Membrane-Permeant, Bioactivatable Derivatives of ... - ACS Publications

Chapter 15

Membrane-Permeant, Bioactivatable Derivatives of Inositol Polyphosphates and Phosphoinositides 1

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Carsten Schultz , Marco T. Rudolf , Hartmut H. Gillandt , and Alexis E. Traynor-Kaplan 2,3

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Institut für Organische Chemie,Abt.Bioorganische Chemie, Universität Bremen, UFT, 28359 Bremen, Germany Department of Medicine, The Whittier Institute, University of California at San Diego, La Jolla, CA 92093

Downloaded by CORNELL UNIV on October 18, 2016 | http://pubs.acs.org Publication Date: December 15, 1998 | doi: 10.1021/bk-1999-0718.ch015

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The extracellular application of membrane-permeant, bioactivatable derivatives of myo-inositol 1,4,5-trisphosphate and phosphatidyl-myo -inositol 3,4,5-trisphosphate to living cells mimicked the intracellular action of these second messengers, such as elevation of intracellular Ca -levels in PC12 cells and inhibition of calcium-mediated chloride secretion in T cells, respectively. 3,6-Di-O-butyryl-myo-inositol 1,2,4,5-tetrakisphosphate hexakis(acetoxymethyl) ester and 1',2'dipalmitoyl-6-O-butyryl-phosphatidyl-myo-inositol 3,4,5-trisphosphate heptakis(acetoxymethyl) ester were prepared by total synthesis. 2+

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The field of intracellular signal transduction is certainly one of the most rapidly expanding areas in the life sciences to date. To better understand the complex signaling pathways there is an urgent need for agents to specifically modulate intracellular levels of signaling molecules. While in the past those compounds were mostly agonists or antagonists directed towards the extracellular moiety of membrane-located receptors, many of the more recent research tools are extracellularly applicable compounds, lipophilic enough to pass through cellular membranes. Some of these tools generated a tremendous boost in particular research areas: for instance the discovery of the ATPase inhibitor thapsigargin (7) for the calcium field, or wortmannin as an inhibitor of phosphatidylinositol 3-kinase (Ptdlns 3-kinase) in tyrosine kinase-mediated signaling (2). However, a more direct manipulation of intracellular messenger levels is generally difficult because many of the known second messengers carry negative charges in the form of phosphates, which results in membrane-impermeability. This is especially true of the highly charged inositol polyphosphates of which some 20 are abundant in living cells. Not surprisingly, up to not long ago, for only one of them, Ins(l,4,5)P (1), a clear physiological function was assigned. Therefore, membrane-permeant derivatives of the naturally occurring inositol phosphates should be highly beneficial to serve as tools for studying the functions of inositol phosphates. Similarly attractive appears the possibility of synthesizing chemically modified derivatives of second messengers and converting them into membrane-permeant derivatives. These compounds should allow investigation of pharmacological properties of these derivatives in the natural environment of the living cell. This methodology should also be applicable when the 3

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Current address: Inologic Inc., 652 First Avenue South, Suite 601, Seattle, WA. 232

©1999 American Chemical Society

Bruzik; Phosphoinositides ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

233 binding proteins still remain unknown, generating the intriguing possibility to map proteins prior to their first isolation. The information extractable from these experiments might subsequently help in designing the affinity resins, photoaffinity probes or even antagonists with certain physiological functions leading to potential intracellularly acting pharmaceuticals.


