A ballistic semiconductor device of the present invention comprises a n-type emitter layer (102), a base layer (305) made of n-type InGaN, a n-type collector layer (307), an emitter barrier layer (103) interposed between the emitter layer (102) and the base layer (305) and having a band gap larger than that of the base layer (305), and a collector barrier layer (306) interposed between the base layer (305) and the collector layer (307) and having a band gap larger than that of the base layer (305), and operates at 10 GHz or higher.

Several semiconductor devices have been proposed as a hot electron transistor (HET) using hot electrons. A first prior art is a resonant-tunneling hot electron transistor device (RHET) proposed by Yokoyama et al. (see Japanese Journal of Applied Physics Letters, Vol. 24, No. 11, p. L853, (1985))
FIG. 11 and FIGS. 12(a), 12(b), and 12(c) are views showing a device structure and a principle of operation shown in FIGS. 1 and 3 of the cited reference by Yokoyama et al. In the first prior art, after an AlGaAs layer 21 is grown on a n+-GaAs substrate 20 to be 300 nm thick, a Si doped n+-GaAs layer 22, an AlGaAs barrier layer 23, a GaAs well layer 24, an AlGaAs barrier layer 25, and a Si doped n+-GaAs layer 26 are grown to be 100 nm, 5 nm, 5.6 nm, 5 nm, and 50 nm, respectively.
The first prior art comprises a resonant-tunneling structure within an emitter region of the HET, and a device operation at 77K has been reported. The operation is as follows. When a base 12 and an emitter 14 are equipotential, electron energy of the emitter is lower than a quantum level (E1) of a quantum well 13 provided between the emitter 14 and the base 12 as shown in FIG. 12(a), so that no current flows through the emitter.
Upon a voltage being applied between the base and the emitter, the electron energy of the emitter conforms to the quantum level of the quantum well as shown in FIG. 12(b), thereby generating a resonant-tunneling effect.
More specifically, the electron energy of the emitter has a certain distribution, and, only electrons having energy that conforms to the quantum level are emitted to the base by the resonant-tunneling effect. Since the emitted electrons have high energy, they pass through the base layer at a high speed substantially without scattering (ballistic conduction), go over an energy barrier (qΦc) between a base layer and a collector barrier layer, and are injected into the collector barrier layer.
The injected electrons travel through the collector barrier layer substantially without scattering, and reach a collector layer. Since the electrons are not substantially scattered throughout the above-described process, it is expected that the device operates at a speed higher than that of a general transistor device depending on scattering and diffusion.
However, the first prior art has a problem that an operating temperature is limited at a room temperature, device gain is small, and an operation speed is not so high as expected. For example, in the first prior art, operation at 77K is reported, but the operation at a room temperature and improvement of the operation speed is not described.
Accordingly, an InP based HET operating at a room temperature (second prior art) has been reported (see IEEE Electron Device Letters, Vol. 14, No. 9, pp. 441-443, September, (1993)).
However, the second prior art has a problem that the device gain is smaller than that of a general transistor, and the operation speed is not especially high.

Matsushita Electric Industrial Co., Ltd.


A circuit having a first circuit portion configured to receive a pulse-width modulated first signal and a second signal, and configured to generate third and fourth signals each responsive to the first and second signals; a second circuit portion configured to receive the third and fourth signals and to generate a fifth signal responsive to both the third and fourth signals; and a third circuit portion configured to control an on/off state of a first switch in response to the fifth signal, wherein the second signal is present at a load path terminal of the first switch. Also, various related methods.

Switching regulators with half-bridge circuits commonly use a bootstrap circuit having a diode and a bootstrap capacitor connected in series. The bootstrap circuit is typically connected to a node between a high-side transistor and a low-side transistor. To minimize chip size, the size of the bootstrap capacitor is kept small, thereby also keeping the capacitance small. With the reduced capacitance of the bootstrap capacitor, the power consumption of the floating logic being supplied by the bootstrap capacitor becomes more relevant. Ideally, to alleviate the problems associated with a small bootstrap capacitance, the charge stored in the boot strap capacitor should be maximized and the power consumption of the logic circuits supplied by the bootstrap capacitor should be minimized.
The charge stored in the boot strap capacitor can be increased if the diode connected between the supply voltage and the bootstrap capacitor is replaced by a device having a lower voltage drop than a silicon diode. For this reason it is known to employ a Schottky diode instead of a silicon diode. However, this approach is unfavorable, because when using common chip fabrication processes Schottky diodes cannot be easily integrated and so have to be realized as an external component.
For various reasons, the high-side transistor is not driven directly by a level-shifted version of an input signal, but rather via a floating latch which is turned on and turned off by short pulses. In order to reduce a degradation of the signal due to noise, low resistor values are used in the level-shifter resulting in a high load of the bootstrap capacitor. The average load of the bootstrap capacitor can be reduced by triggering only short pulses which are then level shifted. This level shifted pulses trigger the floating latch, thus restoring the signal controlling the switching state of the high-side switch.
There is a general need for a circuit arrangement and for a method for driving a high-side transistor. Such a circuit arrangement may allow for a relatively simple solution which may provide a fail-safe operation without the necessarily using additional refresh circuits, and may further allow integration into a single chip without necessarily using external components.

Circuits and methods for controlling an on/off state of a switch


The present disclosure pertains to a system configured to protect flows in piping systems using minimal spare components. Some embodiments may provide: a first piping subsystem configured to receive a portion of the input flow; a second piping subsystem configured to receive the portion of the input flow by substituting for the first subsystem; a test subsystem configured to detect whether each of the first and second subsystems is able to vent when at least one, in the each subsystem, of a respective pressure and a respective pressure rate satisfies first and second criteria, respectively; and first and second pilots configured to detect a maximum pressure and a maximum pressure rate, respectively, of the portion of the first and second subsystems.

Guards against system damage associated with fluid processing, transfer, and storage, such as pressure surges (e.g., water hammer or other scenarios comprising pressurized fluids), are known. For example, rates of change that are too great or too little in an enclosed flow of a fluid may dangerously affect pressure. Surge of pressure can be generated by any system component. Known surge-relief systems implement valves to relieve, dissipate, or otherwise attenuate excessive pressure transients to a storage vessel, fluid of which being potentially returned to the product line. Known systems also may comprise costly spare subsystems that run parallel to each of the piping runs, thus inefficiently requiring much spare equipment.

This patent is subject to a terminal disclaimer.

Systems and methods for providing surge relief


A fixed pattern noise removal method for an image sensor includes steps of calculating each compensation value corresponding to each pixel column according to a plurality of compensation pixel values of the each pixel column; and sampling a plurality of active pixel values in an active pixel area and compensating the plurality of active pixel values according to the each compensation value of the each pixel column, to generate a plurality of compensated active pixel values. A plurality of active pixels sense light to generate the plurality of active pixel values.

1. Field of the Invention
The present invention relates to a fixed pattern noise removal method, and more particularly, to a fixed pattern noise removal method in an image sensor which only needs to calculate each compensation value corresponding to each pixel column for compensation, so as to save memory usage and reduce computation complexity.
2. Description of the Prior Art
Generally for an image sensor, the pixels used for image sensing are not uniform in dimension, spacing, and efficiency. Also, a row sampler such as the correlation double sampling (CDS) sampling active pixel values row by row may result in deviations. That is, a specific sampler is used for a specific column pixel, and thus if samplers in a row are not uniformly designed or fabricated, sampled pixel values of pixel columns may have deviation, which causes noise in vertical lines of the image due to the sampling non-uniformity. Thus, the image captured by the image sensor may have a non-random fixed pattern noise (FPN) induced by the drawback of the hardware architecture.
For example, please refer to FIG. 1A and FIG. 1B, which illustrate schematic diagrams of a real image IMG and a noise image NIMG with a column fixed pattern noise, respectively. As shown in FIG. 1A and FIG. 1B, since the specific sampler is used for a specific column pixel, and samplers in a row are not uniformly designed and fabricated, the sampled pixel values of the pixel columns have fixed pattern noises in vertical lines of the image (the vertical stripes as shown in FIG. 1B) due to the sampler non-uniformity.
In such a situation, a conventional technology takes a real picture and an optical black picture separately under the same condition, and then subtracts the optical black picture from the real picture to remove the fixed pattern noise. However, this method requires the image sensor to record and store the whole optical black picture for different parameter control (e.g., temperature, gain value, exposure time), which consumes a huge memory. Thus, there is a need for improvement of the prior art.


A refrigeration apparatus includes an air chiller, a storage enclosure defining a compartment, a duct system, and a valve system. The air chiller blows chilled air into the duct system. The compartment has a first and a second opening, each of which is coupled to the duct system. The valve system has valves that can be moved to route the chilled air so that it enters into the first opening and exits the second opening, or vice versa. In one implementation, the first opening is at the top of the compartment and the second opening is at the bottom of the compartment, and the valve system is controlled by a control circuit that periodically switches the valves (via an actuator) to change the direction of the chilled air. This effectively maintains a relatively uniform temperature throughout the compartment.

Maintaining a relatively uniform temperature is important in any refrigeration system, but it is particularly important in the context of food and beverage refrigeration. Without proper temperature distribution, some food in a refrigerator will be too cold, resulting in unwanted freezing and some will be too warm, which raises the risk of spoilage. In most contexts, a uniform temperature is not only desirable, but is mandated by regulations. For example, depending upon the aircraft operating authority certain types of food served on passenger airlines is required to be maintained at a maximum temperature of no more than 7° C. and in some countries 4° C.
Typically, pre-prepared airline food is stored in galley carts prior to serving to passengers. However, current galley cooling systems have to force air just above freezing either into the galley carts or into insulated compartments containing several galley carts just to ensure that the temperature does not exceed the required temperature in any portion of the carts. This is due to the temperature increase as the air passes through or over the galley carts to remove the heat entering the galley cart or compartment. The lower maximum temperature requirement of 4° C. means that the current cold air source is less efficient resulting in the need to use more powerful and heavier systems that use more electrical power. Thus, it can be seen that there is a need for a new method and apparatus for maintaining a uniform temperature in a refrigeration system.

