U.S. patent number 4,816,571 [Application Number 07/058,179] was granted by the patent office on 1989-03-28 for chemical capping by phosphitylation during oligonucleotide synthesis.
This patent grant is currently assigned to Applied Biosystems, Inc.. Invention is credited to William A. Andrus, J. William Efcavitch, Lincoln J. McBride.
United States Patent |
4,816,571 |
Andrus , et al. |
March 28, 1989 |
Chemical capping by phosphitylation during oligonucleotide
synthesis
Abstract
A method is provided for capping failure sequences in
oligonucleotide synthesis by phosphitylation. A phosphite monoester
is reacted with the 5' or 3' hydroxyl of the failure sequence
between successive condensation steps in a synthesis procedure to
form a 5' or 3' phosphite diester with the failure sequence. The
phosphite diester substituent is inert with respect to subsequent
reaction steps in the synthesis of the desired oligonucleotide
product.
Inventors: |
Andrus; William A. (San
Francisco, CA), Efcavitch; J. William (Belmont, CA),
McBride; Lincoln J. (Redwood City, CA) |
Assignee: |
Applied Biosystems, Inc.
(Foster City, CA)
|
Family
ID: |
22015191 |
Appl.
No.: |
07/058,179 |
Filed: |
June 4, 1987 |
Current U.S.
Class: |
536/25.3;
536/25.34; 987/223 |
Current CPC
Class: |
C07F
9/142 (20130101); C07H 21/00 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C07F 9/00 (20060101); C07F
9/142 (20060101); C07H 019/10 (); C07H
019/20 () |
Field of
Search: |
;536/27,28,29 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Matteucci et al., J. Am. Chem. Soc., 103 (11), 3185-3191 (1981).
.
Garegg et al., Tetrahedron Letters, 27 (34), 4055-4058 (1986).
.
Froehler et al., Tetrahedron Letters, 27 (4), 469-472 (1986). .
Setlow and Hollaender, "Genetic Engineering Principles and
Methods", vol. 4, pp. 1-17. .
"Oligonucleotide Synthesis a Practical Approach", Chapter 3 by
Atkinson and Smith entitled, Solid-Phase Synthesis of
Oligodeoxyribonucleotides by the Phosphitetriester Method, pp.
35-53. .
Marvin H. Caruthers, "Gene Synthesis Machines: DNA Chemistry and
Its Uses", Oct. 18, 1985, pp. 281-185, Science, vol. 230. .
Giles and Morrison, "An Economical System for Automated DNA
Synthesis", Mar./Apr. 1987, pp. 16-24..
|
Primary Examiner: Griffin; Ronald W.
Assistant Examiner: Tou; Jenny
Attorney, Agent or Firm: Smith; Joseph H.
Claims
We claim:
1. A method of capping failure sequences in solid phase
oligonucleotide synthesis, the method comprising the step of
condensing a capping agent with a hydroxyl of a failure sequence,
the capping agent being defined by the formula: ##STR5## wherein R
is straight-chain, branch, or cyclic lower alkyl containing from 1
to 6 carbon atoms, electron withdrawing substituted lower alkyl,
lower alkyl-substituted or halo-substituted aryl, or a nitrogen-,
oxygen-, or sulfur-containing heterocycle having from 5 to 8 carbon
atoms.
2. The method of claim 1 wherein said step of condensing includes
reacting said capping agent with said hydroxyl in the presence of a
sterically hindered acid chloride.
3. The method of claim 2 wherein R is a straight-chain, branched,
or cyclic alkyl containing from 1 to 6 carbon atoms, morpholinyl,
thiomorpholinyl, piperidinyl, piperazinyl, or a
beta-electron-withdrawing substituted ethyl, and wherein said
sterically hindered acid chloride is defined by the formula:
##STR6## wherein R' is tert-butyl, sec-butyl, cyclohexyl,
adamantyl, norbornyl, or phenyl.
4. The method of claim 3 wherein said capping agent is a salt
defined by the formula: ##STR7## wherein X.sup.+ is selected from
the group consisting of ammonium, lower alkylammonium, pyridinium,
lutidinium, cyclohexylammonium, and metal salt cations.
