Note: Descriptions are shown in the official language in which they were submitted.
~3~5~
--1--
Description
Defined Sequence Single Strand Oligonucleotides
Incorporating Reporter Groups, Process for the Chemical
Synthesis Thereof, and Nucleosides Useful in Such Synthesis
conical Field
The present invention relates Jo defined sequence
single strand oligonucleotides having a length of fewer
than 200 base units and which contain one or more
nucleotide.unlts to the base of which is attached a
substituent group which has bound thereto one or more
detectable reporter groups; such oligonucleotides being
useful in the identification, localization and detection
of complementary nucleic acid sequences of interest in
cellular or cell-free systems.
Background Art
The enzymatic production of.labelled, double stranded
deoxypolynucleotides has been accomplished with prior art
techniques involving the incorporation of radioisotopes
into doubles Rand DNA by the nick translation protocol
of P. Rugby et at, J. Mol. Blot. 113: 237-251 (1977),
or the gap-filling reaction described by G. Bourguignon
et at, . Viral. 20: 290-306 (1976). Specifically, a
-
nick is introduced via DBase, and then translated along
the DNA strand using a DNA polymers. During the nick
translation procedure, DNA polymers from E. golf pull It
will in the presence of added deoxynucleoside troughs-
plates, condense knuckleheads to the 3' hydroxyl terminus
in a single strand nick region of double-stranded DNA.
Simultaneously the enzyme will remove nucleotides Roy
-2-
the 5' end of the nick. I f one or more of the added
triphos~hates is labeled, for example with a- P-
phosphate, the label will be incorporated into the new
strand by Pot I. Following the gap-filling procedure,
recessed ends left after restriction ~ndonuclease cutting
can be filled in with Clown fragments from Pot I or
To DNA Polymers.
Both the nick translation and the ~ap-filling
procedures will yield double-stranded labeled DNA.
The length of the product depends upon how much DBase I
is added to the reaction. This is usually optimized so
that thirty percent of the label is incorporated and
the strands are 400 to 800 nucleated units in length.
The product length is unpredictably heterogeneous within
the 400 to 800 unit range. In order to conserve enzyme
as well as labeled nucleated, only one microgram of
DNA is usually labeled in each reaction vessel.
Double-stranded polynucleotides which incorporate
pyrimidine bases modified by carbon chains at the C-5
position have been prepared enzymatic ally in a similar
manner. This has been done by enzymatic elongation of
a homopolymeric primer-template as reported by J. Sari
et at., Bloc em. BiosPhys. Act. 6~6: 196-201 (1980). or
by nick translation/gap filling by DNA polymerizes using
2'-deoxyuridine 5'-triphosphate covalently linked to boo-
tin as reported by P. Lunger et at, Pro. Nat. Aged. Sat.
USA 78:6633-6637 (1981), the button being capable of
acting as a recognition site for aiding
Enzymatic methods described by Rugby, et at,
Bour~ui~non, et at. and Lunger, et at, result in products
having similar physical characteristics. Such enzymatic-
ally prepare polynucleotides are 400-800 units in length,
--3--
require double-stranded polynucleotides as starting
materials, produce double-stranded PolYnucleotides
in all cases, and do not allow Labeling at preselected
sites.
In addition, all enzymatic methods modify both
strands of the Polynucleotide. and such product strands
cannot be isolated from one another. During such pro-
cusses, enzymes replace either all units with modified
units or, when provided with a mixture of modified
and naturally-occurrin~ nucleoside triphosphates,
randomly insert modified units. Furthermore, the envy-
matte process described by Lunger, et at, is incapable
of producing polynucleotides incorporating such reporter
groups as fluorescent, luminescent, or antigenic reporter
groups. None of this art is capable of producing oily-
nucleotides of defined length, defined sequence or single-
stranded character, either with or without reporter groups.
Moreover, by the prior art methods, modified bases having
reporter groups attached thereto cannot be incorporated
in a polynucleotide at preselected sites.
Double-stranded polynucleotides which incorporate
adenine bases modified at the C-8 position have also
been prepared enzymatic ally. This has been done by
incorporating 8-aminohexylamlno-ATP (a ribonucleotide)
onto DNA fragments, as reported by C. Vincent et at,
Null. Acids Rest 10:6787-6796 ~1982). The method is
limited in scope, however, allowing only end
labeling with triphosphates of adenine rabbinical-
tides. No modified pyrimidine nucleosides can be
incorporated. Furthermore, as with other enzymatic
methods, both strands of a double-stranded polynucleo-
tide are labeled and no short (~100 units) oligonucleo-
tides of defined sequence can be produced.
~23~5~:1
The prior art enzymatic methods referred to above
require the chemical synthesis of a substituted
nucleoside 5'-tripho'sphate, and demand subsequent
enzymatic recognition and incorporation of the unnatural
5 substrate into nicked' double stranded DNA. Such methods
are incapable of producing polynucleotides of any pro-
selected length or sequence, and the polymerizes and
DNases used therein are expensive. Moreover, these
methods are time consuming, inefficient, demand sub Stan-
10 trial enzymatic activity and are limited to double-stranded
DNA. Only small amounts, i.e., micrograms, of ill-defined
polynucleotides, usually restriction fragments, are
produced, and these must be tediously isolated from
natural sources. Moreover, the scope of modifications
lo obtainable in the polynucleotide product is severely
limited, since the DNA polymers cannot recognize or in-
corporate potentially useful reporter groups such as
fluoresce in or dinitrophenyl.
Attachment of one fluorescent molecule to the 3' end
20 of long polyribonucleotide molecules (RNA) for limited
biological application is disclosed by GO Bagman et
at, J. Histochem. Cy~ochem. 29:238 (1981~. This approach
also used very small amounts (microgram quantities) of
RNA tediously isolated prom natural sources using envy-'
25 matte methodology, and cannot be applied to DNA since
both 2' and 3' hydroxyls are required therefore The much
greater chemical instability of RNA relative to DNA also
minimizes the scope ox application of the polyribonucleo-
tide produced by this method.
The non-enzymatic synthesis of defined sequence
oligonucleotides incorporating naturally-occurring nucleic
acid bases has been reported or reviewed by SPA. Nearing
et at, Mesh. Enamel 65:610 (1980), R. Let singer, J.
Chum. 45:2715 (1980), M. Mattocks et at, J. Amer. Chum.
2 3
--5--
Sock 103:3185 (~982), and G. Alvarado-Urbina et at,
Science 214:270 (1981). Such synthesis usually in-
valved chain extension by coupling an activated
nucleated monomer and a free hydroxyl-bearin~
terminal unit of a growing nucleated chain. The
coupling is effected through a phosphorus-containing
group, as in the phosphate trimester method reviewed
by Nearing et at, or one of the phosphate trimester
methods. Of the latter, those of Let singer et at
and Alvarado-Urbina et at use phosphochloridite
chemistry, and that of Mattocks et at uses phosphor
amidite chemistry.
The aforementioned chemical synthesis of oligo-
nucleotides has incorporated only unmodified or
naturally-occurring nucleic acid bases, and the end
product there of is in short fragments and resembles
unmodified or naturally-occurring RNA or DNA. It is
also worthy of note that the end product of such
synthesis does not incorporate any labels or reporter
groups.