Masking Hydrophilic Groups - A Prodrug Approach The introduction of masking groups for phosphates, hydroxyl or amino groups is meant to increase lipophilicity, thus allowing passive transport across plasma membranes. On one hand, the bioactivatable protecting groups should exhibit sufficient stability in extracellular media to allow application for longer periods of time. On the other hand and most importantly, they need to be substrates for intracellular endogenous enzymes like esterases or lipases to ensure a rapid cleavage of protecting groups, and hence a fast delivery of the biologically active compound. Although the products should be impermeant again after the cleavage and might therefore accumulate inside cells, the ratio of the rates of delivery and metabolism of the deprotected product is crucial for the amount of biologically active compound being generated at a particular time point. Butyrates masking hydroxyl and amino groups have been used by Posternak et al. as early as 1962 to increase the lipophilicity of the intracellular messenger c A M P (3). Butyrates were also used to mask hydroxyl groups in inositol polyphosphates. Additionally, these groups could serve as protecting groups during total synthesis, and should prevent phosphate migration during the unmasking procedure (see below). Masking negative charges is expected to have the largest effect on increasing permeability. The most widely used group for this purpose is certainly the acetoxymethyl (AM) ester group, originally employed to convert carboxylates like penicillin (4) or ion indicators (5,6) into uncharged bioactivatable derivatives. This strategy has subsequently been applied to a variety of organic phosphates (7,8) and phosphonates (9,10) including cyclic phosphate nucleotides like c A M P , cGMP and their derivatives (11-13). The challenging task of converting inositol polyphosphates into biologically active membrane-permeant derivatives was first successfully comleted in 1994. In combining butyrate masking groups for the hydroxyls and AM-esters for the phosphates, 1,2-di-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (BÎnsfe,4,5,6)?/AM) was prepared by total synthesis, and helped to establish the messenger function of Ins(3,4,5,6)P as a negative regulator of transepithelial ion fluxes in colon epithelial cells (14). In the meantime, a variety of modified derivatives of Ins(3,4,5,6)P have been used in a similar way (75), and the compounds are currently under investigation in in-vivomapping experiments as mentioned above (16,17). 4

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Membrane-Permeant Derivatives of Ins(l,4,5)P

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The potential of the AM-ester methodology can be best demonstrated on the most prominent inositol phosphate, Ins(l,4,5)P (1), with its well-defined function in regulating intracellular calcium release (18). First attempts to prepare a membranepermeant derivative of Ins(l,4,5)P resulted in the synthesis of racemic 2,3,6-tri-Obutyryl-myo-inositol 1,4,5-trisphosphate hexakis(acetoxymethyl) ester (2) (79). However, the initial success (20) in elevating intracellular C a levels ([Ca P proved to be unreliable, probably because the generated Ins(l,4,5)P , or one of its active metabolites, were metabolized faster than the respective compound could be delivered to the cytosol. The cleavage of the butyrates was suspected to be the rate3

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234 limiting step. Therefore, a derivative without butyrates, myoinositol 1,4,5trisphosphate hexakis(acetoxymethyl) ester, was prepared but proved inactive, this time probably due to its lack in exhibiting sufficient lipophilicity. Tsien and co workers finally increased the membrane-permeability by using propionoxymethyl and butyroxymethyl esters, respectively, and the resulting derivatives were very active in elevating [Ca ]j when applied extracellularly to astrocytoma cells. However, there was evidence that some phosphate migration occurred (79). 2+

Metabolically Stable, Membrane-Permeant Ins(l,4,5)P Derivative 3

A l l previous attempts were aimed at artificial elevation of the intracellular levels of authentic Ins(l,4,5)P . Most valuable should be such a derivative which is able to trigger intracellular calcium release, but is no substrate for the dominant metabolic enzymes responsible for the rapid decay of Ins(l,4,5)P . This kind of tool would generate a Ins(l,4,5)P signal free of potential effects of metabolites like Ins(l,3,4,5)P and Ins(l,3,4)P . Recently, Potter and co-workers presented the synthesis of Ins(l,2,4,5)P (3) and showed that this compound represents a very potent agonist of intracellular calcium release (27). In addition, Ins(l,2,4,5)P was neither a substrate for the 5-phosphatase nor the 3-kinase. This made Ins(l,2,4,5)P a promising candidate for conversion into a metabolically stable membranepermeant AM-ester derivative. To avoid phosphate migration we relied on butyrate functions covering the hydroxyl groups. Therefore, the desired compound was 2,6di-O-butyryl-myo-inositol l,2,4,5-octakis(acetoxymethyl) ester (Bt Ins(l,2,4,5)P / A M , 4) (Figure 1). 3