Method and apparatus for maintaining a uniform temperature in a refrigeration system


A lancet integrated test element (LIT) includes an incision forming member that has a cutting end configured to form an incision in tissue. A test element is attached to the incision forming member to test fluid from the incision. The test element has a sampling end with a sample opening through which the fluid is collected. The test element is bendable from a first state where the cutting end of the incision forming member is retracted from the sampling end of the test element to a second state where at least a portion of the cutting extends past the sampling end of the test element to form the incision in the tissue.

The present invention generally relates to bodily fluid sampling devices and more specifically, but not exclusively, concerns a lancet integrated test element with a flexible test element that bends during lancing in order to allow a lancet to pierce the skin or other tissue.
The acquisition and testing of bodily fluids is useful for many purposes and continues to grow in importance for use in medical diagnosis and treatment, such as for diabetes, and in other diverse applications. In the medical field, it is desirable for lay operators to perform tests routinely, quickly, and reproducibly outside of a laboratory setting, with rapid results and a readout of the resulting test information. Testing can be performed on various bodily fluids, and for certain applications is particularly related to the testing of blood and/or interstitial fluid. Such fluids can be tested for a variety of characteristics of the fluid, or analytes contained in the fluid, in order to identify a medical condition, determine therapeutic responses, assess the progress of treatment, and the like.
The testing of bodily fluids basically involves the steps of obtaining the fluid sample, transferring the sample to a test device, conducting a test on the fluid sample, and displaying the results. These steps are generally performed by a plurality of separate instruments or devices. Performing these steps can be difficult for patients, especially for patients with limited hand dexterity, such as the elderly, or those suffering the affects of their condition, like diabetes. Diabetics suffer many symptoms that can make self-monitoring difficult. For example, diabetics can sometimes experience numbness or tingling in their extremities, such as their hands, and also wounds tend to heal more slowly for diabetics. In a typical procedure, the patient first creates an incision in the skin by lancing the skin with a lancet. In order to ensure that a sufficient number of capillaries are cut for supplying an adequate bodily fluid sample, the incision has to usually be deep, which can be rather painful for the patient. Often the incision still does not provide an adequate amount bodily fluid for the sample, and the patient then must resort to expressing the fluid from the incision. If during expression of the fluid the patient is not careful, smearing of the fluid can occur, which may result in rendering the sample useless. Once a sufficient amount of fluid collects as a droplet on the skin, the patient has to position a test strip over the site such that the test strip contacts and absorbs a sufficient amount of the droplet for testing. Usually the droplet of fluid is quite small, and patients, especially ones with hand motor control problems, may experience great difficulty in positioning the test strip so as to collect a sample from the droplet. As should be appreciated, patients can become frustrated by this procedure, and consequently, they may perform the test less often or may even quit testing altogether.
Recently, lancet integrated test elements, or LITs, have been developed in which a test strip is integrated with a lancet so as to form a single disposable unit. While these integrated units have somewhat simplified the collection and testing of fluid samples, there are still a number of issues that need to be resolved before a commercial unit can be implemented. One issue concerns the interaction between the lancet and the test strip during fluid collection. In one type of design, the lancet is fixed relative to the test strip and extends past the edge of the test strip. During lancing, the entire integrated lancing test strip is fired by a lancing mechanism to form an incision, and after forming the incision, the entire integrated lancing test strip is typically retracted from the skin so that the blade is removed from the incision in order to promote blood flow as well as to dull the pain.
With the lancet fixed relative to the strip, a number of difficulties in sampling the fluid are created. For instance, as noted before, the lancet typically extends from the test strip near the capillary opening for the test strip. At such a position, the blade of the lancet can interfere with the collection of body fluid by smearing the droplet of blood on the skin and/or by drawing blood away from the capillary channel. Further, the distance that the capillary has to be retracted is directly proportional to the length of the lancet blade that extends from the test strip. The greater penetration depth created by longer lancet blades usually increases the amount of blood that is bled from the incision, but the greater length of the lancet necessitates that the test strip be retracted farther away from the skin, which in turn can reduce the chances that the blood will be successfully drawn into the capillary channel of the test strip. Conversely, shorter lancets reduce the distance of the test strip from the skin, but shorter lancets normally produce smaller fluid sample sizes from the incision. Moreover, retraction of the entire integrated device is sometimes inconsistent, thereby leading to some undesirable consequences. If the integrated device is retracted too far from the skin, the capillary channel might not be able to contact the fluid droplet on the skin, thereby resulting in an incomplete test or insufficient sample size for testing. The fixed lancet can also interfere with fluid collection because the fluid will tend to wick up the lancet during fluid collection.
To alleviate some of these difficulties, LITs have been developed in which the lancet is moveable relative to the test element. In one typical design, the lancet is coupled to a test strip via a metal spring. After the lancet is fired and the incision is formed, the spring is used to retract the lancet from the incision. However, such designs still have a number of drawbacks. For example, the flexible nature of the spring can complicate manufacturing by creating greater component variance when aligning the lancet with the test element. Also, packaging and maintaining the sterility of the lancet and test strip is complicated by the spring. With the spring, the lancet can easily extend, which can lead to accidental cuts and exposure of the lancet. Moreover, forming the spring adds expense to manufacturing, and the spring can be prone to damage during handling. If the spring is damaged or the lancet is misaligned, the lancet might not fully retract, which can lead to a number of difficulties. For instance, part of the lancet might remain in the incision after lancing, which can be quite painful to the user. Even when the lancet does not remain in the incision, the lancet tip can still extend past the end of the test element so as to disrupt fluid collection. With the lancet tip extending past the end of the test element, the body or biological fluid, such as blood, will tend to first wick up the lancet, thereby wasting the fluid. Due to the rigid nature of the test strip, if the test strip is pressed too hard against the skin, fluid flow from the incision can become constricted. Thus, needs remain for further contributions in this area of technology.


Sequence-specific oligonucleotides are provided having substantially pure chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp phosphotriester, and chiral Rp phosphotriester linkages. The novel oligonucleotides are prepared via a stereospecific SN2 nucleophilic attack of a phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate anion on the 3xe2x80x2 position of a xylonucleotide. The reaction proceeds via inversion at the 3xe2x80x2 position of the xylo reactant species, resulting in the incorporation of phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate linked ribofuranosyl sugar moieties into the oligonucleotide.