Direct modification of homopolymeric polynucleo-
tides has been reported in systems of polyuridylic
acid rBigge, et at, J. Curb., Nucleosides, Nucleotides
8:259 (1981)]. The reported procedure is of limited
scope and is not productive of useful products.
Treatments described cause extensive polynucleotide
cleavage and degradation, result in product irrever-
silly contaminated with metal ions, and are capable
of modifying only citizen residues of a DNA pylon-
clouted. The method is incapable of producing defined sequence oligonucleotides of specific
lengths, cannot modify thiamine or Purina bases, and
cannot modify at previously selected sites.
I I
From the foregoing it will be apparent that
labeled, defined sequence polynucIeol:ides have been
produced heretofore only by enzymatic methods which
have the disadvantages pointed out herein, portico-
laxly those relating to time, cost, product length sequence and yield. Moreover, such methodology is
productive of only double-stranded products. Such
double-stranded polynucleotides can be denatured by
alkali or heat to cause spontaneous short-term swooper-
lion of strands in solution. However, the individual single strands cannot be physically isolated from each
other, and removal of the denaturing conditions results
in rapid renatura~ion to double-stranded form. Since
conditions productive of hybridization are also pro-
ductile of renaturation, subjecting the denaturedpolynucleotide to hybridization conditions results in
; return of the polynucleotide to its original double-
stranded configuration in which hybridization of either
of the strands to a target polynucleotide is limited
I by competition from the other strand.
Lo
--7--
odif~a~ion of nucleosides has been undertaken,
for example, in the synthesis of anti viral C-5 sub-
stituted pyrimidine nucleosides disclosed by Bergstrom,
et at, J. Amer. Chum. Sock 98:1587-1589 (1976); Ruth,
5 et at, J. Org. Chum. 43:2870-2876 (1978); and Bergstrom,
et at, Us S. Patent Nos. 4,247,544 and 4,267,171, or
in the synthesis of C-8 substituted adenine derivatives
disclosed by Zappelli, et at, U. S. Patent Nos.
4,336,188 and 4,199,498. These nucleosides, and
10 others reported by Lunger, et at, and D. Ward, European
Patent Application No. 0063879, are not useful in
the process of the present invention. Such reported
nucleosides are highly reactive at undesired sites,
and, if used in oli~,onucleo~ide synthesis ~,~; chemical
15 methods, would result in undesired side proxy unwon-
troll able synthesis, and no desired product. Moreover,
such reported nucleosides do not contain wrier sites
; in the substituent group, cannot be modified by the
attachment of reporter groups nor do they contain
20 masked reactive functionalities. No such nucleosides
are useful in the process of the present invention.
No chemical synthesis of defined sequence oligo-
; nucleotides incorporating modified bases of any kind,
either with or without reporter groups, has been
2, disclosed in the prior art.
There is an urgent need for high quality labelled,defined sequence single strand oligonucleotides which
do not involve hazardous and unstable radioisotopes,
and satisfaction of this need is a Principal object of
Lo 3
--8--
the present invention. This object is accomplished by
a chemical,. i.e., non enzymatic., process which provides
a predictable, superior product in high yield. More
particularly, the process of the present invention
accomplishes the chemical incorporation into defined
sequence oligonucleotides of nucleotides modified with
a wide variety of selected detectable reporter groups,
such oligonucleotides being useful, for example, for
the identification, localization, isolation and/or
quantitation of complementary sequences of interest.
Another object of the invention is to provide
novel nucleosides useful in the chemical synthesis of
labeled, defined sequence single strand oligonucleo-
tides.
The process of the present invention accomplishes
the de nova chemical synthesis of labeled, defined
sequence oligonucleotides and is superior to prior
art enzymatic methods in a number of respects. Gore
specifically the process of the present invention makes
possible the synthesis of labeled, defined sequence
single-stranded oligonucleotides of homogeneous pro-
dictably defined length, preferably having fewer than
200 base units, in contrast to the production of
heterogeneous unpredictable populations of 400 to
10,000 base units of double-stranded character produced
by prior art enzymatic methods.
The yield of product produced by the process of
the present invention is of the order of hundreds to
tens of thousands of micrograms, in contrast to the
yield of a few micrograms provided by the prior art
enzymatic methods. Moreover, the product oligonucleo-
tides of the present invention are single-stranded,
rather than the double-stranded products of enzymatic
Lo
go
methods. The single strand configuration of the
product oligonucleotldes avoids the competition
from a complementary strand inherent in reburied-
ration of double-stranded polynucleotides.
Disclosure of Invention
The invention comprises a defined sequence single
strand oligonucleotide of fewer than about 200 base
units in length which comprises at least one nucleon
tide unit having attached to the base thereof at a
starkly tolerant site (such as C-5 of pyrimidines
and C-8 of urines) a substi~uent group which has bound
thereto one or more detectable reporter groups. The
invention Allah comprises a process for the chemical,
i.e., non enzymatic, synthesis of a defined sequence
single strand oligonucleotide which comprises coupling
an activated nucleated monomer and a free hydroxyl-
bearing terminal unit of a growing nucleated chain,
at least one of the monomer and terminal unit having
its base modified at a circle tolerant site by
attachment thereto of a substituent group capable of
binding one or more detectable reporter groups. The
invention additionally comprises novel nucleosides and
nucleated useful in this synthetic process.
The oligonucleotide produced by the process of the
present invention may include one or more pyrimidine-
based or purine-based units which may be ribonucleotides
or deoxyribonucleotide, and prior to the synthesis
thereof, the reporter groups are preselected, as are
the particular nucleated units to which the reporter
groups are attached.
s;
. . .
~L~3~6~1
-10-
Best Mode of Carrying Out the Invention
The chemical process by which the defined sequence
single strand oli~onucleotides of the present invention
are preferably synthesized comprises coupling an anti-
voted nucleated monomer and a free hydroxy-bearing
terminal unit of growing knucklehead chain, at least
one of said monomer and terminal unit having its base
modified at a starkly tolerant site by attachment
thereto of a substituent group capable of binding one
or more detectable reporter groups.
The substituent groups of the present invention
which are capable of binding reporter groups can be
generally characterized as those which exhibit nucleon
Philip properties with respect to such reporter groups.
Exemplary of such substituent groups are those which
contain primary or aromatic amine, carboxylic acids,
hydroxyls and the like.
The bates of the nucleated monomer and terminal
unit are selected to provide the predetermined sequence
of nucleated units desired in the end product oligo-
nucleated. Such bases can take the form of the urines
adenine (A), guanine (Go, or hypoxanthine (Ho, or of the
pyrimidines Ursula (U), citizen (C), or thiamine (T).
Such bases may also take the form of any other base which
can be isolated from natural sources.
A starkly tolerant site on the nucleated unit can
be defined as a position on the nucleic acid base of the
unit at which modification of the unit can be effected
by attachment whereto of the substituent group without
causing significant interference with hybridization of
the product oligonucleotide and complementary nucleic
acid component, and without starkly preventing the
substituent group from binding one or more reporter
groups. Circle tolerant sites are found at the C-8
~L~3~5~
position of porn and at the C-5 position of pyrimi-
dines. Since the oligonucleotid~s of the present
invention are particularly useful as hybridization
probes, modifications which attach substituent and/or
5 reporter groups should not be at sites on the pyrimi-
dine or Purina bases which are necessary for specific
hybridization Sites which should not be modified
include No and ,6 of adenine bases; No, No, and ox
of guanine bases; No and No of citizen bases. Goner-
ally, substitution a any heteroatom (N or O) should be avoided.