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Synthesis. The synthesis has been described recently (22). In brief, it starts from the well-known l,2:4,5-di-0-cyclohexylidene-fnyo-inositol (rac-S) as is summarized in Figure 2. Exhaustive butyrylation and subsequent cleavage of the ketals gave racemic 3,6-di-O-butyryl-myo-inositol which was phosphitylated using a classic phosphite approach, followed by oxidation to the fully protected 1,2,4,5tetrakis(dibenzyl)phosphate (rac-6). At this stage, the enantiomers 6 and ent-6 were separated by preparative H P L C on a chiral stationary phase (Chiradex, 10 μπι, Merck). It should be mentioned here that many inositol poly(dibenzyl)phosphates can be separated on this phase (unpublished results). The absolute configurations were verified by preparing the dicamphanates of 5, separating the diastereomers, and then following the synthetic pathway to the fully protected tetrakisphosphate 6. The latter and its enantiomer were deprotected, and the resulting free acids were alkylated with acetoxymethyl bromide in the presence of diisopropylethylamine in dry acetonitrile. Purification of the octakis(acetoxymethyl) esters Bt2lns(l,2,4,5)P /AM (4) and Bt Ins(2,3,5,6)P /AM (ent-4) by preparative reverse phase HPLC yielded more than 0.5 g of each enantiomer. 4

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Measurement of intracellular Ca -levels in PC12 cells. PC 12 cells were loaded with the fluorescent calcium indicator fura-2 and transferred into a cuvette for ratio measurements with excitation at 340 / 380 nm, and emission at 505 nm as described before (23). Bt2lns(l,2,4,5)P /AM in the stock solution containing D M S O and pluronic 127 (1% and 0.04 % final concentration, respectively) was added to the gently stirred cell suspension. The addition of 1.3 mM Bt2lns(l,2,4,5)P /AM (4) led to a smooth rise in [Ca 1 within about 30 min to reach a plateau of about 700 n M [Ca 1 as is shown in Figure 3. The subsequent addition of the natural agonist bradykinin (2 μΜ) gave no further effect. Smaller doses (0.9 mM) exhibited halfmaximal responses with a sharp transient after a dose of bradykinin. Control experiments with similar doses of the enantiomer Bt2lns(2,3,5,6)P /AM (ent-4) had 4

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HO 1θΡΟ(ΟΗ)

BtO I OPO(OCHOAc)

2

2

OPO(OH)

OPO(OCHOAc)

2

2

1 : lns(1,4,5)P

2

2: Bt3lns(1 A5)PÂM

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(HO)jOPO |OPO(OH)

(AcOCH,0)zOPO I OPCKOCHjOAc^

2

->^.OPO(OH) OPO(OH) 2

3: lns(1,2 4,5)P >

2

4

V 2

^-^^TOPO

OPO(OBn) (BnO) OPOj

2

2

LTO

^T^-^V.OBt

(BnO) OPO ^J0PO(OBn) 2

2

BtO OB

OPO(OBn) OPO(OBn)

(BnO)OPCK (BnO) OPO eni-S

;

2

2

2

1.H /Pd-C (10%) 2

I*

AcOCH Br, DIEA, MeCN

(AcOCH 0) OPO (AcOCH 0) OPO| I t O ^ ^ ^ OBt BtO (AcOCH 0) OPO (AcOCH 0) OPO 2

2

2

2

2

2

OPO(OCH OAc) OPO(OCH OAc)

2

2

2

2

2

2

BtO OPO(OCHj:OAc) OPO(OCH OAc)

2

2

2

2

Figure 2. A brief summary of the synthetic pathway to Bt2lns(l,2,4,5)P /AM. 4


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no effect on [Ca ]j, nor was the response to a subsequent dose of bradykinin altered. The results showed that Bt2lns(l,2,4,5)P /AM was able to enter the cells, and that intracellular endogenous enzymes deprotected the compound to give Ins(l,2,4,5)P or a biologically active derivative thereof. The constant rise in [Ca ]j suggested that Ins(l,2,4,5)P was metabolically stable inside the cells, as was verified by H P L C analysis of cell extracts (24). 4

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Membrane-Permeant Derivative of PtdIns(3,4,5)P

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Phosphatidyl-myoinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P , 7) has attracted enormous attention since its first discovery in 1988 (25). Nevertheless, its actual second messenger function still remains to be shown directly, because most of the knowledge about its potential effects results from studies with the enzyme which forms PtdIns(3,4,5)P : phosphatidylinositol 3-kinase (PI 3-kinase, 26). We therefore set out to prepare a membrane-permeant derivative which would allow to deliver PtdIns(3,4,5)P to the interior of the cell without disrupting the plasma membrane, and circumventing the tyrosine kinase receptors usually involved in activating PI 3kinase. This analog would generate a „clean" PtdIns(3,4,5)P signal and would offer the advantage to study the down-stream effect of PtdIns(3,4,5)P without the interference of other signaling pathways.