This invention is directed to sequence-specific oligonucleotides having chiral phosphorus linkages and to a novel chemical synthesis of these and other oligonucleotides. The invention includes chiral alkylphosphonate, chiral phosphotriester, and chiral phosphoramidate-linked oligonucleotides. The invention further includes chiral phosphorothioate, chiral alkylphosphonate, chiral phosphotriester, and chiral phosphoramidate-linked oligonucleotides that contain at least one modified nucleoside unit. The novel chemical synthesis provides such chiral phosphorothioate, chiral alkylphosphonate, chiral phosphotriester, and chiral phosphoramidate oligonucleotides as well as xe2x80x9cnaturalxe2x80x9d or xe2x80x9cwild typexe2x80x9d phosphodiester oligonucleotides.
Messenger RNA (mRNA) directs protein synthesis. As a therapeutic strategy, antisense therapy strives to disrupt the synthesis of target proteins by using a sequence-specific oligonucleotide to form a stable heteroduplex with its corresponding mRNA. Such antisense oligonucleotides generally have been natural phosphodiester oligonucleotides.
As contrasted to natural phosphodiester oligonucleotides, the use of phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides in antisense therapy provides certain distinguishing features. Each of the phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate phosphorus linkages can exist as diastereomers. Certain of these phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides have a greater resistance to nucleases. Some have solubilities similar to the solubility of natural phosphodiester oligonucleotides. Other have solubilities different from that of the natural phosphodiester oligonucleotides. Some are generally more chemically or thermodynamically stable than the natural phosphodiester oligonucleotides. At least the phosphorothioates have oligonucleotide-RNA heteroduplexes that can serve as substrates for endogenous RNase H.
The phosphorothioate oligonucleotides, like the natural phosphodiester oligonucleotides, are soluble in aqueous media. In contrast, methylphosphonate, phosphotriester, and phosphoramidate oligonucleotides, which lack a charge on the phosphorus group, can penetrate cell membranes to a greater extent and, thus, facilitate cellular uptake. The internucleotide linkage in methylphosphonate oligonucleotides is more base-labile than that of the natural phosphodiester internucleotide linkage, while the internucleotide linkage of the phosphorothioate oligonucleotides is more stable than the natural phosphodiester oligonucleotide linkage.
The resistance of phosphorothioate oligonucleotides to nucleases has been demonstrated by their long half-life in the presence of various nucleases relative to natural phosphodiester oligonucleotides. This resistance to nucleolytic degradation in vitro also applies to in vivo degradation by endogenous nucleases. This in vivo stability has been attributed to the inability of 3xe2x80x2-5xe2x80x2 plasma exonucleases to degrade such oligonucleotides. Phosphotriester and methylphosphonate oligonucleotides also are resistant to nuclease degradation, while phosphoramidate oligonucleotides show some sequence dependency.
Since they exist as diastereomers, phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides synthesized using known, automated techniques result in racemic mixtures of Rp and Sp diastereomers at the individual phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkages. Thus, a 15-mer oligonucleotide containing 14 asymmetric linkages has 214, i.e. 16,384, possible stereoisomers. Accordingly, it is possible that only a small percentage of the oligonucleotides in a racemic mixture will hybridize to a target mRNA or DNA with sufficient affinity to prove useful in antisense or probe technology.
Miller, P. S., McParland, K. B., Jayaraman, K., and Ts""o, P. O. P (1981), Biochemistry, 20:1874, found that small di-, tri- and tetramethylphosphonate and phosphotriester oligonucleotides hybridize to unmodified strands with greater affinity than natural phosphodiester oligonucleotides. Similar increased hybridization was noted for small phosphotriester and phosphoramidate oligonucleotides; Koole, L. H., van Genderen, M. H. P., Reiners, R. G., and Buck, H. M. (1987), Proc. K. Ned. Adad. Wet., 90:41; Letsinger, R. L., Bach, S. A., and Eadie, J. S. (1986), Nucleic Acids Res., 14:3487; and Jager, A., Levy, M. J., and Hecht, S. M. (1988), Biochemistry, 27:7237. The effects of the racemic diastereomers on hybridization becomes even more complex as chain length increases.
Bryant, F. R. and Benkovic, S. J. (1979), Biochemistry, 18:2825 studied the effects of diesterase on the diastereomers of ATP. Published patent application PCT/US88/03634 discloses dimers and trimers of 2xe2x80x2-5xe2x80x2-linked diastereomeric adenosine units. Niewiarowski, W., Lesnikowski, Z. J., Wilk, A., Guga, P., Okruszek, A., Uznanski, B., and Stec, W. (1987), Acta Biochimica Polonia, 34:217, synthesized diastereomeric dimers of thymidine, as did Fujii, M., Ozaki, K., Sekine, M., and Hata, T. (1987), Tetrahedron, 43:3395.
Stec, W. J., Zon, G., and Uznanski, B. (1985), J. Chromatography, 326:263, have reported the synthesis of certain racemic mixtures of phosphorothioate or methyphosphonate oligonucleotides. However, they were only able to resolve the diastereomers of certain small oligomers having one or two diastereomeric phosphorus linkages.
In a preliminary report, J. W. Stec, Oligonucleotides as antisense inhibitors of gene expression: Therapeutic implications, meeting abstracts, Jun. 18-21, 1989, noted that a non-sequence-specific thymidine homopolymer octomerxe2x80x94i.e. a (dT)8-mer, having xe2x80x9call-except-onexe2x80x9d Rp configuration methylphosphonate linkagesxe2x80x94formed a thermodynamically more stable hybrid with a 15-mer deoxyadenosine homopolymerxe2x80x94i.e. a d(A)15-merxe2x80x94than did a similar thymidine homopolymer having xe2x80x9call-except-onexe2x80x9d Sp configuration methylphosphonate linkages. The hybrid between the xe2x80x9call-except-onexe2x80x9d Rp (dT)8-mer and the d(A)15-mer had a Tm of 38xc2x0 C. while the Tm of the xe2x80x9call-except-onexe2x80x9d Sp (dT)8-mer and the d(A)15-mer was  less than 0xc2x0 C. The hybrid between a (dT)8-mer having natural phosphodiester linkages, i.e. octathymidylic acid, and the d(A)15-mer was reported to have a Tm of 14xc2x0 C. The xe2x80x9call-except-onexe2x80x9d thymidine homopolymer octamers were formed from two thymidine methylphosphonate tetrameric diastereomers linked by a natural phosphodiester linkage.
To date, it has not been possible to chemically synthesize an oligonucleotide having more than two adjacent, chirally pure phosphorous linkages. Indeed, even in homopolymers it has been possible to produce only three such adjacent chiral linkages. For an oligonucleotide to be useful as an antisense compound, many nucleotides must be present. While not wishing to be bound by any particular theory, it is presently believed that generally at least about 10 or more nucleotides are necessary for an oligonucleotide to be of optimal use as an antisense compound. Because it has not been possible to resolve more than two or three adjacent phosphorus linkages, the effects of induced chirality in the phosphorus linkages of chemically synthesized antisense oligonucleotides has not been well assessed heretofore.
Except as noted above, the sequence-specific phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides obtained utilizing known automated synthetic techniques have been racemic mixtures. Indeed, it was recently stated in a review article that: xe2x80x9cIt is not yet possible to synthesize by chemical means diastereomerically pure chains of the length necessary for antisense inhibition,xe2x80x9d see J. Goodchild (1990) Bioconjugate Chemistry, 1:165.
The use of enzymatic methods to synthesize oligonucleotides having chiral phosphorous linkages has also been investigated. Burgers, P. M. J. and Eckstein, F. (1979), J. Biological Chemistry, 254:6889; and Gupta, A., DeBrosse, C., and Benkovic, S. J. (1982) J. Bio. Chem., 256:7689 enzymatically synthesized diastereomeric polydeoxyadenylic acid having phosphorothioate linkages. Brody, R. S. and Frey, P. S. (1981), Biochemistry, 20:1245; Eckstein, F. and Jovin, T. M. (1983), Biochemistry, 2:4546; Brody, R. S., Adler, S., Modrich, P., Stec, W. J., Leznikowski, Z. J., and Frey, P. A. (1982) Biochemistry, 21: 2570-2572; and Romaniuk, P. J. and Eckstein, F. (1982) J. Biol. Chem., 257:7684-7688 all enzymatically synthesized poly TpA and poly ApT phosphorothioates while Burgers, P. M. J. and Eckstein, F. (1978) Proc. Natl. Acad. Sci. USA, 75: 4798-4800 enzymatically synthesized poly UpA phosphorothioates. Cruse, W. B. T., Salisbury, T., Brown, T., Cosstick, R. Eckstein, F., and Kennard, O. (1986), J. Mol. Biol., 192:891, linked three diastereomeric Rp GpC phosphorothioate dimers via natural phosphodiester bonds into a hexamer. Most recently Ueda, T., Tohda, H., Chikazuni, N., Eckstein, R., and Watanabe, K. (1991) Nucleic Acids Research, 19:547, enzymatically synthesized RNA""s having from several hundred to ten thousand nucleotides incorporating Rp diastereomeric phosphorothioate linkages. Enzymatic synthesis, however, depends on the availability of suitable polymerases that may or may not be available, especially for modified nucleoside precursors.
Thus, while phosphorothioate, alkylphosphonate, phosphoamidate, and phosphotriester oligonucleotides have useful characteristics, little is known concerning the effects of differing chirality at the phosphorus linkages. It would therefore be of great advantage to provide oligonucleotides having phosphorous linkages of controlled stereochemistry.
Accordingly, it is one object of this invention to provide sequence-specific oligonucleotides having chirally pure phosphorothioate, alkylphosphonate, phosphotriester or phosphoramidate linkages.
It is a further object to provide phosphorothioate, alkylphosphonate, phosphoramidate, and phosphotriester oligonucleotides comprising substantially all Rp or all Sp linkages.
It is another object to provide phosphorothioate, alkylphosphonate, phosphoramidate, and phosphotriester oligonucleotides that have antisense hybridizability against DNA and RNA sequences.
It is still another object of this invention to provide phosphorothioate, alkylphosphonate, phosphoramidate, and phosphotriester oligonucleotides for use in antisense diagnostics and therapeutics.
A further object is to provide research and diagnostic methods and materials for assaying bodily states in animals, especially diseased states.
Another object is to provide therapeutic and research methods and materials for the treatment of diseases through modulation of the activity of DNA and RNA.
It is yet another object to provide new methods for synthesizing sequence-specific oligonucleotides having chirally pure phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkages.
The present invention provides stereoselective methods for preparing sequence-specific oligonucleotides having chiral phosphorous linkages. In certain preferred embodiments, these methods comprise the steps of:
(a) selecting a first synthon having structure (1):
wherein:
Q is O or CH2;
RA and RB are H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
RD is O, S, methyl, alkoxy, thioalkoxy, amino or substituted amino;