A reporter groups can be defined as a chemical group
which may be aromatic and/or polycyclic and which has
a physical or chemical characteristic which can be
readily measured or detected by appropriate physical
or chemical detector systems or procedures. Reporter
groups which are useful in oligonucleotides of the pro-
sent invention are readily detectable. Ready detect-
ability may be provided by such characteristics as
color orange, luminescence, fluorescence, or radio-
activity; or it may be provided by the ability of the
reporter group to serve as a ligand recognition site.
Such groups are termed functionally calorimetric,
luminescent, fluorescent, radioactive or ligand recog-
notion groups. Among such groups are those suitable for ready detection by conventional detection techniques,
such as calorimetric, spectrophotometric, fluorometric
or radioactive detection, as well as those which are
capable of participating in the formation of specific
ligand-ligand complexes which contain groups detectable
by such conventional detection procedures.
A reporter group as used herein may be characterized
as a label which has physical, chemical or other
-12-
characteristics which can be readily measured or de-
tooted by the use of appropriate measurement or detect
lion procedures. A reporter group as defined herein
also includes ligand recognition groups which are capable
5 of initiating one or more ligand-ligand interactions
which ultimately provide a reaction product or a complex
having physical, chemical or other characteristics which
can be readily measured or detected by the use of appear-
private measurement or detection procedures.
Exemplary of the measurable or detectable kirk-
teristics which such groups, reaction products or come
plexus may exhibit or induce are a color change,
luminescence, fluorescence or radioactivity. Such
characteristics can be measured or detected by the use
15 of conventional calorimetric, spectrophotometric,
fluorometric or radioactivity sensing instrumentation.
The interactions which usefully can be initiated
by the reporter group defined herein include appropri-
lately specific and selective ligand-ligand interactions
productive of groups or complexes which are readily
detectable, for example, by calorimetric, spectxophoto-
metric, fluorome~ric, or radioactive detection prove-
Doria. Such interactions may take the form of protein-
ligand, enzyme-substrate, antibody-antigen, carbohydrate-
lath protein-cofactor, protein-effector, nucleic
acid-nucleic acid or nucleic acid-ligand interactions.
Exemplary of such ligand-ligand interactions are
dinitrophenyl-dinitrophenyl antibody, biotin-avîdini
oligonu~leotide-complementary oligonucleotide, DNA-DNA,
RNA-DNA and NADH-dehydrogenase. Either one of each such
ligand-ligand pairs may serve as a ligand recognition
type reporter group. Other useful interactions will
suggest themselves Jo those skilled in the art.
-13-
In the process of thy present invention, a selected
reporter group or groups can optionally be attached to
the nucleotlde monomer before coupling of the monomer
to the terminal unit of the nucleated chain, or it
can be attached to the product oligonucleotide after
formation thereof. The sequence of the nucleated units
in the product oligonucleotide is preselected to provide
such oligonuc1eotide with precise specificity for its
ultimate use. The oligonucleotides of the present
invention are useful tools in recombinant DNA and other
protocols involving nucleic acid rehybridization tech-
piques. Among such uses are identification, focalize-
lion, isolation and/or quantitation of complementary
sequences of interest in cellular or cell-free systems.
More specifically, such uses may include diagnostic
applications or any fundamental biological event in-
valving hybridization of nucleic acid components, or
purification of complementary sequences by affinity
chromatography when the product oligonucleotide is
attached to a solid support through the modifications
at a starkly tolerant site, with or without sub-
sequent detection.
The nucleated units in the product oligonucleotide
may be Purina or pyrimidine-based and may comprise units
having naturally~o~curring bases intermixed with units
having modified bases. Such units may be rabbinical-
tides or deoxyribonucleotide. The coupling step pro-
fireball involves coupling of a monomer unit activated
at the 3' position with a free 5' hydroxyl of the ton-
final unit of the growing nucleated chain. Alterna-
lively such coupling can involve coupling of a monomer
unit activated at the 5' position with a free 3' hydroxyl
of the terminal unit of the nucleated chain. The
- 1 4 - I I
terminal unit may be the initial or only unit in the
rowing nucleated chain at the time of coupling
thereto of the nucleated monomer or it may be the
terminal one of a plurality of nucIeotide units.
The process of the present invention produces
defined sequence oligonucleotides of the following
generic formula:
5, B
HO - _ B
OPT I
HO R
FORMULA I
Therein n is 1 to about 199, preferably about 5 to.
about 60, and most preferably about 10 to about 40, R'
is hydrogen or hydroxy, and B it any of the naturally-
occurring Purina or pyrimidine bases adenine, guanine,
citizen, Ursula, thiamine, or any other naturally-
occurring base, the nucleated units having naturally-
occurring bases being independently intermixed with owner more nucleated units having modified bases (By).
The modified pyrimidine bases (Pam) are substituted at
the C-5 position, and typical examples thereof are the
Ursula and citizen bases illustrated by the following
generic formulas:
- 15~ I
NH"
p m I Hi Jo or NOR
OWN OWN
modified Ursula base modified citizen base
The modified Purina bases (Put) are substituted at the
C-8 position, and typical examples thereof are the
modified adenine and guanlne base striated by the
5 following ~en~lric formulas:
put ,. No R or NJ~Z
modifies Rdenine base modified Gwen base
.
The substituent group R is characterized by its ability
to bind one or more reporter groups. In the modified
pyrimidine bases the substituent group R comprises two
or more carbon atoms, whereas in the modified Purina
base R comprises one or more carbon atoms. In this context,
'''";~
-16~ 6
R preferably take the form of one of the following
unctionalized carbon chains:
R OH ~Rl~2, CH2GHRlR2, Cruller,
o '1
I! '
-CH=CRl-~NHR2, or -CH=CRl-~-R~
wherein Al is hydrogen or alkali R2 us alkyd, alkenyl,
aureole, or functionalized alkyd, alkenyl, Rowley wherein
functional groups include one or more amine 9 asides,
nitrites, carboxylic acids and esters,hydroxy, dint-
trophenyl, aminobenzenesulfonates, or the like; and Z
is a polyval~nt heteroatom such as nitrogen, oxygen
or sulfur. In addition, R2 may be attached to a solid
support, or to one or more reporter groups which lung-
lion, for example, as a calorimetric, fluorescent,
luminescent, radioactive, or ligand recognition group.