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Design. Membrane-permeability was intended to be provided by masking the phosphates with acetoxymethyl esters as was successfully shown for inositol polyphosphates (see above). Ideally, the two hydroxy groups should be converted to butyrates to avoid phosphate migration during enzymatic AM-ester hydrolysis. However, this pathway would require alkylation of 2,6-di-0-butyryl-PtdIns(3,4,5)P with acetoxymethyl bromide. We suspected that this precursor would be insoluble in moderately polar solvents (such as MeCN) necessary for the reaction. Alternatively, the AM-esters could be first generated from 2,6-di-0-butyryl-Ins(3,4,5)P , and the product then coupled with an activated diacylglycerol derivative, e.g. an activated phosphite. Unfortunately, these coupling reactions usually need the help of nucleophilic bases, and we did not trust the bis(acetoxymethyl) triester groups to survive such treatment. We therefore developed an alternative route which combines the lipid coupling and the AM-alkylation in one-pot reaction. The starting point for this pathway was our assumption that during formation of inositol phosphate acetoxymethyl ester, cyclic 5-membered-ring phosphate triesters will be formed in high yields when an unprotected hydroxy group is cw-vicinally located to the phosphate (C. Stadler, C. Schultz, unpublished data). We have shown before (27) that these cyclic phosphate are readily opened by alcohols to give predominantly the thermodynamically more stable equatorial phosphate triester (Figure 5). Therefore, we aimed for a precursor which would first allow the intermediate formation of a 1,2-cyclic phosphate acetoxymethyl ester, and could subsequently be ring-opened by diacylglycerol to give the phospholipid linkage. We chose dipalmitoylglycerol for this purpose to finally yield l\2 -dipalmitoyl-6-0-butyryl-phosphatidyl-myi?-inositol 3,4,5-trisphosphate heptakis-(acetoxymethyl) ester, DiC -6-Bt-PtdIns(3,4,5)P /AM (8, Figure 4). 3

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Synthesis. The synthetic pathway (Figure 6) started from the well-described racemic 3-0-benzyl-l,2:5,6-di-0-cyclohexylidene-myo-inositol (9). After butyrylation of the 6-hydroxy group, the ketals were removed by acidic hydrolysis. The resulting tetrol 10 was phosphorylated employing O^-dibenzyl-NJV'-diisopropylphosphoramidite followed by oxidation with peracetic acid at -40°C to give the fully protected 2,3,4,5tetrakisphosphate (11). Hydrogenolysis with Pd on carbon in glacial acetic acid



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ο

Figure 4. Structures of PtdIns(3,4,5)P and its membrane-permeant derivative DiC -6-Bt-PtdIns(3,4,5)P /AM. 3

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ο W Ο-^Ί "°

JC

Ο

Ρ

OH

/

O-R

2

O-R

1


O

R = AM ester 1

R*BDAG

Figure 5. Cyclic intermediates generated by the introduction of AM-esters allow the subsequent lipid / inositol phosphate coupling.

~?

1.Bt-0,DMAP

OPO(OBn) (BnOJ.OPOÂ^ H Pd/C

O H

2

2f

Ο'Ύ^ΟΗ

HO^YÔBt OH

(BnOÔPO" γ OBt OPO(OBn)

1 0

n

2

Ο

ΟΡΟίΟΗ),

Γ

(HOfeOPO^ (HOÔPO'^YÔBt OPCKOH), 1 2

Ο

Ρ-ο^ο^

dlpalmitoylglycerol

1

(AcOCHjOfeOPOÂÔ OBt OPO(OCHOAc) (AcOCHjOÔPO^Y^î 2

2

(AcOCHÔPO^Xô' (AcOCHÔPO'

Ό ^ OPO(OCHOAc) 2

2

Figure 6. Synthetic pathway to DiC -6-Bt-PtdIns(3,4,5)P /AM. 16

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240 afforded the free acid of the tetrakisphosphate (12). This compound has the desired free hydroxy group at the 1-position, ready to receive the cyclizing 2-O-phosphate. The alkylation with acetoxymethyl bromide in dry M e C N in the presence of an excess of diisopropylethylarnine (DDEA) was performed in a N M R tube to follow the reaction by P N M R . Formation of the expected pair of diastereomers could be monitored by appearance of resonances at 13.7 and 14.3 ppm. After 7 hours no more changes were observed and the mixture was transferred into a dry flask. The volatile compounds including DIEA were removed and the oily residue was dissolved in toluene. The excess of 1,2-dipalmitoyl-sw-glycerol fSigma) was added and the mixture was stirred for 24 h at 4°C. The subsequent T N M R analysis showed no more signal in the 14 ppm region. The mixture was brought to dryness and the residue was extracted with toluene and hexane to give the desired DiC -6-BtPtdIns(3,4,5)P /AM (8) in moderate yield. A l l compounds were examined by N M R and F A B - M S . 31

