNase A by 4 is not complete. f i r prolonged (2 h ) exposure of RNase A to saturating 4, the enzyme still retains 33% of its ability to catalyze the cleavage of UpA. Addition of a fresh aliquot of inhibitor caused no additional inactivation, suggesting that inhibition is not caused by a n advantageous impurity present at low concentrations. The fact t h a t inactivation is incomplete suggests that t h e covalent modification does not occur to Hisl2, H i s l l 9 , or Lys41, because mutation of any one of these residues to alanine causes a >lo4fold decrease in t h e catalytic activity of the enzyme.8,20 Structural and site-directed mutagenesis studies reveal which residues a r e likely to be candidates for covalent modification by 4. The three-dimensional structure of a crystalline RNase A*d(ApTpApApG) complex indicates that 4 may modify a residue in t h e enzymic B2 or P2 subsitea21 The B2 subsite, which binds to t h e adenosine base in t h e UpA substrate, consists of Gln69, Asn71, and G l u l l l . The P2 subsite, which binds to t h e phosphoryl group in a n RNA substrate proceeding t h e scissile P-05' bond, consists of Lys7 a n d ArglO. Replacing Lys7 or ArglO with an alanine or glutamine r e ~ i d u e ,or ~ Gln69 ~,~~ or G l u l l l with an alanine residue,24results in a