5. The method of claim 4 wherein X.sup.+ is selected from the group
consisting of triethylammonium, tetrabutylammonium,
diisopropylethylammonium, pyridinium, lutidinium, and
cyclohexylammonium.
6. The method of claim 5 wherein X.sup.+ is selected from the group
consisting of triethylammonium, tetrabutylammonium, and
diisopropylammonium.
7. The method of claim 6 wherein R is a straight-chained or
branched alkyl of 1 to 4 carbon atoms, phenylethyl,
beta-cyanoethyl, morpholino-piperidino, thiomorpholino, or
beta-nitroethyl.
8. The method of claim 6 wherein said sterically hindered acid
chloride is present in an equimolar amount as said capping
agent.
9. A method of synthesizing an oligonucleotide of a predetermined
sequence on a solid support, the method comprising the steps
of:
(a) deprotecting a 5'-protected oligonucleotide attached to the
solid support to form a deprotected oligonucleotide;
(b) reacting a 5'-protected nucleotide monomer with the deprotected
oligonucleotide to form either a 5'-protected oligonucleotide or a
failure sequence, the failure sequence having a 5' hydroxyl;
(c) capping the failure sequence by reacting a capping agent with
the 5' hydroxyl of the failure sequence, the capping agent being
defined by the formula: ##STR8## wherein R is lower alkyl,
electron-withdrawing substituted lower alkyl, or lower alkyl- or
halo-substituted aryl; and
(d) repeating steps (a)-(c) until the oligonucleotide of the
predetermined sequence is obtained.
10. The method of claim 8 wherein R of said capping agent is lower
alkyl; beta-cyano-, beta-nitro-, beta-phenylsulphonyl-, or
beta-phenylester-substituted ethyl; lower alkyl- or
halo-substituted phenyl; lower alkyl- or halo-substituted benzyl;
morpholino; thiomorpholino; or piperidino; and wherein said step of
capping includes reacting said capping agent with said 5' hydroxyl
of said failure sequence in the presence of a sterically hindered
acid chloride of the formula: ##STR9## wherein R' is tert-butyl,
sec-butyl, cyclohexyl, adamantyl, norbornyl, or phenyl.
11. The method of claim 9 wherein R' is tert-butyl, norbornyl, or
adamantyl.
12. The method of claim 10 wherein said capping agent is a salt
defined by the formula: ##STR10## wherein X.sup.+ is
triethylammonium, tetrabutylammonium, or
diisopropylethylammonium.
13. The method of claim 6 wherein R is a straight-chained or
branched alkyl of 1 to 4 carbon atoms.
14. The method of claim 6 wherein R is morpholino, piperidino, or
thiomorpholino.
15. The method of claim 9 wherein R is a straight-chained or
branched alkyl of 1 to 4 carbon atoms.
16. The method of claim 9 wherein R is morpholino, piperidino, or
thiomorpholino.
Description
The invention relates generally to methods for synthesizing
oligonucleotides, and more particularly, to the use of phosphite
monoesters to chemically cap failure sequences in either DNA or RNA
synthesis.
Genes and gene control regions can now be routinely characterized
and studied at the molecular level. This has been made possible by
recent advances in the technology associated with analyzing,
modifying, and synthesizing DNA and RNA. Of particular importance
has been the development of machines for the automated synthesis of
support-bound single stranded DNA, e.g. Matteucci and Caruthers, J.
Amer. Chem. Soc., Vol. 103, pgs. 3185-3191 (1981); and Gait, ed.,
Oligonucleotide Synthesis: A Practical Approach (IRL Press,
Washington, D.C., 1984).
The methods of choice for conducting automated DNA synthesis are
the phosphoramidite and hydrogenphosphonate chemistries, e.g.
Beaucage and Caruthers, Tetrahedron Letters, Vol. 22, pgs,
1859-1862 (1981); McBride and Caruthers, Tetrahedron Letters, pgs.
245-248 (1983); Froehler and Matteucci, Tetrahedron Letters, Vol.
27, pgs. 469-472 (1986); Garegg et al, Tetrahedron Letters, Vol.
27, pgs, 4051-4054 (DNA synthesis) and pgs. 4055-4058 (RNA
synthesis)(1986); and Froehler et al, Nucleic Acids Research, Vol.