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Membrane-permeant PtdIns(3,4,5)P derivatives mimic E G F in its effect of inhibiting carbachol induced CI*-secretion of T cells. It has been shown before that epidermal growth factor (EGF) inhibits calcium-mediated chloride secretion (CaMCS) in T cells, a human colonic epithelial cell line (28). This effect is most likely mediated by the activation of Ptdlns 3-kinase, because the well-known PtdJns 3-kinase inhibitor wortmannin abolished the inhibitory response (29). We therefore chose the well-established Cl-secretion assay to investigate whether our membranepermeant PtdIns(3,4,5)P derivative could mimic the effect of E G F mentioned above. T cells, grown to confluency on snap-well inserts, were preincubated with DiC -6Bt-PtdIns(3,4,5)P /AM (8) for 30 min. To avoid solubility problems, 8 was dissolved in D M S O containing 4% pluronic 127 (BASF, Germany) prior to use. The final DMSO concentration did not exceed 1%. After mounting the snap-wells into Ussing chambers chloride, secretion was monitored as short circuit current (Δΐ^) as described before (30,31). Stimulation by the muscarinic agonist carbachol (100 μΜ) 10 min after mounting resulted in a transient elevation of Cl-secretion (Figure 7). Cells preincubated with 100 μΜ DiC, -6-Bt-PtdIns(3,4,5)P /AM showed only an about 50% response to carbachol indicating that CaMCS is inhibited by D i C PtdIns(3,4,5)P , or by one of its active metabolites generated through the intracellular hydrolysis of the bioactivatable protecting groups. Hence, we can conclude that the E G F signaling pathway employs PtdIns(3,4,5)P (or one of its metabolites) as a second messenger in T cells. In control experiments neither 1,2dipalmitoyl-jw-glycerol nor PtdIns(3,4,5)P itself exhibited any effect on CaMCS (data not shown). In a more detailed study we showed that compounds with shorter fatty acid chains such as lauroyl or octanoyl were equally potent, and that the effect of E G F , as well as that of DiC -6-Bt-PtdIns(3,4,5)P /AM, could be reversed by doses of a membrane-permeant derivative of Ins(l,4,5,6)P , thus restoring C l secretion (32). Unfortunately, we cannot exclude the possibility that the signal generated by DiC -6-Bt-PtdIns(3,4,5)P /AM is mediated by a downstream metabolite of PtdIns(3,4,5)P . Therefore, membrane-permeant derivatives of other 3-phosphorylated phosphatidylinositides would be highly desirable for future experiments. 3

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Acknowledgment We are in indebted to the Deutsche Forschungsgemeinschaft and the NIH (to A.E.T.) for financial support.



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Figure 7. Extracellular doses of DiC -6-Bt-PtdIns(3,4,5)P /AM inhibit chloride secretion of cells, thus mimicking the effect of EGF. 16

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242 Literature Cited 1. 2. 3. 4. 5. 6. 7.