RE is O or S;
RX is H, OH, or a sugar derivatizing group;
BX is a naturally occurring or synthetic nucleoside base or blocked nucleoside base; and
Y is a stable blocking group, a solid state support, a nucleotide on a solid state support, or an oligonucleotide on a solid state support;
(b) selecting a second synthon having structure (2):
wherein:
RF is a labile blocking group; and
L is a leaving group or together L and Bx are a 2-3xe2x80x2 or 6-3xe2x80x2 pyrimidine or 8-3xe2x80x2 purine cyclonucleoside;
(c) adding the second synthon to the first synthon in the presence of a base to effect nucleophilic attack of the 5xe2x80x2-phosphate of the first synthon at the 3xe2x80x2-position of the second synthon to yield a new first synthon having structure (3):
xe2x80x83via a stereospecific inversion of configuration at the 3xe2x80x2 position of the second synthon; and
(d) treating the new first synthon with a reagent to remove the labile blocking group RF.
Additional nucleotides are added to the new first synthon by repeating steps (b), (c), and (d) for each additional nucleotide. Preferably, RF is an acid-labile blocking group and said new first synthon in step (d) is treated with an acidic reagent to remove said acid-labile RF blocking group.
The present invention also provides sequence-specific oligonucleotides comprising a plurality of nucleotides linked by chiral phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides linkages wherein at least one of the nucleosides is a non-naturally occurring nucleoside. Preferably, the nucleosides are connected via linkages selected from the group consisting of chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp chiral phosphotriester or chiral Rp phosphotriester linkages. In one embodiment of the invention each of the linkages of the oligonucleotide is a substantially pure chiral phosphorous linkage. In other embodiments less than all of the phosphate linkages are substantially pure chiral phosphorous linkages. In further embodiments, the oligonucleotides of the invention form at least a portion of a targeted RNA or DNA sequence.
The present invention also provides oligonucleotides comprising nucleoside units joined together by either all Sp phosphotriester linkages, all Rp phosphotriester linkages, all Sp phosphoramidate linkages, or all Rp phosphoramidate linkages. Also provided are oligonucleotides having at least 10 nucleoside units joined together by either all Sp alkylphosphonate linkages or all Rp alkylphosphonate linkages. Preferably such alkylphosphonate linkages are methylphosphonate linkages. Each of these oligonucleotides can form at least a portion of a targeted RNA or DNA sequence.
In preferred embodiments of the invention, the oligonucleotides include non-naturally occurring nucleoside units incorporated into the oligonucleotide chain. Such nucleoside units preferably have structure (4) or structure (5):
wherein Q, RB, RG, and BX are defined as above and RC is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl, SOMe, SO2Me, ONO2, NO2, N3, NH2, NH-alkyl, substituted NH-alkyl, OCH2CHxe2x95x90CH2, OCHxe2x95x90CH2, OCH2CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, poly-alkylamino, substituted silyl, an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide.
In preferred embodiments, BX is a pyrimidinyl-1 or purinyl-9 moiety such as, for example, adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.
In further preferred embodiments, the modified nucleosides include nucleosides having structures (6)-(11):
wherein:
G and K are, independently, C or N;
J is N or CR2R3;
R1 is OH or NH2;
R2 and R3 are H, NH, lower alkyl, substituted lower alkyl, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocycloalkyl, heterocycloalkylamino, aminoalkylamino, hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
R4 and R5 are, independently, H, OH, NH2, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
R6 and R7 are, independently, H, OH, NH2, SH, halogen, CONH2, C(NH)NH2, C(O)O-alkyl, CSNH2, CN, C(NH)NHOH, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide; and
X is a sugar or a sugar analog moiety where said sugar analog moiety is a sugar substituted with at least one substituent comprising an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide.
The present invention also provides compounds which are useful in forming the oligonucleotides of invention. Such compounds have structure (12):
wherein Q, RA, RD, RE, RX, L, and BX are defined as above and RF is H or a labile blocking group.
The oligonucleotides of the invention are useful to increase the thermodynamic stability of heteroduplexes with target RNA and DNA. Certain of the oligonucleotides of the invention are useful to elicit RNase H activity as a termination event. Certain other oligonucleotides are useful to increase nuclease resistance. The oligonucleotides of the invention are also useful to test for antisense activity using reporter genes in suitable assays and to test antisense activity against selected cellular target mRNA""s in cultured cells.
As will be recognized, adjacent nucleosides of a naturally occurring or wild type oligonucleotide are joined together by phosphodiester linkages, i.e. diesters of phosphoric acid. The natural phosphodiester linkages in oligonucleotides are at the same time both non-chiral and pro-chiral sites. Substitution of one of the oxygen atoms of the phosphate moiety of a nucleotide with another atom yields an asymmetric center on the phosphorus atom. Since a nucleotide unit already contains a first asymmetrical center within its sugar moiety, further asymmetry at the phosphorus atom of the nucleotide yields a diasymmetric nucleotide. Such a diasymmetric nucleotide is a chiral nucleotide having Sp and Rp diastereomers.
Substitution of one of the oxygen atoms of the phosphate moiety of a nucleotide with a sulfur atom yields Sp and Rp diastereomeric phosphorothioate analogs. Similarly, substitution of a phosphate oxygen atom by an alkyl moiety yields diastereomeric alkylphosphonate analogs. Substitution with an alkoxy group yields diastereomeric Sp and Rp phosphotriesters. Substitution with a thioalkoxy group yields a mixed triesterxe2x80x94a phosphodiesterthioester. Substitution with an amine or a substituted amine (including heterocyclic amines) yields diastereomeric Sp and Rp phosphoramidates.
It will be appreciated that the terms xe2x80x9cphosphatexe2x80x9d and xe2x80x9cphosphate anionxe2x80x9d as employed in connection with the present invention include nucleotides and oligonucleotides derived by replacement of one of the oxygen atoms of a naturally occurring phosphate moiety with a heteroatom, an alkyl group or an alkyoxy group. Thus, the terms xe2x80x9cphosphatexe2x80x9d or xe2x80x9cphosphate anionxe2x80x9d include naturally occurring nucleotides, phosphodiesters of naturally occurring oligonucleotides, as well as phosphorothioate, alkylphosphonate, phosphotriester, and phosphoamidate oligonucleotides.
Since there exist numerous phosphodiester linkages in an oligonucleotide, substitution of an oxygen atom by another atom such as, for example, sulfur, nitrogen, or carbon in one or more of the phosphate moieties yields a racemic mixture unless such substitution occurs in a stereospecific manner. As a practical matter, see Stec, W. J., Zon, G., and Uznanski, B. (1985), J. Chromatography, 326:263, above. Separation of the diastereomers of racemic mixtures of non-stereospecific synthesized oligonucleotides is only possible when there are a minimum of diasymmetric sites, for example, two diasymmetric sites. Since the diasymmetric substituent group at each diastereomeric phosphorus atom could have steric, ionic or other effects on conformation, binding, and the like at each such site, sequence-specific oligonucleotides having all Sp or all Rp chiral phosphorus linkages are desirable.
In accordance with this invention, sequence-specific oligonucleotides are provided comprising substantially pure chiral phosphate linkages such as, for example, phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkages. In contrast to prior art synthetic oligonucleotides, at least certain of the chiral phosphorous linkages of the present oligonucleotides are not racemic in nature but, rather, possess relatively high enantiomeric purity. As will be recognized by those skilled in the art, enantiomeric purityxe2x80x94also known as chiral purityxe2x80x94is manifested for a chemical compound by the predominance of one enantiomer over the other. Thus, an oligonucleotide can be said to possess a substantially pure chiral phosphate linkage where, for example, the Sp form of that linkage greatly predominates over the Rp form. In accordance with the present invention, at least certain of the chiral phosphate linkages present in an oligonucleotide should have chiral purity greater than about 75%. Preferably such linkages have chiral purity greater than about 90%, more preferably greater than about 95%, even more preferably about 100%. Chiral purity may be determined by any of the many methods known in the art, including but not limited to x-ray diffraction, optical rotary dispersion, and circular dichroism.
The oligonucleotides of the invention are expected to exhibit one or more efficacious properties such as, for example, hybridization with targeted RNA""s and DNA""s, cellular absorption and transport, or improved enzymatic interaction. At the same time, it is expected that these improvements to the basic oligonucleotide sequences will not significantly diminish existing properties of the basic oligonucleotide sequence. Thus, the present improvements are likely to lead to improved drugs, diagnostics, and research reagents.
Further improvements likely can be effected by making one or more substitutions or modifications to the base or the sugar moieties of the individual nucleosides employed to prepare the chiral oligonucleotides of the invention. Such substitutions or modifications generally comprise derivation at a site on the nucleoside base or at a site on the nucleoside sugar, provided such derivation does not interfere with the stereoselective syntheses of the present invention by, for example, blocking nucleophilic attack of the 5xe2x80x2-phosphate of a first synthon at the 3xe2x80x2-position of a second synthon. In certain embodiments, one or more of the nucleosides of the chiral oligonucleotides of the invention include a naturally occurring nucleoside unit which has been substituted or modified. These non-naturally occurring or xe2x80x9cmodifiedxe2x80x9d nucleoside units preferably have either structure (4) or structure (5):
wherein:
Q is O or CHRG;
RA and RB are H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
RC is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl, SOMe, SO2Me, ONO2, NO2, N3, NH2, NH-alkyl, substituted NH-alkyl, OCH2CHxe2x95x90CH2, OCHxe2x95x90CH2, CH2CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, polyalkylamino, substituted silyl, an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide;
RG is H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide; and
BX is a nucleoside base, a blocked nucleoside base, a nucleoside base analog, or a blocked nucleoside base analog.
In preferred embodiments BX is a pyrimidinyl-1 or purinyl-9 moiety as for instance adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine. Preferably, BX is selected such that a modified nucleoside has one of the structures (6)-(11):