14, pgs. 5399-5407 (1986). A synthetic cycle is repeated under
computer control to add one nucleoside monomer unit at a time to
achieve the desired sequence and length which defines the
oligonucleotide. For example, within the phosphoramidite, or
phosphite triester, synthetic cycle several reactions are
necessary:
I. Deprotect the reactive functionality (usually a 5' hydroxyl) on
the growing chain;
II. Achieve coupling by the addition of a monomer and
activator;
III. Cap unreacted 5' hydroxyls to prevent further coupling to
failure sequences; and
IV. Oxidize the newly formed internucleotide phosphorous linkage to
the naturally occurring pentacoordinate state.
The phosphoramidite method is highly optimized, allowing the
construction of oligonucleotides as much as 175 nucleotides in
length, Efcavitch, S. W., pgs. 65-70 in Biophosphate and Their
Analogues: Synthesis, Structure, Metabolism, and Activity, Bruzik
and Stec, eds. (Elsevier, Amsterdam, 1987). Such performance
requires an average yield per cycle of greater than 99%. An
essential feature of the synthesis cycle is an effective capping
reaction to permanently remove unreacted growing chains from
participation in subsequent cycles. Without capping, failure
sequences or deletion sequences, those oligonucleotides missing one
or more monomeric nucleotides with respect to the desired sequence,
will attain a greater average length than they would with capping.
The utility of capping is to minimize the length and presence of
failure sequences. With capping, a higher concentration of
monomeric nucleotide is available to the correctly growing
sequences of DNA. Moreover, with an efficient capping reation
performed each cycle, the correct sequence DNA, or product, is more
easily located, and thus purified by conventional means, such as
gel electrophoresis or HPLC. The presence of failure sequences
having nearly identical size and composition as the product makes
purification extremely difficult.
During phosphoramidite DNA synthesis, failure sequences are capped
by acetylation, effected by the concurrent delivery of acetic
anhydride and dimethylaminopyridine (DMAP) to the synthesis column.
The resulting 5' acetate ester cap prevents the sequence of DNA
from participatinag in subsequent condensation reactions in the
synthesis. Unfortunately, however, the acetate ester cap is removed
during the post-synthesis ammonia cleavage/deprotection step, which
makes failure sequence contaminants available to participate in a
variety of enzymatic reactions for which the complete sequences
were prepared. Such participation, for example, could measurably
reduce the efficiency by which DNA linkers are constructed making
their use in recombinant vectors more difficult. The availability
of a cap which survived the post-synthesis cleavage/deprotection
step would be highly useful.
For the hydrogen-phosphonate method, capping by acetylation is not
possible. Acetylation capping of the unreacted 5' hydroxyls of
failure sequences occurs at a useful rate only by catalysis with a
strong base, such as DMAP, N-methylimidazole, or triethylamine. The
internucleotide hydrogen phosphonate linkage is modified by
phosphorous acetylation under the influence of these strong bases.
The phosphorous acetylated residues are then susceptible to
cleavage during the post-synthesis cleavage/deprotection step,
resulting in internucleotide scission.
It has been claimed that a discrete capping step is unnecessary in
the hydrogen-phosphonate method due to acylation of unreacted
5'hydroxyls of failure sequences during the condensation step, e.g.
Froehler and Matteucci (cited above) and Froehler et al (cited
above). Acylation can occur by esterificaion of 5' hydroxyls by the
commonly used acid chloride activators or by the reactive coupling
intermediate. The acid chloride activator is present during the
coupling reaction to form the reactive coupling intermediate with
monomers. However, it has been demonstrated that coupling and
acylation can be incomplete during the condensation step, leaving a
certain amout of 5' hydroxyl available for increasing the size of
failure sequences during subsequent cycles of synthesis. An
effective capping operation for hydrogen-phosphonate DNA synthesis
is clearly desirable.
SUMMARY OF THE INVENTION
The invention is a method of capping failure sequences in
oligonucleotide synthesis by phosphitylation. Preferably, the
method involves solid phase, or support-bound, oligonucleotide
synthesis by phosphoramidite, phosphotriester, and/or nucleoside
hydrogen phosphonate chemistries. Capping is achieved by reacting a
phosphite monoester capping agent with the 5' or 3' hydroxyl of the
failure sequences between successive condensation steps in the
synthesis procedure. The 3' or 5' phosphite diester substituent of
the failure sequence is inert with respect to subsequent reaction
steps in the synthesis of the desired oligonucleotide product.