8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Thastrup, O.; Linnebjerg, H.; Bjerrum, P. J.; Knudson, J. B.; Christensen, S. B . Biochem. Biophys. Acta 1987, 927, 65-73. U i ,M.;Okada, T.; Hazeki, K.; Hazeki, O. Trends Biochem. Sci. 1995, 303-307. Posternak, T.; Sutherland, E. W.; Denion, W. F. Biochem. Biophys. Acta 1962, 65, 558-564. Jansen, Α. Β. Α.; Russell, T. J. J. Chem. Soc. 1965, 2127-2132. Tsien, R. Y . Nature 1981, 290, 527-528. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 3440-3450. Sastry, J. K.; Nehete, P. N.; Khan, S.; Nowak, B . J.; Plunkett, W.; Arlinghaus, R. B.; Farquhar, D. Mol. Pharmacol. 1992, 41, 441-445. Freed, J. J.; Farquhar, D.; Hompton, A . Biochem. Pharmacol. 1989, 38, 31933198. Iyer, R. P.; Phillips, L . R.; Biddle, J. Α.; Thakker, D. R.; Egan, W.; Aoki, S.; Mitsuga, H . Tetrahedron Lett. 1989, 30, 7141-7144. Saperstein, R.; Vicario, P. P.; Strout, Η. V . ; Brady, E.; Slater, Ε. E.; Greenlee, W. J.; Ondeyka, D. L.; Patchett, Α. Α.; Hangauer, D. G. Biochemistry 1989, 28, 5694-5701. Schultz, C.; Vajanaphanich, M.; Barrett, Κ. E.; Sammak, P. J.; Harootunian, A . T.; Tsien, R. Y . J. Biol. Chem. 1993, 268, 6316-6322. Schultz, C.; Vajanaphanich, M.; Genieser, H.-G.; Jastorff, B.; Barrett, Κ. E.; Tsien, R. Y . Mol. Pharmacol. 1994, 46, 702-708. Kruppa, J.; Keely, S. J.; Schwede, F.; Schultz, C.; Barrett, Κ. E.; Jastorff, B . Bioorg. Med. Chem. Lett. 1997, 7, 945-948. Vajanaphanich, M.; Schultz, C.; Rudolf, M. T.; Wasserman, M.; Enyedi, P.; Craxton, Α.; Shears, S. B.; Tsien, R. Y . ; Barrett, Κ. E.; Traynor-Kaplan, A . E. Nature 1994, 371, 711-714. Roemer, S.; Stadler, C.; Rudolf, M. T; Jastorff, B.; Schultz, C. J. Chem. Soc. Perkin Trans. I, 1683-1694. Rudolf, M . T.; Li, W.; Wolfson, N . ; Traynor-Kaplan A . E . ; Schultz, C. submitted. Rudolf, M. T.; Wolfson, N . ; Traynor-Kaplan, A . E.; Schultz, C. submitted. Berridge, M. J., Nature 1993, 361, 315-325. L i . W.; Schultz, C.; Llopis, J.; Tsien, R. Y . Tetrahedron 1997, 53, 12017-12040. Schultz, C.; Tsien, R. Y . FASEB J. 1992, 6(5), Abstract A1924. Mills, S. J.; Safrany, S. T.; Wilcox, R. Α.; Nahorski, S. R.; Potter, Β. V . L . Bioorg. Med. Chem. Lett. 1993, 3, 1505-1510. Gillandt, Η. H.; Berg, I.; Guse, A . H.; Mayr, G. W.; Schultz, C. submitted. Benters, J.; Schäfer, T.; Beyersmann, D.; Hechtenberg, S. Cell Calcium 1996, 20, 441-446. Berg, I.; Guse, A . H.; da Silva, C. P.; Gillandt, H . H.; Schultz, C.; Mayr, G . W. in preparation. Traynor-Kaplan, A . E.; Harris, A . L.; Thompson, B . L . ; Taylor, P.; Sklar, L . Nature 1988, 334, 353-356. Toker, Α.; Cantley, L . C. Nature 1997, 387, 673-676. Schultz, C.; Metschies, T.; Gerlach B.; Stadler, C.; Jastorff, B . Synlett 1990, 1, 163-165. Uribe, J. M.; Gelbmann J. M.; Traynor-Kaplan, A . E.; Barrett, Κ. E. Am. J. Physiol. 1996, 271, C914-C922. Uribe, J. M.; Keely, S. J.; Traynor-Kaplan, A . E.; Barrett, Κ. E. J. Biol. Chem. 1996, 271, 26588-26595. Dharmsathaphorn, K.; Pandol, S. J. J. Clin. Invest., 1986, 77, 348-354.


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31. Wasserman, S. I.; Barrett, Κ. Ε.; Huott, Ρ. Α.; Beuerlein, G.; Kagnoff, M.; Dharmsathaphorn, K. Am. J. Physiol 1988, 254, C53-C62. 32. Eckmann, L.; Rudolf, M. T.; Ptasznik, Α., Schultz, C.; Jiang, T.; Wolfson, N.; Tsien, R. Y., Fierer, J.; Shears, S. B.; Kagnoff, M. F.; Traynor-Kaplan, A . E . Proc. Natl. Acad. Sci. USA 1997, in press.


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