Functionally fluorescent groups include fluoresce ins,
radiomen, an the like or adduces thereof; function-
ally luminescent groups include luminous, assuredness,
Lucifer ins, dioxetanes, dioxamides, and the like or
adduces thereof. Ligand recognition groups include
vitamins (such as button or adduces thereof, including
iminobiotin and desthiobiotin), antigens such as donator-
phenols, carbohydrates and other functional groups or
adduces of such groups which can be recognized by ligand-
like interactions with proteins or from which such ligand-
like interactions can be elicited. Another oligonucleotidecapable of interaction with nucleic acids is illustrative
of a group from which a ligand-like interaction can be
elicited. Ligand recognition groups may also serve as
functionally calorimetric reporter groups when recog-
notion results in dye formation. For example, whendinitrophenyl is used as a reporter group, known detection
systems using an antidinitrophenyl antibody coupled to
peroxides can be used as a detection system, resulting
in a color change. Functionally radioactive groups
incorporate a radioactive element in the chosen reporter
group.
7 -
. .
~3~5
- 1 7 -
When reference is made herein to the use of Purina
or pyrimidine bases, such expressions are intended to
include analogs of such bases. Among such analogs are
the analogs of Purina bases, such as the deazaadenosines
(tubercidins, formycins, and the like), and the analogs
of pyrimidine bases, such as desirously, deazacytosine,
azauracils, azacytosines, and the like.
Oligonucleotides of Formula I are best prepared by
chemical synthesis from monomer nucleated analog units
10 of the formula:
(~) s PYRE
or '
R joy I = OPERA
O Where:
R6 = methyl, sheller
phenol
FORMULA II X - sheller, dialkyl-
amino, morpholino
wherein R3 is tritely (triphenylmethyl), dimethoxytrityl,
or other appropriate masking group for the 5'~hydroxyl;
B and R' sure masked, if appropriate; and represents
15 a phosphorus-containing group suitable for internucleo-
tide bond formation during chain extension in synthesis
of a product oligonucleotide. The phosphorus-containing
groups suitable for internucleotide bond formation
are preferably alkyd phosphomonochloridites or alkyd
20 phosphomonoamidites, Alternatively phosphate trimesters
may be employed for this purpose. The monomer unit may
, .
-18- .
alternatively have R3 attached at the 3' hydroxyl and
attached at the 5'-hydroxyl.
Generally, the term "masking group" or "blocking
group" it a functional expression referring to the
5 chemical modification our "blocking" of an integral
functional group by attachment of a second moiety to
disguise the chemical reactivity of the functional
group and prevent it from reacting in an undesired
manner. Such modification is reversible, and allows
10 subsequent conversion back to the original functional
group by suitable treatment. In many cases, such mask-
in formally inter converts structural functionality,
e.g., a primary amine masked by acetylation becomes a
substituted aside which can be later converted back to
15 the primary Camille by appropriate hydrolysis.
The compounds of Formula I include the acceptable
conjugate acid salts thereof. Conjugate acids which
may be used to prepare such salts are those containing
n~nreactive cations and include, for example, nitrogen-
20 containing bases such as ammonium salts, moo-, do-,
in- or tetra-substituted amine salts, and the like, or
suitable metal salts such as those of sodium, potassium,
and the. like.
The process steps of the present invention will now
25 be generally described and illustrated diagra~atically.
Thereafter, the.i~ention will be illustrated more specie
call and detailed examples thereof provided. Since the
invention Ritz Jo oligonucleotides incorporating ought
pyrimidine-based and purine-based nucleated units, the
30 use of both pyrimidine and purine-based compounds in the
synthetic process will be illustrated. The specific
pyrimidine and purine-based compounds illustrated are
only exemplary of the respective pyrimidine and Purina
classes, and it is to be understood that any other member
--19- '
of the respective class can be substituted therefore
in the process and the product oligonucleotide, when
ever suitable or desired. While deoxyribonucleotide
compounds are shown for the most part, it is under-
5 stood that ribonucleotide compounds are also con-
template by the invention and can be substituted for
the deoxyribonucleotide compounds wherever rabbinical-
tide compounds are desired in the product oligonucleo-
tide.
One of the more important aspects of the invention
is the provision of a new class of nucleosides which
are essential as intermediates in the process for sync
the sizing the new oligonucleotides. Such nucleosides
each have a base which is modified by a substituent
15 group comprising a functionalized carbon chain and one
or more asides, the nitrogen of the asides being attached
to a starkly tolerant site on the base through the
carbon chain. In the case of pyrimidine-based nucleon
sides, the carbon chain it attached at the C-5 position,
20 and in the case of the purine-based nucleosides, the
carbon chain is attached at the C-8 position through
a polyvalent heteroatom, such as nitrogen, oxygen or
sulfur. In addition, such nucleosides are chemically
blocked at the 5' position (or the 3' position) with
I a group, such as dimethoxytrityl, appropriate for the
chemical synthesis of oligonucleotides.
-20- 3 I
In the new class of nucleosides the substituent
group may be chosen from CH2CHRlCn on . 1 n on '
CHRl~nH2nY, OH Curl C NHCnH2n , 1 n Zen
wherein Al is hydrogen or Clue lower alkali n is
5 0 to 20 and Y it one or more amino, substituted amino,
substituted amino or substituted aminoalkylphenyl groups.
O
More specifically, Y may include one or more -NH~CX3
wherein X it hydrogen, fluorine or chlorine. Synthesis
of these nucleosides, as well as of the masked forms
10 thereof described hereinafter in Examples I, II, IV,
VI, VII, VOW, X, XII, and XIII. Preferred nucleosides
incorporate the substituent group -CH=CHG NCHnH2nY at
the C-5 of pyrimidine nucleosides wherein n = 3 to 12
15 and Y is -NHCCX3. Most preferred are such nucleosides
wherein the pyrimidine base is Ursula.
The process of thy present invention may be initiated
by the preparation of the selected knucklehead. Generally 9
the most preferred nucleoside~ are best prepared in the
20 following manner. methyl 3-acrylyl)-2'-deoxyuridine
is prepared from 2'-deoxyuridine by the method of
Bergstrom and Ruth [J. Amer. Chum. Sock 96:1587 (1976)].
The nucleoside is then treated with 1.05 equivalents of
dimethoxytrityl chloride in pardon for 4 hours to
25 block the 5' hydroxyl with dimethoxytrityl (DOT). The
resulting product is purified by silica chromatography
eluding a gradient of 0-10% methanol in chloroform
containing 2Z triethylamine. The purified 5'-DMT-5-
(methyl 3-acrylyl)-2'-deoxyuridine is treated with
30 1 N KOCH for 24 his. at ambient temperature to hydrolyze
the methyl ester.