As used herein, the term capping refers to reacting either the free
5' hydroxyl of a 3' to 5' growing nucleotide chain or the free 3'
hydroxyl of a 5' to 3' growing nucleotide chain with a capping
agent to render the chain incapable of participating in subsequent
condensation steps. The preferred capping agents of the invention
are phosphite monoesters of the form: ##STR1## wherein R, either
alone or together with the oxygen to which it is attached, is
unreactive with the reagents used in solid phase oligonucleotide
synthesis, particularly phosphoramidites or nucleoside hydrogen
phosphonates. Preferably, R represents a lower alkyl, an
electron-withdrawing substituted lower alkyl, a lower alkyl- or
halo-substituted aryl, or a heterocycle containing nitrogen,
oxygen, or sulfur and from 5-8 carbon atoms. More particularly, R
is methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,
tert-butyl, n-pentyl, cyclopentylmethyl, isopentyl, neopentyl,
n-hexyl, neohexyl, isohexyl, cyclohexylmethyl,
betacyclopentylethyl, lower alkyl- or halo-substituted phenyl,
lower alkyl- or halo-substituted benzyl, or lower alkyl- or
halo-substituted phenylethyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, beta-electron-withdrawing-substituted
ethyl, or the like. In further preference, the electron-withdrawing
substituent of beta-electron-withdrawing-substituted ethyl is
cyano, nitro, phenylsulphonyl, or phenylester. Most preferably, the
beta-electron-withdrawing-substituted ethyl is beta-cyanoethyl. In
further preference, the lower alkyl- or halo-substituents of the
lower alkyl- or halo-substituted phenyl and benzyl are methyl,
chloro, or bromo. In further preference, morpholinyl,
thiomorpholinyl, and piperidinyl are morpholino, thiomorpholino,
and piperidino, respectively.
As used herein, the term lower alkyl refers to straight-chaind,
branched, or cyclic alkyls containing from 1 to 6 carbon atoms.
"Electron-withdrawing" denotes the tendency of a substituent to
attract valence electrons of the molecule of which it is apart,
i.e. it is electronegative, March, Advanced Organic Chemistry, pgs.
16-18 (John Wiley, New York, 1985).
As used herein, the term oligonucleotide refers to a single
stranded chain of either deoxyribonucleotides or ribonucleotides
having from a few, e.g. 2-20, to many, e.g. 20 to several hundred
or more, nucleotides.
The chemical structures illustrated by Formula I are referred to in
the literature as both phosphites and phosphonates. Reflecting the
approximate usage in the literature, throughout the structures will
be referred to as phosphites, except when R is a nucleoside. In
such cases the structure will be referred to as a hydrogen or
H-phosphonate.
The present invention overcomes deficiencies in both the nucleoside
hydrogen phosphonate and the phosphite triester methods of
oligonucleotide synthesis. Use of the capping step in the
nucleoside hydrogen phosphonate synthesis process significantly
enhances yields by reducing the average length of failure
sequences. In both the nucleoside hydrogen phosphonate method and
the phosphite triester method, attachment of the capping agents of
the invention renders the failure sequences incapable of
participating in subsequent biological experiments for which the
complete-sequence products are destined, e.g. 5' enzymatic
phosphorylation, either for labeling with .sup.32 P, or as a
pretreatment for subsequent ligation to other pieces of DNA.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 presents data illustrating the relative purity of reaction
products from hydrogen phosphonate syntheses of 34-mer
oligonucleotides withou (lane 1) and with (lane 2) the capping step
of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The invention includes a method for capping failure sequences in
oligonucleotide synthesis, and methods of synthesizing
oligonucleotides which include the capping method of the invention
as a step. As illustrated by Formula II, the capping method of the
invention comprises reacting a phosphite monoester defined by
Formula I, 1, with the free 5' or 3' hydroxyl of a failure
sequence, 2, in the presence of a sterically hindered acid
chloride, 3, to form a phosphite diester, 4, between the failure
sequence and a group which is inert to subsequent reaction steps.