wherein:
G and K are, independently, C or N;
J is N or CR2R2;
R1 is OH or NH2;
R2 and R3 are H, NH, lower alkyl, substituted lower alkyl, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocycloalkyl, heterocycloalkylamino, aminoalkylamino, hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
R4 and R5 are, independently, H, OH, NH2, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
R6 and R7 are, independently, H, OH, NH2, SH, halogen, CONH2, C(NH)NH2, C(O)O-alkyl, CSNH2, CN, C(NH)NHOH, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide; and
X is a sugar or a sugar analog moiety where said sugar analog moiety is a sugar substituted with at least one substituent comprising an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide. It is preferred that X have the general structure (4) or (5).
For the purposes of this invention, improving pharmacodynamic properties means improving oligonucleotide uptake, enhanced oligonucleotide resistance to degradation, and/or strengthened sequence-specific hybridization with RNA and improving pharmacokinetic properties means improved oligonucleotide uptake, distribution, metabolism or excretion. RNA cleaving moieties are chemical compounds or residues which are able to cleave an RNA strand in either a random or, preferably, a sequence-specific fashion.
Exemplary base moieties of the invention are any of the natural pyrimidinyl-1- or purinyl-9- bases including uracil, thymine, cytosine, adenine, guanine, 5-alkylcytosines such as 5-methylcytosine, hypoxanthine, 2-aminoadenine, and other modified bases as depicted in the formulas above. Exemplary sugars include ribofuranosyl, 2xe2x80x2-deoxyribofuranosyl, their corresponding five membered ring carbocyclic analogs as well as other modified sugars depicted in the formulas above. Particularly preferred modified sugars include 2xe2x80x2-fluoro and 2xe2x80x2-O-methyl-2xe2x80x2-deoxyribofuranosyl, i.e. 2xe2x80x2-fluoro and 2xe2x80x2-O-methyl-xcex2-D-erythro-pentofuranosyl.
Alkyl groups of the invention include but are not limited to C1-C12 straight and branched chained alkyls such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, isopropyl, 2-butyl, isobutyl, 2-methylbutyl, isopentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl. Alkenyl groups include but are not limited to unsaturated moieties derived from the above alkyl groups including but not limited to vinyl, allyl, crotyl, propargyl. Aryl groups include but are not limited to phenyl, tolyl, benzyl, naphthyl, anthracyl, phenanthryl, and xylyl. Halogens include fluorine, chlorine, bromine, and iodine. Suitable heterocyclic groups include but are not limited to imidazole, tetrazole, triazole, pyrrolidine, piperidine, piperazine, and morpholine. Carbocyclic groups include 3, 4, 5, and 6-membered substituted and unsubstituted alkyl and alkenyl carbocyclic rings. Amines include amines of all of the above alkyl, alkenyl, and aryl groups including primary and secondary amines and xe2x80x9cmasked aminesxe2x80x9d such as phthalimide. Amines are also meant to include polyalkylamino compounds and aminoalkylamines such as aminopropylamine and further heterocyclo-alkylamines such as imidazol-1, 2 or 4-yl-propylamine. Substituent groups for the above include but are not limited to other alkyl, haloalkyl, alkenyl, alkoxy, thioalkoxy, haloalkoxy, and aryl groups as well as halogen, hydroxyl, amino, azido, carboxy, cyano, nitro, mercapto, sulfides, sulfones, and sulfoxides. Other suitable substituent groups also include rhodamines, coumarins, acridones, pyrenes, stilbenes, oxazolo-pryidocarbazoles, anthraquinones, phenanthridines, phenazines, azidobenzenes, psoralens, porphyrins, cholesterols, and other xe2x80x9cconjugatexe2x80x9d groups.
Methods of synthesizing such modified nucleosides are set forth in copending applications for United States Letters Patent, assigned to the assignee of this invention, and entitled Compositions and Methods for Modulating RNA Activity, Ser. No. 07/463,358, filed Jan. 11, 1990; Sugar Modified Oligonucleotides That Detect And Modulate Gene Expression, Ser. No. 07/566,977, filed Aug. 13, 1990; and Compositions and Methods for Modulating RNA Activity, U.S. Ser. No. 91/00243, filed Jan. 11, 1991, the entire disclosures of which are incorporated herein by reference.
The chirally pure phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides of the invention can be evaluated for their ability to act as inhibitors of RNA translation in vivo. Various therapeutic areas can be targeted for such antisense potential. These therapeutic areas include but are not limited to herpes virus (HSV), the TAR and tat regions of HIV, the codon regions of Candida albicans chitin synthetase and Candida albicans xcex2 tubulin, papilloma virus (HPV), the ras oncogene and protooncogene, ICAM-1 (intercellular adhesion molecule-1) cytokine, and 5xe2x80x2-lipoxygenase. A targeted region for HSV includes GTC CGC GTC CAT GTC GGC (SEQ ID NO:1). A targeted region for HIV includes GCT CCC AGG CTC AGA TCT (SEQ ID NO:2). A targeted region for Candida albicans includes TGT CGA TAA TAT TAC CA (SEQ ID NO:3). A targeted region for human papillomavirus, e.g. virus types HPV-11 and HPV-18, includes TTG CTT CCA TCT TCC TCG TC (SEQ ID NO:4). A targeted region for ras includes TCC GTC ATC GCT CCT CAG GG (SEQ ID NO:5). A targeted region for ICAM-1 includes TGG GAG CCA TAG CGA GGC (SEQ ID NO:6) and the sequence CGA CTA TGC AAG TAC (SEQ ID NO:7) is a useful target sequence for 5-lipoxygenase. In each of the above sequences the individual nucleotide units of the oligonucleotides are listed in a 5xe2x80x2 to 3xe2x80x2 sense from left to right.
The phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides of the invention may be used in therapeutics, as diagnostics, and for research, as specified in the following copending applications for United States Letters Patent assigned to the assignee of this invention: Compositions and Methods for Modulating RNA Activity, Ser. No. 07/463,358, filed Jan. 11, 1990; Compositions and Methods for Detecting and Modulating RNA Activity, Ser. No. 07/463,358, filed Jan. 11, 1990; Antisense Oligonucleotide Inhibitors of Papilloma Virus, Ser. No. 07/445,196 Filed Dec. 4, 1989; Oligonucleotide Therapies for Modulating the Effects of Herpesvirus, Ser. No. 07/485,297, filed Feb. 26, 1990; Reagents and Methods for Modulating Gene Expression Through RNA Mimicry Ser. No. 07/497,090, filed Mar. 21, 1990; Oligonucleotide Modulation of Lipid Metabolism, Ser. No. 07/516,969, filed Apr. 30, 1990; Oligonucleotides for Modulating the Effects of Cytomegalovirus Infections, Ser. No. 07/568,366, filed Aug. 16, 1990; Antisense Inhibitors of the Human Immunodeficiency Virus, Ser. No. 09/521,907, filed May 11, 1990; Nuclease Resistant Pyrimidine Modified Oligonucleotides for Modulation of Gene Expression, Ser. No. 07/558,806, filed Jul. 27, 1990; Novel Polyamine Conjugated Oligonucleotides, Ser. No. 07/558,663, filed Jul. 27, 1990; Modulation of Gene Expression Through Interference with RNA Secondary Structure, Ser. No. 07/518,929, filed May 4, 1990; Oligonucleotide Modulation of Cell Adhesion, Ser. No. 07/567,286, filed Aug. 14, 1990; Inhibition of Influenza Viruses, Ser. No. 07/567,287, filed Aug. 14, 1990; Inhibition of Candida, Ser. No. 07/568,672, filed Aug. 16, 1990; and Antisense Oligonucleotide Inhibitors of Papillomavirus, Ser. No. PCT/US90/07067, filed Dec. 3, 1990. These patents disclose a number of means whereby improved modulation of RNA and DNA activity may be accomplished through oligonucleotide interaction. To the extent that the specific sequences disclosed therein may be used in conjunction with the present invention, the disclosures of the foregoing United States patent applications are incorporated herein by reference.
The oligonucleotides of the invention preferably are prepared via the process shown in Scheme 1, wherein a selected nucleotide is coupled to another nucleotide or to a growing oligonucleotide chain via a nucleophilic displacement reaction. As will be recognized, this process is also applicable to the preparation of oligonucleotides comprising non-chiral phosphodiester linkages. In Scheme 1, RF is a phosphate blocking group, BX is a suitable heterocyclic nucleoside base (blocked or unblocked), RX is a sugar derivatizing group and Q is an oxygen or methylene group. Together RD and RE are the necessary oxygen, sulfur, nitrogen, substituted nitrogen, alkoxy or thioalkoxy groups that form the phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linking groups. For Scheme 1, the Y group of the above formulas is depicted as a CPG (Controlled Pore Glass) group. Other Y groups also may be used.
In Scheme 1, a first nucleotide (13) is attached to a solid state CPG support via its 3xe2x80x2-hydroxyl group in a standard manner, yielding compound (14). Compound (14), which forms a first synthon, is treated with an appropriate base, producing anionic compound (15). Compound (15) is reacted with a first unit of compound (2)xe2x80x94a xylofuranosyl nucleotide bearing blocking group RF on its phosphate functionality and leaving group L at its 3xe2x80x2 position. Compound (2) is a second synthon. The BX moiety of structure (2) may be the same as or different than the BX moiety of compound (15), depending on the sequence of the desired oligonucleotide. The anion of compound (15) displaces the leaving group at the 3xe2x80x2 position of compound (2). The displacement proceeds via inversion at the 3xe2x80x2 position of the second synthon, forming compound (16) with n=1.
The resulting phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkage of compound (16) extends from the 5xe2x80x2 position of the first synthon (compound (14)) to the 3xe2x80x2 position of the second synthon (compound (2)). The inversion at the 3xe2x80x2 position of the second synthon results in the final configuration at the 3xe2x80x2 nucleotide derived from the second synthon being a normal ribofuranosyl sugar conformation. Compound (16) on the solid state CPG support is now washed to free it of any unreacted compound (2).
The second synthon carries a phosphate blocking group RF on its phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate phosphorus group. After coupling of the second synthon to the first synthon to yield compound (16) wherein n=1 and washing, the phosphate blocking group RF is removed with an acid, yielding compound (17) wherein n=1. Compound (17), which represents a new first synthon, is now treated with base to generate a further anionic, compound (18) with n=1. Compound (18) is suitable for nucleophilic attack on a further unit of compound (2) (the second synthon) to form a new compound (16) wherein n=2. In this further unit having compound (2), the BX moiety may be the same or different from the BX moiety of either of the nucleotides of compound (16) wherein n=1, depending on the desired sequence.
Compound (16) wherein n=2 is washed and then treated with acid to deblock the RF blocking group, yielding a further new first synthon, compound (17) wherein n=2. This new first synthon, is now ready to be further cycled by treatment with base to yield compound (18) wherein n=2, which is now reacted with a further unit having compound (2) to yield a further unit of having structure (16) wherein n=3. Again, BX may be the same or different than previously BX moieties. The cycle is repeated for as many times as necessary to introduced further nucleotides of the desired sequence via compound (2).