-21~
The resulting 5'-DMT-5-(3~acrylyl)-2'-deoxyuridine
is treated with excess dicyclohexylcarbodiimide and
hydroxybenztriazole in pardon. After 4 hours, a
2-5 fold excess of,yl,~-diaminoheptane is
5 added, and the reaction stirred overnight. After
12-20 hours, a 10-20 fold excess of trifluoroacetic
android is added, and the reaction stirred at room
temperature for 4 hours. The product is purified
by silica chromatography eluding a gradient of 0-10%
10 methanol in chloroform containing 2% triethylamine,
followed by exclusion chromatography using Sephadex
LH-20 eluding 1% triethylamine in methanol. Appear-
private fractions are combined to yield 51DMT-5-
[N-(7-trifluoroacetylaminoheptyl)-l-acrylamido]-2''-
15 deoxyuridine; such product is appropriate for oligo-
nucleated synthesis by the phosphochloridite prove-
dune described in Examples XV and XVIII. Alternatively,
such a compound can be prepared by the combination of
methods described in Examples II and III. Replacing
20 diaminoheptane in this process with other Damon-
alikeness (e.g., diaminopropane, diaminohexane, Damon-
dodecane) is productive of other compounds of varying
substituent length wherein n = 3, 6, Go 12 and
P
R = -C~l=CHC NHCnH~nNHCCX3. Two such nucleosides, one
25 pyrimidine (uracil~based and the other Purina
(adenine)-based, are shown at the top of the diagram
below illustrating the process. Reactive sites on
the bases of the nucleosides are then masked, as shown
in Reaction 1, by attachment of, for example, a
3C bouncily group (By) to the amine at the
-22 I
6 position of the adenine-based nucleoside. Such
masking it generally described in "Synthetic Pro-
seeders in Nucleic Acid Chemistry", Vol. 1, W. Zorbach
and R. Tip son ens. (Wiley - Intrusions, NAY., 1968)
Unprotected amine on the substituent group are masked,
for example, by attachment thereto of triflu~roacetyl
groups (A), a also shown in Reaction 1.
The selected 3' or 5' hydroxyl of the nucleoside
is then masked by attachment thereto of a dimetho-
10 xytrityl (DOT) group. In Reaction 2 illustrated below the 5'-hydroxyl is masked, leaving the 3' hydroxyl
free or available for reaction. Alternatively, the 3'
hydroxyl could be masked, leaving the 5' hydroxyl free.
The nucleoside is then converted Jo an activated
nucleated monomer, preferably by attachment to its 3'
hydroxyl Off a phosphorus-containing group which includes
an activating moiety. When the modified nucleoside is
properly blocked, modifications of the procedures de-
scribed by Let singer, et at, Mattocks, et at, or as
reviewed by Nearing, et at can be utilized for oligo-
nucleated synthesis. The use of phosphochloridite
chemistry such as that disclosed by Letsingf2r en at,
is detailed in Examples XVI-XVIII. In order to. use
phosphoamidite chemistry, a modification of the prove-
dune of Mattocks, et at, is used, phosphitylatingthe protected modified nucleoside with methyl sheller
(N, N-diisopropyl)phosphoamidite or methyl sheller-
phosphomorpholidite, a in the improved procedure of
Dormer, et at [Nucleic Acids Rest 11:2575(1983)]. Al-
fry r
i f,' ,:
' of
-23-
ternatively, the protected modified nucleoside can be
phosphorylated with 1~2 en. chlorophenyl dichlorophos~
plate in trimethylphosphate at room temperature lot-
lowed by quenching with water to give the sheller-
5 phenol phosphate adduce of Lye modified nucleoside, suchadducts being useful in a modification of the phosphor
trimester approach as illustratively reviewed by Nearing,
et at. The diagram illustrates in Reaction 3 the sync
thesis of activated monomer nucleated units of Formula
10 II by attachment to the nucleoside 3' hydroxyl of a pros-
phomonochloridite group in which the chlorine functions
as an activating moiety.
Coupling or condensation of the selected activated
nucleated monomer, i.e. the uracil-based monomer or
15 the adenine-based monomer, to the terminal unit of a
growing nucleated chain is illustrated in Reaction 4
in the diagram. The nucleated chain is shown as in-
eluding in its right hand end a nucleoside unit having
a naturally occurring base and having a solid support
o'er masking group R4 attached to its 3' hydroxyl. The
illustrated chain also includes one or more (n') nucleon
tide units having naturally-occurring bases, said units
being coupled to the 5' hydroxyl of the nucleoside unit,
the terminal one of the nucleated units having a free
25hydroxyl at the 5' position. In the coupling reaction
~'~ I 5
-24-
the chlorine of the monomer reacts with the hydrogen of
the free hydroxyl of the terminal unit and is displaced,
50 that the oxygen of the terminal unit couples to the
phosphorus of the monomer as shown, and the monomer
thereby becomes the new terminal unit of the nucleated
chain.
The DOT 5' blocking group is then removed to permit
further extension of the nucleated chain by sequential
coupling thereto of additional activated nucleated
monomer units. The nucleated units added to the chain
can be preselected and may have either naturally-occur-
ring or modified bases. The diagram shows in Reaction
pa the further extension of the chain by the addition
of one or more (n") nucleated units having naturally-
occurring bases.
When an oligonucleotide of the selected length and sequence has been synthesized, the DOT group is removed
from the terminal unit thereof, and the masked reactive
groups are unmasked. Examples of modified Ursula and
adenine bases with their reactive groups unmasked are
also shown diagrammatically at Reaction 5. If the
initial nucleated unit of the chain is bound to a solid
support R4, the chain is then removed from such solid
support. The appropriate order of unmasking can be pro-
selected.
Reporter groups R5 appropriate for the
.
it ! ' ' , i
-25-
intended use of the product oligonucleo~ide can then
be bound to such substituent groups as exemplified
in Reaction 6, which illustrates the respective bases
with reporter groups I bound to the respective
5 substituent group thereof.
Illustration of Synthetic Process
a ~N~CH2~7N~2 SHEEHAN
Ho 0 ¦ Starting Ho 0
Nucleosides
Ho Ho
Ursula based adenine-based
. Reaction 1:
Mooney of reactive sites
, on nucleoside base 8
H~a~CH2~NHC:C: FJ I N H C C I
HO HO
Jo HO ( By bouncily
so
-26-
Reaction 2
Masking of 5'-hydroxyl
. I (DOT = d.~methoxytrityl) r
HN~a~cH2~NHCC lFJ No\> N~CH2~NHCC FJ
DMT-0~ DMT-0
HO HO
reaction 3
Activation to phosphor
~chloridite r
HN~H~CH2~NHCCF3 ~N~CH2~NHCCF,
DMT-O DMT-O
O Monomer nucleated O
SHOP- units of Formula II SHOP:
-27-
Reaction 4
Condensation to terminal unit
of growing oligonucleo~ide chain
COO _
/ OR
Growing oligonucleotide chain \ ,
(x = amine masking groups
. on A, C, G, or sub-
\ stituent grow
\ '
Box --r
DMT-O~
o ox 1 X
O
Lo H3 _
OR 4
I 3
-28-
I
Reaction pa
¦ Further chain eon-
gushiness
r
BY r
DMT-O--~'~J
of
O Box
lo H O
Pi 1 BY
O Pow
OR
. Reaction 5
HO - P Deb locking, unmasking,
r removal from R4
. I
O By
OPT
OWE I
PRODUCT B
POW
OLIGONUCLEOTIDE _ _ ,~" Y
n ' + n" = O to about 198 Jo
where By modified base (see
pug 29)
I 3
-29-
where, for example, By in product oligonucleotide
(pug 28) is:
C3 No
Hl.J~N~C~12~NH2 ~N~CH2~NH2
Ursula base adenine base
(modified) (modified)
To the product oligonucleotide a variety of useful
reporter groups (R5) may be attached. For example:
I
Product oligonucleotide Product oligonucleotide
with above modified with above modified
. Ursula be, ye adenine base
Reaction 6
N-hydroxy- Attachment of FIT
; succinimidyl reporter groups (R5)
buttonhole-
aminocaproic NH2
acid ~cH2~6NHcNH-fluor0~cein
r '.