##STR2##
Preferably, the capping agents of the invention (1 in Formula III
below) are prepared by alkaline hydrolysis of the symmetrical
phosphite diesters, 5, as described by Gibbs et al in Synthesis,
pgs. 410-413 (1984), which is incorporated by reference. The
phosphite monoester 1 can be used directly as a salt after
evaporating volatile by products of the reaction or after
purification by conventional means. ##STR3##
In the sterically hindered acid chloride 3, R' is preferably
tert-butyl, sec-butyl, cyclohexyl, adamantyl, norbornyl, phenyl,
aryl, or the like. More preferably, R' is tert-butyl, norbornyl, or
adamantyl. Most preferably, R' is adamantyl.
Preferably, X.sup.+ is ammonium, lower alkylammonium, pyridinium,
lutidinium, cyclohexylammonium, a metal salt cation such as
Na.sup.+, K.sup.+, Li.sup.+, Ba.sup.+, Mg.sup.+, or the like. More
preferably, X.sup.+ is triethylammonium, tetrabutylammonium,
diisopropylethylammonium, pyridinium, lutidinium, or
cyclohexylammonium. Most preferably, X.sup.+ is triethylammonium,
tetrabutylammonium, or diisopropylammonium.
Preferably, prior to delivery to the synthesis column bearing the
oligonucleotide, a phosphite monoester of the invention and its
cationic counter ion are dissolved in a solution comprising an
aprotic polar solvent, such as acetonitrile, tetrahydrofuran,
dichloromethane, or the like, or some combination thereof, and a
mild base such as pyridine, picoline, lutadine, collidine, or the
like. Pyridine is the most preferred mild base. Preferably, the
concentration of the phosphite monoester is between about 0.1 to
1.0 molar. Likewise, the sterically hindered acid chloride (3 in
Formula II), prior to delivery to the synthesis column, is
dissolved in a solution comprising an aprotic polar solvent, such
as acetonitrile, tetrahydrofuran, dichloromethane, or the like, or
some combination thereof, and a mild base such as pyridine,
picoline, lutadine, collidine, or the like. Pyridine is the most
preferred mild base. The respective solutions are delivered
concurrently to the synthesis column bearing the growing
oligonucleotide so that approximately equimolar amounts of the
phosphite monoester and sterically hindered acid chloride are
present in the reaction mixture. This operation can be readily
performed by an automated DNA synthesizer, such as the Applied
Biosystems models 380A, 380B, or 381A. The capping procedure of the
invention is performed as a step in each cycle, after the coupling
reaction, to render the failure sequences inert. Preferably, the
synthesis column is immersedin the reaction mixture for about
20-120 seconds at room temperature, after which the reagents are
flushedfrom the column with a solvent, such as acetonitrile,
tetrahydrofuran, dichloromethane, pyridine, or the like, or some
combination thereof. All vessels within the instrument must be
maintained rigorously free of moisture and oxygen under an
atmosphere of an inert gas, such as argon.
Detailed procedures for the phosphite triester and hydrogen
phosphonate methods of oligonucleotide synthesis are described in
the following references, which are incorporated by reference:
Caruthers et al, U.S. Pat. Nos. 4,458,066 and 4,500,707; Matteucci
et al, J. Amer. Chem. Soc., Vol. 103, pgs. 3185-3191 (1981);
Caruthers et al, Genetic Engineering, Vol. 4, pgs. 1-17 (198);
Jones, chapter 2, and Atkinson et al, chapter 3, in Gait, ed.,
Oligonucleotide Synthesis: A Practical Approach (IRL Press,
Washington, D.C., 1984); Froehler et al, Tetrahydron Letters, Vol.
27, Pgs. 469-472 (1986); Garegg et al, Tetrahedron Letters, Vol.
27, pgs. 4051-4054 and 4055-4058 (1986); and Froehler et al,
Nucleic Acids Research, Vol. 14, pgs. 5399-5407 (1986).
The following examples serve to illustrate the present invention.
The concentrations of reagents, temperatures, and the values of
other variable parameters are only to exemplify the invention and
are not to be considered limitations thereof.