If it is desired to have the 5xe2x80x2 terminal end of the final oligonucleotide as a phosphate group, then the last compound (17) is appropriately removed from the CPG support. If it is desired to have the 5xe2x80x2 terminal end as a hydroxyl group, then the penultimate nucleotide is added as compound (22), it is converted to compounds (16), (17), and (18) and the resulting compound (18) is reacted with a xylofuranosyl nucleoside, compound (19). Compound (19), like compound (2), includes a leaving group L within its structure. Reaction of compound (19) with compound (18) yields oligonucleotide, compound (20), which is released from the CPG support with concentrated ammonium ion to yield the desired oligonucleotide, compound (21). The ammonium ion treatment will also remove any base blocking groups as is standard in automated oligonucleotide synthesis.
In summary, as shown in Scheme 1, a phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate 5xe2x80x2 nucleotide (or the 5xe2x80x2-terminal nucleotide of a growing oligonucleotide) functions as a first synthon. This is converted to an anion with a base. This anion displaces a leaving group at the 3xe2x80x2 position of a xylofuranosyl nucleotide. The xylofuranosyl nucleotide comprises a second synthon. The displacement proceeds via inversion at the 3xe2x80x2 position of the second synthon with the resulting phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkage that is formed extending from the 5xe2x80x2 position of the first synthon to the 3xe2x80x2 position of the second synthon. The inversion at the 3xe2x80x2 position of the second synthon results in the final configuration at the 3xe2x80x2 nucleotide derived from the second synthon being a normal ribofuranosyl sugar conformation. It has a 3xe2x80x2 to 4xe2x80x2 trans orientations (a ribofuranosyl sugar conformation) that is identical to natural or wild type oligonucleotides.
The second synthon carries a phosphate blocking group on its phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate phosphorus group. After coupling of the second synthon to the first synthon, this phosphate blocking group is removed, generating a new first synthon having an anion at its 5xe2x80x2 phosphate suitable for nucleophilic attack on a further second synthon. Thus, after coupling of the first and second synthon, the newly joined first and second synthons now form a new first synthon. The oligonucleotide is elongated nucleotide by nucleotide via the nucleophilic attack of a phosphate anion at the 5xe2x80x2 end of the growing oligonucleotide chain on the leaving group at the 3xe2x80x2 position of the soon-to-be-added xylofuranosyl configured second synthon nucleotide.
It is presently preferred that the phosphate blocking group be a base stable, acid labile group. Such a phosphate blocking group maintains the phosphate moiety of the second synthon in a protected form that cannot react with the leaving group of the second synthon. This inhibits polymerization of the second synthon during the coupling reaction.
The nucleophilic coupling of the first and second synthons is a stereoselective coupling process that maintains the stereospecific configuration about the phosphorus atom of the first synthon. Thus the particular Sp or Rp diastereomeric configuration of a resolved phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate moiety at the 5xe2x80x2 end of a starting second synthon nucleotide or the 5xe2x80x2 terminal end of a growing first synthon oligonucleotide is maintained. While the sugar portion of the second synthon undergoes inversion about its 3xe2x80x2 position as a result of the coupling process, the phosphate portion of the second synthon retains its stereospecific configuration. After coupling of the second synthon to the first, the phosphate moiety (for example, the phosphorothioate, alkylphosphate, phosphoamidate or phosphotriester moiety) of the second synthon retains its original stereochemistry. That is, an Rp diastereomeric second synthon retains the Rp configuration of its phosphate moiety, while an Sp diastereomeric second synthon retains the Sp configuration of its phosphate moiety.
For example, to form an Rp chiral phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotide a first Rp diastereomeric nucleotide is chosen as the first synthon. It is coupled with an Rp diastereomeric second synthon nucleotide. The resulting dinucleotide maintains the Rp orientation at the inter-nucleotide linkage between the first and second nucleotides. When a third nucleotide is next coupled to the first two, the Rp diastereomeric phosphate moiety from the second nucleotide forms the inter-nucleotide linkage between the second and third nucleotides and is maintained in the Rp orientation. If the third nucleotide was also an Rp diastereomeric nucleotide then when a fourth nucleotide is added to the growing oligonucleotide chain, the inter-nucleotide linkage between nucleotides three and four is also an Rp diastereomeric linkage. If each added xe2x80x9csecond synthonxe2x80x9d is also an Rp diastereomer, then the resulting oligonucleotide will contain only Rp inter-nucleotide linkages. If an oligonucleotide having Sp inter-nucleotide linkages is desired, then the first nucleotide and each of the added subsequent nucleotides are selected as Sp diastereomeric nucleotides.
The first synthon can be a first nucleotide or a growing oligonucleotide chain. If it is desired that each of the nucleotides of the oligonucleotide be ribofuranoside configured nucleotides, then the first nucleotide is selected as a ribofuranoside configured nucleotide. Each added second synthon, while added as a xylofuranoside configured nucleotide, after inversion is converted to a ribofuranoside configured nucleotide.
The 3xe2x80x2 position of the first nucleotide is either blocked if a solution reaction is practiced or is coupled to a solid state support if a solid state reaction (as for instance one utilizing a DNA synthesizer) is practiced. Each additional nucleotide of the oligonucleotide is then derived from a xylofuranosyl nucleotide, i.e. a second synthon. Because the first nucleotide of the oligonucleotide can be a xe2x80x9cstandardxe2x80x9d ribofuranosyl nucleotide coupled via its 3xe2x80x2 hydroxyl to a solid state support, the standard solid state supports known in the art, such as controlled pore glass (CPG) supports, can be utilized and the second synthons added as reagents to the growing oligonucleotide on a standard DNA synthesizer such as, for example, an Applied Biosystems Inc. 380B Nucleic Acid Synthesizer. However, unlike standard DNA synthesizer techniques, nucleotide coupling is not achieved using activated phosphoamidate chemistry. Instead, the above-noted nucleophilic displacement reaction of a phosphate anion on a 3xe2x80x2 leaving group of a xylofuranosyl nucleotide is utilized as the coupling reaction. Even when chiral phosphoramidate phosphate linkages are being incorporated into the sequence-specific chiral oligonucleotides of the invention, the phosphoramidate groups of individual nucleotides are not directly used to effect the coupling reaction between nucleotides. Rather, as with phosphorothioates, alkylphosphonates, and phosphotriesters, an anion is generated at the phosphate moiety. It is this anionxe2x80x94not an activated amidate speciesxe2x80x94that is the activated species for effecting coupling.
Once the first nucleotide is loaded on a solid state support utilizing standard techniques, the anion necessary for nucleophilic attack is generated via treatment of the first nucleotide, i.e. the first synthon, with a base. Suitable bases include but are not limited to sodium hydride, methylmagnesium chloride, and t-butylmagnesium chloride. These are used in a suitable solvent such as acetonitrile, tetrahydrofuran or dioxane.
The second synthon is added either concurrently with the base or subsequent to it. After coupling, the growing oligonucleotide is washed with a solvent and then treated with a reagent to effect deblocking of the phosphate blocking group of the second synthon. If a preferred acid-labile blocking group is used to block the phosphate of the second synthon, deblocking is easily effected by treating the growing oligonucleotide on the solid state support with an appropriate acid.
Suitable acid-labile blocking groups for the phosphates of the second synthon include but are not limited to t-butyl, dimethoxytrityl (DMT) or tetrahydropyranyl groups. Suitable acids for deblocking the second synthon phosphate blocking group include but are not limited to acetic acid, trichloroacetic acid, and trifluoromethane sulfonic acid. Such acids are suitably soluble in solvents such as tetrahydrofuran, acetonitrile, dioxane, and the like.
Following treatment with an appropriate deblocking reagent to effect deblocking of the phosphate protecting group, the growing oligonucleotide is then washed with an appropriate solvent such as tetrahydrofuran, acetonitrile or dioxane. The oligonucleotide is now ready for the addition of a further nucleotide via treatment with base to generate an anion on the 5xe2x80x2 terminal phosphate followed by the addition of a further second synthon. Alternatively, the anion can be generated concurrently with addition of a further second synthon. Suitable leaving groups for inclusion at the 3xe2x80x2 position of the xylofuranosyl second synthon include but are not limited to the group consisting of halogen, alkylsulfonyl, substituted alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate. A more preferred group includes chloro, fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl, and 2,4,6-trichlorophenyl. These leaving groups are subject to SN2 displacement reactions with inversion about the 3xe2x80x2 position of the sugar to provide the required 3xe2x80x2-4xe2x80x2 trans ribofuranosyl configuration after inversion. The ionized oxygen atom of the phosphate moiety of the first synthon displaces these leaving groups to effect the coupling of the first synthon to the second synthon.
A further class of leaving groups include certain sugar-base cyclonucleosides. These include 2,3xe2x80x2 or 6,3xe2x80x2-cyclopyrimidines and 8,3xe2x80x2-cyclopurine nucleosides. These nucleosides are alternately known as anhydro nucleosides. Since the sugar-heterocycle bond of such cyclonucleosides is xe2x80x9csynxe2x80x9d with the heterocycle base, nucleophilic addition with inversion at this site also yields the desired ribofuranoside configuration of the added nucleotide after addition of the second synthon to the first synthon. The linking atom between the 3xe2x80x2 position of the sugar and the 2 or 6 position of a pyrimidine base or the 3xe2x80x2 position of the sugar and the 8 position of the purine base can be oxygen, sulfur or nitrogen.