O O `
HNJ~N~cH2~NH~cH2 ~NH-biotin
I
PRODUCT OLIGONUCLEOTIDES WITH REPORTER GROUPS (where
R5 = blown or fluoresce in)
-30- 3
Having discussed the process of the present
invention in general terms and illustrated the same
diagrammatically 9 each of the reactions referred to
will now be discussed more specifically.
I With reference Jo reaction 1, masking ox chemically
reactive amine such as No of citizen, No of adenine,
N of guanine, and alkyd or aureole amine of the modified
bases with suitable masking groups can be conveniently
accomplished in suitable solvents such as alcohols, pyre-
10 dines, letdowns, chloroform, and the like, by reaction of the nucleosides with an excess of appropriate acid
androids for about 1 to 24 hours at temperatures in
the range of OKAY to 110C, generally 20C to 80C.
Appropriate acid androids include acetic android,
15 trifluoroacetic android, bouncily android, anisoyl
android, and the like. Preferred are acutely, in-
fluoroacetyl, bouncily, and isobutyryl android.
Masking of the 5'-hydroxy in Reaction 2 can be
conveniently effected by reaction of the nucleosides
20 with a slight excess of appropriate acid-labile masking
reagents, such as tritylchlorides, monomethoxytrityl
chloride, dimethoxytrityl chloride (DMTCl),trimethoxy-
tritely chloride and the like. Preferred is dimethoxy-
tritely chloride. Typical reactions are carried out in
25 suitable solvents, such as pardon, letdowns, in-
alkylamines, and the like, at temperatures in the range
of -20C to 120C, generally 20C to 100C, for about 1
to 48 hours. The preferred reaction utilizes 1.1
equivalents of DMTCl in pardon at room temperature
30 for 2 hours.
It is generally preferred that the respective pro-
ducts of each recline described hereinabove be swooper-
ted and/or isolated prior to use as a starting material
-31~
for a subsequent reaction. Separation and isolation
can be effected by any suitable purification procedure
such as, for example, evaporation, filtration, crystal-
ligation, column chromatography, thin layer chromatog-
5 rough, etc. Specific illustrations-of typical swooper-
Zion and isolation procedures can be had by reference
to the appropriate examples described hereinbelow; how-
ever, other equivalent separation procedures can, of
course, also be used. Also, it should be appreciated
10 that, where typical reaction conditions (e.g., tempera-
lures, mole ratios, reaction times) have been given,
conditions both above and below the typical ranges can
also be used, though generally less conveniently.
Activation to the phosphate analog illustrated in
15 Reaction 3 can be most conveniently effected by treat-
mint of the nucleoside compounds with suitable phosphi-
tilting agents in appropriate solvents at temperatures
in the range of -90C to 60C for 1 minute to 2 hours.
Suitable phosphitylating agents include methylphospho-
20 dichloridite, o-chlorophenylphosphodichloridite, p-chlo-
rophenylphosphodichloridite, methylphospho(dialkylamino~
monochloridite 9 and the like. Appropriate solvents in-
elude pyridlne, letdowns, acetonitrile, tetrahydro-
Furman, Dixon 9 chloroform and the like containing 0-20%
25 appropriate base generally 1-5 vow %) such as letdowns,
colliding triakylamines and the like. Preferred pros-
phitylating agents are methylphosphodichloridite, o-
chlorophenylphosphodichloridite, and methylphospho-
(di-isopropylamino)-monochloridite. Preferred phosphy-
30 tilting conditions are with 0.9 equivalents of methylphosphodichloridite in pardon or acetonitrile contain-
in 5% letdown for 5 to 10 minutes at room tempera-
lure or below.
-32-
The chemical incorporation of the modified nucleon
tide analog monomers into a growing nucleated chain to
produce defined sequence nucleotides is illustrated in
Reactions 4 and pa. Typical condensations are in appear-
5 private solvents at temperatures in the range of -20~C
to 50C~ preferably at ambient temperature, for about
1 to 60 minutes. Appropriate solvent mixtures include
pardon, letdowns, acetonitrile, tetrahydrofuran,
Dixon, chloroform and the like containing 0-20% appear-
lo private base (generally 1 to 5 volume %) such as lutidines,collidines, trialkylamines and the like. The growing
chain may be Swahili, insoluble, or attached to a suit-
able solid support by appropriate chemical methods known
in the art. Preferred is attachment to a solid support.
15 Furthermore, the growing chain may or may not have pro-
piously incorporated one or more modified nucleoside
analogs.
After condensation of the activated monomer to the
growing chain, in Reaction 4, the initial product is
20 treated with suitable reagents to accomplish oxidation
of the intermediate phosphitetriester, optional capping
to block unrequited 5'-hydroxyls on the oligonucleotide
chain, and removal of the 5'-DMT group. Oxidation of
the phosphate trimester can be accomplished by treatment
25 with 0.1-5 wavily % iodine in suitable solvents, for
example, tetrahydrofuran/water/lutidine mixtures. Comma-
eel capping of unrequited 5'-hydroxyls can be accomplished
by acetylation or acylation with, for example, acetic
android and ~dimethylaminopyridine in tetrahydrofuran/
I letdown mixtures. Removal of the blocking group,
usually DOT, is most conveniently effected by treatment
with mild organic acids in nonerotic solvents, such as
mild acids including, for example 9 1-5 vow % dichloro-
acetic or trichloroace~ic acid in chloroform or
-33-
dichloromethane. The growing nucleated chain, after
removal of DOT, can now serve as acceptor for subset
quint elongation by sequential reaction with activated
monomers to eventually produce the oligonucleotide of
5 desired length and sequence, as shown in Reaction pa.
After an oligonucleotide of desired sequence is
produced, Reaction 5 is accomplished to provide the
product oligonucleotlde. To this end, thiophenol treat-
mint is used to remove methyl masking groups from pros-
10 plate trimesters, and suitable aqueous alkali or ammonia treatment is used to remove bouncily, acutely, isobutyl,
trifluoroacetyl, or other groups from the protected
amine and/or to remove the product from the solid sup-
port. Removal ox DOT from the oligonucleotide product
15 is accomplished by the appropriate treatment with a mild
acid, such as aqueous acetic acid at ambient temperature
to 40C for 10 to 60 minutes. Such reactions may be
accomplished before or during final purifications. Final
purification is accomplished by appropriate methods, such
20 as polyacrylamide gel electrophoresis, high pressure
liquid chromatography PLUCK), reverse phase or anion
exchange on DEAE-cellulose, or combinations of these methods.
The process described herein for synthesis of oligo-
nucleotides can utilize modified deoxyribonucleosides
worry R' is H) or modified ribonucleosides (where R' is
hydroxyl). When ribonucleosides are used, the 2'-hydroxyl
is masked by an appropriate masking group such as, for
example, that afforded by silylethers. Other rubs
-34- 3
analogs, including Arabians and 3'-deoxyribose, can
also be accommodated in the process to produce the
desired oligonucleotide.