EXAMPLES
Example I
Synthesis of Isopropylphosphite Triethylammonium Salt ##STR4##
Diisopropylphosphite (10.0 g, 0.06 moles), triethylamine (14.6 g,
0.14 moles), isopropanol (20 ml), and water (10 ml) were mixed in a
flask under an argon atmosphere and heated at 60.degree. C. for 48
hours. The volatile components were removed under vacuum, leaving a
viscous, clear oil. The resulting product was produced in 95% yield
(12.8 g) and had the following spectral data:
.sup.1 H nmr (acetone d6, chemical shifts relative to TMS):
10.1+3.3 (d, 1H, J=610 Hz), 4.4 (m, 1H), 3.15 (q, 6H, J=7 Hz), 1.35
(d, 6H, J=7 Hz), 1.20 (t, 9H, J=7 Hz)
.sup.31 P nmr (acetone d6, chemical shift relative to H.sub.3
PO.sub.4): 1.10 ppm J=610 Hz.
Example II
Synthesis of Ethylphosphite Triethylammonium Salt
The triethylammonium salt of ethylphosphite was synthesized by the
same procedure as Example I to give a product having the followng
spectral data:
.sup.1 H nmr (acetone d6): 10.0+3.4 (d, 1H, J=599 Hz), 3.85 (q, 2H,
J=7 Hz), 3.15 (q, 6H, J=7 Hz), 1.32 (t, 9H, J=9 Hz), 1.20 (t, 3H,
J=7 Hz)
.sup.31 P nmr (acetone d6): 0.64 ppm J=599 Hz
Example III
Reaction of Triethylammonium isopropyl phosphite with Thymidine
Attached to a Solid Support
A solution consisting of 0.1M triethylammonium isopropyl phosphite
in 1:1 acetonitrile:pyridine, and a solution consisting of 0.1M
1-adamantane carboxylic acid chloride in 1:1 acetonitrile:pyridine
were delivered concurrently to 1.0 micromole of thymidine linked
via a 3' succinate to a controlled-pore glass support in an Applied
Biosystems model 380B DNA synthesizer. After approximately 30
seconds, the solutions were removed from the column, and the column
was washed with acetonitrile. After oxidation of phosphite diester
linkage, the product was cleaved from the support with ammonia and
subjected to HPLC analysis. By comparison with an authentic sample,
it was determined that the major component of the product was
5'-isopropylphosphate thymidine.
Example IV
Synthesis of a 34 Base Oligonucleotide by the Hydrogen Phosphonate
Method With and Without Capping
The same 34-mer oligonucleotide,
5'-AGGGCCGAGCGCAGAACTGGTCCTGCAACTTTAT, was twice synthesized by the
hydrogen phosphonate method on an Applied Biosystems model 380B DNA
synthesizer following the procedure described by Froehler et al
(cited above), once including the capping step of the invention,
and once excluding the capping step. The capping step was performed
using the reagents and reaction conditions of Example III.
FIG. 1 illustrates the results of the gel electrophilic separation
of the material cleaved from the respective columns: lane 1
contains the material produced without capping, and lane 2 contains
the material produced with capping. The material in both lanes was
visualized by UV shadowing. It can be readily seen that the
material in lane 2 contains fewer failure sequences near the 34-mer
product, as determined by the intensity of lower molecular weight
bands near the 34-mer on the gel.
Example V
Synthesis of an 18 Base Oligonucleotide by the Hydrogen Phosphonate
Method With and Without Capping
The 18-mer oligonucleotide, 5'-TCACAGTCTGATCTCGAT, was synthesized
twice by the hydrogen phosphonate method, once with capping and
once without capping, following the same procedure as Example IV.
The material cleaved from each column was analyzed by HPLC and the
ratio of the correct sequence product to the most prevalent class
of failure sequences (17-mers) was determined from the areas under
the respective peaks on the chromatograms. The ratio with capping
was 33.9. The ratio without capping was 4.9.
The foregoing disclosure of preferred embodiments of the invention
has been presented for purpores of illustration and description. It
is not intended to be exhaustive or to limit the invention to the
precise form disclosed, and obviously many modifications and
variations are possible in light of the above teaching. The
embodiments were chosen and described in order to best explain the
principles of the invention and its practical application, to
thereby enable others skilled in the art to best utilize the
invention in various embodiments and with various modifications as
are suited to the particular use contemplated. It is intended that
the scope of the invention be defined by the claims appended
hereto.
* * * * *