Since a basic environment is created during coupling of the first synthon to the second synthon and an acidic environment (utilizing the preferred acid-labile phosphate blocking group) is created during deblocking of the phosphate blocking group from the nucleotide derived from the second synthon, if blocking groups are utilized on the base or sugar portions of the nucleotides such base or sugar blocking groups must be stable to both acidic and basis conditions. Suitable blocking groups for the heterocyclic base or the sugar are selected to be stable to these conditions. One type of blocking groups that can be used are acid-base stable, hydrogenolysis-sensitive blocking groups; that is, blocking groups which can be removed with molecular hydrogen but not with acid or base. A benzyl blocking group is such a suitable hydrogenolysis-sensitive blocking group.
Other heterocycle base or sugar blocking groups are those that require more pronounced acid or base treatment to de-block than may be experienced during the basic activation of the nucleophilic displacement reaction of the second synthon blocking group or the acidic removal of the phosphate blocking group. Two such blocking groups are the benzoyl and isobutyryl groups. Both of these require strong basic conditions for their removal. These basic conditions are more stringent than that required to generate the phosphate anion for the nucleophilic displacement reaction. This allows the use of such benzoyl or isobutyryl blocking groups for the 6-amino group of adenine, the 2-amino group of guanine, and the 4-amino group of cytosine. Suitable precursor molecules for the second synthons include the xylo derivatives of the common nucleosides. Certain of these xe2x80x9cxylo nucleosidesxe2x80x9d are commercially available and others are known in the nucleoside literature.
Xylo nucleosides include but are not limited to xylo derivatives of adenosine, guanosine, inosine, uridine, cytidine, thymidine, 5-methylcytidine, and 2-aminoadenosine, i.e. 9-(xcex2-D-xylofuranosyl)adenine, 9-(xcex2-D-xylofuranosyl)guanine, 9-(xcex2-D-xylofuranosyl)hypoxanthine, 1-(xcex2-D-xylofuranosyl)uracil, 1-(xcex2-D-xylofuranosyl)cytosine, 1-(xcex2-D-xylofuranosyl)thymine, 5-methyl-1-(xcex2-D-xylofuranosyl)cytosine, and 2-amino-9-(xcex2-D-xylofuranosyl)adenine. They also include the xylo equivalents of the common 2xe2x80x2-deoxy nucleosides such as 9-(xcex2-D-2xe2x80x2-deoxy-threo-pentofuranosyl)adenine, 9-(xcex2-D-2xe2x80x2-deoxy-threo-pentofuranosyl)guanine, 9-(xcex2-D-2xe2x80x2-deoxy-threo-pentofuranosyl)hypoxanthine, 1-(xcex2-D-2xe2x80x2-deoxy-threo-pentofuranosyl)uracil, 1-(xcex2-D-2xe2x80x2-deoxy-threo-pentofuranosyl)cytosine, 1-(xcex2-D-2xe2x80x2-deoxy-threo-pentofuranosyl)thymine,5-methyl-1-(xcex2-D-2xe2x80x2-deoxy-threo-pentofuranosyl)cytosine, and 2-amino-9-(xcex2-D-2xe2x80x2-deoxy-threo-pentofuranosyl)adenine.
Other preferred nucleosides that are suitable precursors for the second synthon include but are not limited to 2xe2x80x2-fluoro, 2xe2x80x2-methoxy, 2xe2x80x2-O-allyl, 2xe2x80x2-methyl, 2xe2x80x2-ethyl, 2xe2x80x2-propyl, 2xe2x80x2-chloro, 2xe2x80x2-iodo, 2xe2x80x2-bromo, 2xe2x80x2-amino, 2xe2x80x2-azido, 2xe2x80x2-0-methyl, 2xe2x80x2-O-ethyl, 2xe2x80x2-O-propyl, 2xe2x80x2-O-nonyl, 2xe2x80x2-O-pentyl, 2xe2x80x2-O-benzyl, 2xe2x80x2-O-butyl, 2xe2x80x2-O-(propylphthalimide), 2xe2x80x2-S-methyl, 2xe2x80x2-S-ethyl, 2xe2x80x2-aminononyl, 2xe2x80x2-aralkyl, and 2xe2x80x2-alkylheterocyclo such as propylimidazoyl derivatives of the above 2xe2x80x2-deoxy-threo-pentofuranosyl nucleosides. Representatives of this group include but are not limited to 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-fluoro-threo-pentofuranosyl)adenine, 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-fluoro-threo-pentofuranosyl)guanine, 9-(xcex2-D-2xe2x80x2-deoxy-2-fluoro-threo-pentofuranosyl)hypoxanthine, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-fluoro-threo-pentofuranosyl)uracil, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-fluoro-threo-pentofuranosyl)cytosine, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-fluoro-threo-pentofuranosyl)thymine, 5-methyl-1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-fluoro-threo-pentofuranosyl)cytosine, 2-amino-9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-fluoro-threo-pentofuranosyl)adenine, 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-methoxy-threo-pentofuranosyl)adenine, 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-methoxy-threo-pentofuranosyl)guanine, 9-(xcex2-D-2xe2x80x2-deoxy-2-methoxy-threo-pentofuranosyl)hypoxanthine, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-methoxy-threo-pentofuranosyl)uracil, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-methoxy-threo-pentofuranosyl)cytosine,1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-methoxy-threo-pentofuranosyl)thymine, 5-methyl-1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-methoxy-threo-pentofuranosyl)cytosine,2-amino-9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-methoxy-threo-pentofuranosyl)adenine, 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-allyl-threo-pentofuranosyl)adenine, 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-allyl-threo-pentofuranosyl)guanine, 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-allyl-threo-pentofuranosyl)hypoxanthine, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-allyl-threo-pentofuranosyl)uracil, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-allyl-threo-pentofuranosyl)cytosine,1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-allyl-threo-pentofuranosyl)thymine, 5-methyl-1-(xcex2-D-2xe2x80x2-deoxy-2-O-allyl-threo-pentofuranosyl)cytosine,2-amino -9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-allyl-threo-pentofuranosyl)adenine, 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-methyl-threo-pentofuranosyl)adenine, 9-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-chloro-threo-pentofuranosyl)guanine, 9-(xcex2-D-2xe2x80x2-deoxy-2-amino-threo-pentofuranosyl)hypoxanthine, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-nonyl-threo-pentofuranosyl)uracil, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-benzyl-threo-pentofuranosyl)cytosine, 1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-bromo-threo-pentofuranosyl)thymine,5-methyl-1-(xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-butyl-threo-pentofuranosyl)cytosine, and 2-amino-9-[xcex2-D-2xe2x80x2-deoxy-2xe2x80x2-O-(propylphthalimide)-threo-pentofuranosyl)adenine. The 2xe2x80x2-deoxy-2xe2x80x2-fluoro-threo-pentofuranosyl, and 2xe2x80x2-deoxy-2xe2x80x2-methoxy-threo-pentofuranosyl nucleosides are particularly preferred in that the 2xe2x80x2-fluoro and 2xe2x80x2-methoxy groups give improved nuclease resistance to oligonucleotide bearing these substituents on their respective nucleotides.
Further preferred nucleosides that are suitable precursors for the second synthon include but are not limited to thexylofuranosyl or 2xe2x80x2-deoxy-threo-pentofuranosyl derivatives of 3-deaza purine and 2-substituted amino purine nucleosides including but not limited to 3-deaza-2xe2x80x2-deoxyguanosine, 3-deaza-3-nonyl-2xe2x80x2-deoxyguanosine, 3-deaza-3-allyl-2xe2x80x2-deoxyguanosine, 3-deaza-3-benzyl-2xe2x80x2-deoxyguanosine, 3-deaza-3-nonyl-2xe2x80x2-deoxyguanosine, N2-[imidazol-1-yl-(propyl)]-2xe2x80x2-deoxyguanosine, and 2-amino-N2-[imidazol-1-yl(propyl)]adenosine.
Another preferred group of nucleoside precursors for the second synthon include the carbocyclic nucleosides, i.e. nucleosides having a methylene group in place of the pentofuranosyl ring oxygen atom. Such carbocyclic compounds may exhibit increased stability towards chemical manipulation during activation of the xylo nucleosides for nucleophilic attack.
The xylo nucleoside or derivatized xylo nucleoside is reacted with a suitable phosphorylating agent to phosphorylate the second synthon precursor. Various phosphorylation reactions are known in the art such as those described in Nucleotide Analogs, by Karl Heinz Scheit, John Wiley and Sons, 1980, Chapter Fourxe2x80x94Nucleotides with Modified Phosphate Groups and Chapter Sixxe2x80x94Methods of Phosphorylation; Conjugates Of Oligonucleotides and Modified Oligonucleotides: A Review Of Their Synthesis and Properties, Goodchild, J. (1990), Bioconjugate Chemistry, 1:165; and Antisense Oligonucleotides: A New Therapeutic Principle, Uhlmann, E. and Peyman, A. (1990), Chemical Reviews, 90:543.
Preferred phosphorylating agents include phosphoryl chlorides. Suitable phosphoryl chlorides include but are not limited to thiophosphoryl chloride, t-butoxyphosphoryl chloride, t-butoxy(methyl)phosphoryl chloride, t-butoxy-(methyl)thiophosphoryl chloride, t-butoxy(methoxy)phosphoryl chloride. Other phosphoryl chlorides may include t-butoxy(N-morpholino)phosphoryl chloride, t-butoxy(ethoxy-ethylamino)phosphoryl chloride, t-butoxy(methy-thioxy)phosphoryl chloride, and the like. Such phosphorylating agents are utilized to yield the corresponding phosphorothioate, phosphoramidate, phosphotriester, alkylphosphonates, and phosphodiester xylo nucleotides.
Even enzymatic phosphorylation is possible, as for example the phosphorylation of 9-(xcex2-D-xylofuranosyl)guanine by nucleoside phosphotransferase from Pseudomonas trifolii as per the procedure of Suzaki, S., Yamazaki, A. Kamimura, A., Mitsugi, K., and Kumashior, I. (1970), Chem. Pharm. Bull. (Tokyo), 18:172.
1-(xcex2-D-Xylofuranosyl)uracil 5xe2x80x2-phosphate was identified but not separated from its 3xe2x80x2 isomer as reported by Holy, A. and Sorm, F. (1969), Coll. Czech. Chem. Commun., 34:1929. Also, 9-(2xe2x80x2-O-benzyl-xcex2-D-xylofuranosyl)adenine 5xe2x80x2-phosphate was obtained as an intermediate by Hubert-Habart, M. and Goodman, L. (1969), Chem. Commun., 740. Removable of the benzyl blocking group would give the desired unblocked nucleotide.
Additionally, the alkylphosphonates can be prepared by the method of Holy, A. (1967), Coll. Czech. Chem. Commun., 32:3713. Phosphorothioates have also been prepared by treatment of the corresponding nucleoside with trisimidazolyl-1-phosphinesulfide followed by acid hydrolysis with aqueous acetic acid, Eckstein, F. (1966 and 1970), J. Am. Chem. Soc., 88:4292 and 92:4718, respectively. A more preferred method is by selective thiophosphorylation by thiophosphorylchloride in triethylphosphate, Murray, A. W. and Atkinson, M. R. (1968), Biochemistry, 7:4023.
The appropriate phosphorylated xylo nucleotide is then activated for nucleophilic displacement at its 3xe2x80x2 position by reacting the 3xe2x80x2-hydroxyl group of the xylo compound with an appropriate anhydride, chloride, bromide, acyloxonium ion, or through an anhydro or cyclo nucleoside or the like to convert the 3xe2x80x2-hydroxyl group of the xylo nucleoside to an appropriate leaving group.
In a further method of synthesis, treatment of 2xe2x80x2,3xe2x80x2-anhydroadenosine with sodium ethylmercaptide gives 9-[3-deoxy-3-(ethylthio)-xcex2-D-xylofuranosyl]adenine. Treatment of this compound with a first synthon nucleophile may generate a terminal 2-ethylthio arabinofuranosyl nucleoside that could be desulfurized to yield the corresponding 2xe2x80x2-deoxynucleoside.
If during phosphorylation or conversion of the xylo 3xe2x80x2-hydroxyl to a 3xe2x80x2-activated leaving group stereospecific diastereomers are not obtained, after completion of the phosphorylation or conversion of the 3xe2x80x2-hydroxyl to an activated leaving group, the Rp and Sp diastereomers of these compounds will then be isolated by HPLC. This will yield pure diastereomers in a stereospecific form ready for use as the second synthons of Scheme 1.
Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting.