The substituent group modifying a nucleated
5 base must be capable of binding one or more reporter
groups either prior to or after the chain extension
coupling reaction. In the latter case, selected product
oligonucleo~ides are reacted with suitable agents to
attach such reporter group. For example, when modified
10 bases are incorporated into the oligonucleotide and R2
of the substltuent group contains one or more primary
amine, coupling with amine-reactive groups such as
isocyanate, isothiocyanate, active carboxylic acid
conjugates, epoxies or active aromatic compounds
15 using suitable conditions is productive of aside, urea,
Thor, amine or aromatic amine linkages. For example,
an oligonucleotide which contains an Ursula or adenine
base modified by a substituent group having a primary
amine, as shown in the Reaction 5 diagram, can be
20 reacted with a suitable reagent, such as fluoresce in
isoth~ocyanate or N-hydroxysuccinimidyl 6-biotinylamino-
caproic acid to provide a reporter group R5 (fluoresce in
or button, respectively) bound to the subs~i~uent group
as shown in Reaction 6. Other reporter groups which
25 can be attached in similar manner include a wide
variety of organic moieties such as fluoresce ins,
Rhoda mines, acridinium salts, dinitrophenyls,
benzenesulfonyls, luminous, Lucifer ins, buttons,
vitamins, carboxyhydrates and the like. Suitably
-35~
active reporter groups are available commercially,
or can be synthesized, for example, by processes of
the type generally described in "Bioluminescence and
Chemiluminescence" [M. Delco and W. McElroy, ens.,
5 Aged. Press, New York (1981)~, by D. Russell, et at.,
or H. Schroeder, eta [Moth. Enzymol. LXII, 1978],
and references cited wherein.
Typically, attachment of reporter groups is
conveniently accomplished in predominately aqueous
lo solvents by reaction of the substituent groups of
modified bases wherein R2 = On Hen NH2 wit
of the selected reporter group at temperatures in
the range of about -20C to 50C (preferably 20C
to 40C) for 1 to 24 hours. Suitable solvents are
15 an aqueous buffer and 0-50% organic solvents such
as lower alcohols, tetrahydrofuran, dimethylforma-
mode, pardon, and the like. Preferred reporter
group reactants include fluoresce in iso~hiocyanates,
dinitrophenylisothiocyanates, fluorodinitrobenzene,
20 N-hydroxysuccinimidylbiotin, N-hydroxysuccinimidyl
dinitrobenzoate, isothiocyanates such as aminobutyl
ethyl isoluminol isothiocyanate and the like, active
esters of carboxyfluorescein, rhodamlne, button
adduces, dioxetanes, dioxamides, carboxyacridines,
25 carbohydrates and the like.
Additionally, when the product oligonucleotide
includes modified bases wherein R contains one or more
carboxylic acids, mild condensations with, for example,
primary alkylamines is productive of aside linkages.
30 Typically, this is conveniently effected in Prado-
minutely aqueous solvents by reaction of the oligo-
knucklehead with excess reporter group which contains
a primary amine in the presence of suitable condensing
I
-36-
agents, such as wa~er-soluble carbodiimides, at
temperatures in the range of about -20 C to 50 C
(preferably 20C to 40C) for 6 to 72 hours. Pro-
furred reporter groups of this class include (amino-
5 alkyl)-amino-naphthalene-1,2-dicarboxylic acid hydra-
wide amino-fluoresceins, aminorhodamines, aminoal~yl
luminous, aminoslkylaminobenzenesulfonyl adduces, amino
sugar and the like. Furthermore, the chemical synthesis
of the initial oligonucleotide product may be accomplished
10 with modified nucleated monomers wherein, prior to the
coupling reaction, such reporter groups are attached to
the substituent group. If any such reporter groups
would adversely affect the coupling reaction, they
are appropriately masked to forestall any such adverse
15 effect. On the other hand, certain other reporter groups
are substantially unreactive with respect to the coupling
reaction and therefore do not require masking. For
example, nitrophenyl adduces may be attached to the
substituent group prior to the coupling reaction, and
20 without masking, may be present on the modified nucleon
tide monomer during the coupling reaction without adverse
effect.
Reporter group useful in the method of this invention
generally include aromatic 9 polyaromatic, cyclic, and
25 polycycl~c organic moieties which are further function-
alized by inclusion of heteroatoms such as nitrogen,
oxygen, sulfur and the like.
Product oligonucleotides can include more than
one type of modification or more than one modified
30 base. An illustrative example of an oligonucleotide
of this type is one of the structure:
m G T U Us An . Am A
f o o o o o o o o
I I I I I o \ OWE
':',
-37- I
wherein Cm is 5-(3-aminopropyl) citizen, Us is
5-rN-(4-aminobutyl)-1-acrylamido3uracil, and Am is
8-[6-2,4-dlnitrophenyl)-aminohexyl]aminoadenine.
This product is further modified by reaction with
5 floweriest isothioeyanate to provide a fluoresce in
reporter group on Cm and us.
Such a product oligonucleotide illustrates the
variety of the selection of modified and unmodified
nucleated units in a product oligonucleotide made
possible by the process of the present invention.
More specifically, such oligonucleotide illustrates
the use of more than one type of nucleated unit
having it base modified by substituent groups having
bound thereto reporter groups providing the same or
different types of reporter groups function. Also
illustrated are units whose bases are modified by
substituent groups to which reporter groups are bound
after the coupling reaction, i.e., Cm and us, whereas
Am is illustrative of a unit whose base is modified by
a substituent group to which a dinitrophenyl reporter
group was attached prior to the coupling reaction.
Such oligonucleotide additionally illustrates that it
can include more than one nucleated unit of the same
type, and that it can include units having unmodified
bases intermixed with units having modified bases.
Instead of attaching reporter groups to the
primary amine of the substituent groups as ill-
striated in Reaction 6, such amine or other group can
alternatively be coupled to suitably activated solid
supports. This produces a defined sequence oligo-
nucleated which is covalently bound to such supports
through the modified bases. Such solid supports are
useful in the detection and isolation of complementary
I I
-38-
nucleic cold components. Alternatively, the modified
nucleoside monomers can be coupled Jo solid supports
prior to the chain extension coupling Reaction 4,
to thereby provide solid supports for such monomers
5 during the coupling reaction.
The following specific examples are provided to
enable those skilled in the art to practice the invent
lion. The examples should not be considered limit-
lions upon the scope of the invention, but merely as
10 being illustrative and representative thereof. To
aid in structural clarification, references are made
to the reactions illustrated in the aforementioned
process diagram.
-39-
EXAMPLE I
This example illustrates the synthesis of a mod-
fled nucleoside precursor 5-(3-trifluoroacetylamino-
propenyl)-2'-cleoxyuridine.
5 Chloromercuri-2'-deoxyuridine I g, 7.8 Molly)
is suspended in 200 ml methanol. N-Allyltrifluoro-
acetamide(6.8 ml, 55 Molly) is added, followed by add-
lion of 41 ml of 0.2 N lithium tetrachloropalladate in
methanol. After 18 hours stirring at room temperature,
the reaction is gravity filtered to remove the black
solid palladium, and the yellow methanolic filtrate is
treated with five 200 my portions of sodium bordered,
then concentrated under reduced prowar to solid nest-
due. The residue is purified by flash column chrome-
tography on silica gel eluding 15 vow % methanol in chloroform. Appropriately pure fractions of product
are combined and concentrated under reduced pressure to
give crystalline 5-(3-trifluoroacetylaminopropenyl)-~'-
deoxyuridine (2.4 g). US Max 291 no (~7800), A mix
266 no, (I 4400); TLC (silica eluding 15 vow % methanol
in chloroform) I = 0.4.