Nucleotides having chiral phosphorus linkages


An access device has a distal, expandable member with a low-profile state facilitating insertion and a high-profile state facilitating operation of the device. An obturator can be used to move the expandable member between its two states. Alternatively, the device can have an outer tube and an inner tube connected to respective ends of a cylindrical expansion member, so that co-axial movement of the tubes changes the state of the member. An actuator slideable on a handle facilitates the co-axial movement of the tubes, and a resulting formation of the expandable member in a funnel configuration.

1. Field of the Invention
The present invention relates generally to devices for removing obstructing material from body passages and, more particularly, to an access device configurable between a first small diameter for initial insertion into a body passage and a second larger diameter for effecting the removal of obstructing material.
2. Discussion of the Prior Art
The prior art includes many devices for removing obstructing material from body passages. When the body passage comprises a blood vessel, obstructing materials may include plaque, thrombus, embolus, clots, and fatty deposits. In other cases, obstructions may result from stones and strictures.
Catheters are commonly inserted into vessels for the purpose of dislodging obstructing materials from the vessel walls. In a prevalent technique commonly referred to as an embolectomy/thrombectomy procedure, a balloon tipped catheter is introduced through a surgical incision and into a blood vessel. The balloon tipped catheter is advanced to the location of the obstructing material or occlusion, and the balloon is then inflated at a point within the vessel beyond the point of the obstructing material. The catheter including the attached balloon is then pulled back to the point of insertion. In this manner, the balloon pushes the obstructing material to the point of insertion where it is removed through the incision. When the obstructing material is detached from a vessel wall using this technique, the obstructing material will often have a tendency to disperse and migrate with the flow of blood within the vessel. This dispersion can make collection and removal of the obstructing material difficult, and the migration can put the patient at risk of acute trauma. Thus, a problem associated with this embolectomy technique involves the efficient collection and removal of obstructing material while preventing migration and dispersion of the obstructing material. Other percutaneous procedures exist in the prior art for recanalization of vessels. One percutaneous procedure involves the use of laser energy to vaporize the stenotic material. Another percutaneous procedure, commonly referred to as aspiration embolectomy/thrombectomy, relies on a negative pressure to collect the obstructing material.
Percutaneous or minimally invasive access to a blood vessel in the case of a balloon catheter, for example, requires the catheter to have a very small diameter to fit through a corresponding small incision in the blood vessel. Once the catheter is in the blood vessel, however, portions of the catheter need to assume a large profile removal interface for efficient removal of the obstructing material from the lumen of the blood vessel.
In an attempt to meet this dual functional requirement of small incision diameter and large intraluminal operating diameter, the prior art has incorporated sheaths in conjunction with catheters. U.S. Pat. No. 5,011,488 to Ginsburg discloses the use of an expanding funnel-shaped sheath for use in withdrawing thrombus or embolus (obstructing material) from a blood vessel. The funnel is deployed by extending the expanding funnel-shaped sheath from within a second sheath to thereby allow the compressed funnel to expand radially. Use of this second sheath tends to increase the overall diameter of the device, thus increasing the size of the incision required for insertion of the device. In addition to not achieving an optimally small insertion diameter, this device is also unable to obtain an optimally large intraluminal operating diameter. An optimally large intraluminal operating diameter would allow for better insertion and removal of larger instruments through the sheath. This prior art technique of inserting both the introducer sheath and the pre-shaped funnel sheath into the body passage, and of subsequently removing the introducer sheath, requires the large introduction incision to form a seal around the smaller diameter pre-shaped funnel sheath after the introducer sheath is removed. In other words, the double sheath combination of the prior art requires an initial incision into the body passage large enough to accommodate the introducer sheath and, subsequently, small enough to adequately form a seal around the smaller pre-formed funnel sheath left in place after the introducer sheath is removed. Since the initial incision cannot be subsequently reduced in size to accommodate the funnel sheath, a good seal in this prior art system is difficult to obtain.
Other devices, which provide access to relatively inaccessible regions of blood vessels, are disclosed in U.S. Pat. No. 4,530,698 and U.S. Pat. No. 4,437,859. Needle and sheath combinations for use in drug delivery, blood withdrawal, and dialysis have been proposed by the prior art, but these devices incorporate different structure to solve a problem, which is different than the removal of obstructing material from a body passage. U.S. Pat. No. 5,234,425 to Fogarty discloses a variable diameter sheath constructed of a composite elastomeric material that may be stretched to reduce the diameter. This variable diameter sheath, however, is not used for the removal of obstructing material. Instead, the primary goal of this device is to provide a lining of a body passage with a thin walled single thickness interior sheath, which is introduced into the body passage in a reduced diameter condition and subsequently expanded to snugly fit the interior wall of the body passage. The variable diameter sheath incorporates a tubular braid encapsulated within a coating of high elongation silicone polymer. None of the prior art devices disclose a sheath, which is efficiently configurable between a minimum diameter and a maximum diameter, and which comprises a lumen for accommodating a catheter to facilitate removal of obstructing material from a body passage.

Access device with expandable containment member


Exemplary embodiments of the present invention provide a high efficiency light emitting diode including a semiconductor stack including a first-type compound semiconductor layer, an active layer, and a second-type compound semiconductor layer, a first electrode disposed on the semiconductor stack, and a graphene-metamaterial laminate structure disposed between the first electrode and the semiconductor stack.

1. Field
Exemplary embodiments of the present invention relate to a light emitting diode and, more particularly, to a high efficiency GaN-based light emitting diode capable of preventing optical loss due to electrodes using a metamaterial.
2. Discussion of the Background
Generally, group-III nitrides, such as gallium nitride (GaN), aluminum nitride (AlN), and the like, have attracted attention as materials for light emitting diodes capable of emitting light in the visible and ultraviolet ranges due to excellent thermal stability and direct energy band gap. In particular, blue and green light emitting diodes using indium gallium nitride (InGaN) have been used in a wide range of fields, such as large color flat panel displays, signal lights, indoor lighting, high density light sources, high resolution output systems, optical communications, and the like.
Since it may be difficult to produce a homogeneous substrate capable of growing group-III nitride semiconductor layers, group-III nitride semiconductor layers may be grown on a heterogeneous substrate having a similar crystal structure through metal-organic chemical vapor deposition (MOCVD) or molecular beam epitaxy (MBE). A sapphire substrate having a hexagonal crystal structure may be used as a heterogeneous substrate. However, sapphire is an electrically non-conductive material, which may limit the structure of the light emitting diode. Accordingly, technology for fabricating a vertical-type high efficiency light emitting diode has been developed. In this technology, after epitaxial layers such as nitride semiconductor layers are grown on a heterogeneous substrate such as a sapphire substrate, a support substrate is bonded to the epitaxial layers, followed by separation of the heterogeneous substrate by laser lift-off or the like.
A metal substrate may be used as the support substrate in the vertical-type light emitting diode, thereby providing an excellent current spreading function in a vertical direction. In addition, after separation of the growth substrate, a roughened surface may be formed on an exposed N-plane of the gallium nitride layer through etching such as PEC, thereby providing excellent luminous efficacy.
However, since a vertical-type light emitting diode may include an N-electrode disposed on a light emitting surface, there may be a problem of optical loss due to the N-electrode absorbing or reflecting light generated in the light emitting diode, and preventing the light from being emitted to an outside of the light emitting diode. Although it may be necessary to reduce the area of the N-electrode in order to increase the area of the optical emitting surface, effective reduction in area of the N-electrode may be limited due to adhesive strength or current spreading requirements.
To solve such problems, a reflective metal layer may be disposed under the N-electrode to reflect light entering the N-electrode. However, as the reflected light is emitted to outside after passing through the epitaxial layers, there may be significant loss of light through the epitaxial layers.


A system, method, and computer program product are provided for generating mobile subscriber network experience indicators based on geo-located events. In use, one or more call trace logs are received including protocol flow information between User Equipment and one or more network elements associated with a network. Additionally, one or more call detail records are generated utilizing the one or more call trace logs, each of the one or more call detail records including an associated international mobile subscriber identity (IMSI). Further, the one or more call detail records are geo-tagged with location information associated with the one or more call trace logs. In addition, the one or more geo-tagged call detail records are aggregated based on the international mobile subscriber identity associated with the one or more geo-tagged call detail records, such that a subscriber corresponding to the international mobile subscriber identity is associated with at least one of the aggregated geo-tagged call detail records. Furthermore, a plurality of key performance indicators are identified for the subscriber, utilizing the at least one aggregated geo-tagged call detail record associated with the subscriber. Moreover, a network experience indicator associated with the subscriber is generated utilizing one or more of the plurality of key performance indicators. Still yet, the network experience indicator associated with the subscriber is displayed utilizing a graphical user interface.

Forty percent of an individual mobile subscriber's experience is determined by the quality of the network. Currently, operators do not have access to actionable data relating to the subscriber's network experience that can be used to improve customer care, support intelligent network rollout, drive marketing use cases and prioritise network fault resolution.
Current Customer Experience Management (CEM) systems use probes (intrusive or passive), which collect protocol flow data in the cellular network core (e.g. Gn IRP, between RNC and MSC in UMTS). Due to the nature of the collection point, these probe based systems lack the actual network information that is only available when collecting data at the Radio Access Network (RAN). For example, precision information for geo-tagging and network health cannot be captured is these systems.
There is thus a need for addressing these and/or other issues associated with the prior art.

System, method, and computer program for generating mobile subscriber network experience indicators based on geo-located events


An apparatus and method for providing analysis service based on behavior in a mobile network environment are disclosed. The apparatus includes a control unit configured to control the path of a packet based on predetermined policy information, to block the packet based on a result of an analysis of the packet, or to extract information about the packet and selectively process the extracted information based on the predetermined policy information; a download path and file management engine configured to collect downloaded files corresponding to the URL of the packet, to extract the downloaded files as an app file, and to transfer the extracted app file to a virtual machine; and a virtual machine management engine unit configured to determine whether malware is present in the app file and whether the app file has accessed the resources, and to selectively manage the corresponding app based on a result of the determination.

1. Technical Field
The present invention relates to service for detecting and managing a malicious behavior through the analysis of a vulnerable point in a mobile app in a high-speed network security service.
2. Description of the Related Art
In general, a vulnerable point in an app is handled by a method of blocking the vulnerable point based on a pattern using a mobile terminal vaccine program after the app has been installed in a mobile environment.
In this case, the treatment of infection is highly dependent on the detection ability of a vaccine because malware is isolated and treated after the malware has occurred first and then infection has occurred.
FIG. 1 is the schematic configuration of a system regarding the analysis of a malicious file in conventional network security.
As illustrated in FIG. 1, if a malicious app file is downloaded from a service 116 when a user 110 downloads a mobile app from the service 116, analysis and cooperative tasks are performed via a network traffic analysis system 112 and a virtual machine behavior analysis system 118 in order to analyze such a malicious app. The results of the analysis are transferred to the network traffic analysis system 112 and then managed.
In this case, problems arise in that the time it takes to perform analysis is long and also it is not easy to immediately take measures because the virtual machine behavior analysis system 118 and the network traffic analysis system 112 separately collect and analyze the results of static analysis and dynamic analysis.

Method and apparatus for providing analysis service based on behavior in mobile network environment


A boom mower having a frame, a boom assembly carried by the frame, a cutting mechanism operably engaged with the boom assembly, and at least one translation assembly operably engaged with the boom assembly and the cutting mechanism that moves the cutting mechanism in one or more of the first movement, the second movement, and the third movement. There is a break-away mechanism that enables the cutting mechanism to pivot relative to the at least one translation assembly.

Boom mowers may be attached to landscaping utility vehicles and be utilized to mow and/or cut vegetation such as grasses, shrubs, hedges, and the like. Frequently, the boom mower is mounted to a rear end of the vehicle and is towed behind the vehicle. This arrangement may make it difficult for the operator to monitor the vegetation that is being mowed or cut while driving the tractor forward. In other instances, the boom mower may be mounted to a front end of the vehicle, such as a tractor of around 100 HP or less. Boom mowers are frequently difficult to remove from the vehicle and this limits the use of the vehicle for other purposes. In addition to these issues, previously known boom mowers have had complex operator controls and a limited cutting range.