EXAMPLE I
This example illustrates the synthesis of a modified
nucleoside precursor 5-[N-(trifluoroacetylaminoheptyl)-l-
acrylamido]-2'-deoxyuridine.
5-Chloromercuri-2'-deoxyuridine (3.6 g, 7.8 Molly) is
suspended in 200 ml methanol. N-(7-trifluoroacetylamino-
heptyl)-acrylamide(55 Molly) is added, followed by add-
lion of 41 ml of 0.2 N lithium tetrachloropalladate in
30 methanol. After 18 hours stirring at room temperature,
the reaction is gravity filtered to remove the black
solid palladium, and the yellow methanolic filtrate is
treated with five 200 my portions of sodium bordered,
I $
-40-
then concentrated under reduced pressure to solid nest-
due. The residue is purified by flash column chrome-
tography on silica gel eluding 10 vow % methanol in
chloroform. Appropriately pure fractions of product are
5 combined and concentrated under reduced pressure to give
crystalline 5-[N-(7-trifluoroacetylaminoheptyl)-1-acryl-
amidoJ-2'-deoxyuridine (2.8 g). US Max 302 no
(I 18000), Mooney 230 no, 280 no; TLC (silica eluding
15 vow % methanol in chloroform) Of = 0.3.
EXAMPLE III
This example illustrates masking of 5'-hydroxyl
to produce 5' dimethoxytrityl-5-(3-trifluoroacetyl-
arninopropenyl)-2'-deoxyuridine as illustrated in
Reaction 2.
5-(3-tri~luoroacetylaminopropenyl)-2'-deoxyuri-
dine (2.4 g) is thoroughly evaporated twice from
pardon, then stirred in 40 ml pardon. Dimethoxy-
trityl(DMT)chloride(2.3 g, 6.6 Molly) is added, and the
mixture stirred at room temperature for four hours.
; 2Q After thin layer chromatography (TLC) on silica eluding
10 vow % methanol in chloroform indicates reaction is
complete, the reaction is concentrated to a solid residue.
This residue is purified by column chromatography on
silica eluding chloroform until all faster running
US impurities have eluded, then bringing off product with
5 vow % methanol in chloroform. The residue is then con-
cent rated to give 5'-dimethoxytrityl-5-(3-trifluoroacetyl-
aminopropen-l-yl) -2'-deoxyuridine as a white fluffy
solid (4 g). Product decomposes upon heating; US Max
3Q 291 no, 'I in 266 no; TLC Of 0.6 on silica eluding
10 vow % methanol in chloroform.
-41-
EXAMPLE IV
This example illustrates hydrogenation of excuse-
die double bond and 5'-hydroxyl masking to produce
5'-dimethoxytri..tyl-5-(3-trifluoroacetylaminoproppull'-
5 deoxyurîdine.
Repeating the nucleoside precursor synthesis and
5'-hydroxyl masking procedures of Examples I and III,
but, prior to the addition of the DOT chloride, subject-
in the purified 5-(3-trifluoroacetylaminopropenyl)-2-
deoxyuridine to two atmospheres of hydrogen while stirring at room temperature in methanol over 10%
palladium-on-carbon catalyst is productive of dummy-
thoxytrityl-5-(3-trifluoroacetylaminopropyl)-~'-deeoxyu-
riding.
Examples V to VIII illustrate the synthesis of add-
tonal modified Ursula nucleosides, and subsequent
masking of 5'-hydroxyls as represented by Reaction 2.
EXAMPLE V
Repeating the nucleoside precursor synthesis and
5' hydroxyl masking procedures of Examples I Andy,
but replacing N-allyltrifluoroacetamide with compounds
numbered 1 through 8 below is productive of the come
pounds numbered 1' through 8' below, respectively; i.e.
substitution of.
1 N-(3-butenyl~trichloroacetamide
2 N-(5-hexenyl)trifluoroacetamide
-42~
3 N-(2-methyl-2-propenyl)tri~luoroacetamide
4 N-(4-ethenylphenylmethyl)trifluoroacetamide
N-(l-methyl-3-butenyl)trifluoroacetamide
6 N-(12-trichloroaminododecyl)acrylamide
7 N (pertrifluoroacetylpolylysyl)acrylamide
8 N-(3-trifluoroacetylamidopropyl)acrylamide
is productive of
1'5'-dimethoxytrityl-5-(4-trichloroacetylaminobuten--1-
yl)-2'-deoxyuridine
2'5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen--1-
yl)-2'-deoxyuridine
3'5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-metthy-
propen-l-yl)-2'-deoxyuridine
4'5'-dimethoxvtrityl-5-[2-(4-trifluoroacetylaminometthy-
phenyl)etherl-1-yl]-2'-deoxyuridine
5'5'-dimethoxyltrityl-5-(4-trifluoroacetylamino-4-meethyl-
buten-l-yl)-2'-deoxyuridine
6'5'-dimethoxytrityl-5-[N-(12-trichloroacetylaminodoo-
decyl)-l-acrylamido]-2'-deoxyuridine
I 7'5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylyssol)-
l-acrylamido]-2'-deoxyuridine
8' 5'-dimethoxytrityl-5-[N-~3-trichloroacetylamino-
~ro~yl)-acrylamido~-2'-deoxyuridine
EXAMPLE VI
Repeating the 5' hydroxyl masking procedure of
Example III but replacing 5-(3-trifluoroacetylamino-
propenyl)-2'-deoxyuridine with the 5-substituted-2'-de-
oxyuridines numbered 9 through 18 below is productive
of the products numbered 9' through 18' below, respectively;
i.e. substituting
-aye- I So
9 5-(propen-1-yl)-2'-deoxyuridine
5-(carbmethoxyethyl)-2'-deoxyuridine
'1 5-(3-carbmethoxylprop-1-yl)-2'-deoxyuridine
12 5-(4~carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridiire
5 13 5-(3-cyanopropen-1-yl)-2'-deoxyuridine
14 5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine
15 5-~2-(4-carbmethoxyphenyl)ethen-1-yl~-2'-deoxyuriddine
16 5-(4-acetoxybu~en-1-yl)-2'-deoxyuridine
_ 5-(4-acetoxybut-1-yl)-2'-deoxyuridine
10 18 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxyuridine
is productive of the following 5'-dimethoxytrityl-5-
alkyl-2'-deoxyuridines
9'5'-dimethoxytrityl-5-(propen-l-yl)-2'-deoxyuridinee
10'5'-dimethoxytrityl-5-(2-carbmethoxyethyl)-2'-deoxyy-
uridine
11' 5'-dimethoxytrityl-5-(3-carbmethoxyprop-l-yl)-2'-
deoxyuridine
12'5'-dimethoxytrityl-5-(4-carbmethoxy-2-methylbuten--
l-yl3-2'-deoxyuridine
20 13'5'-dimethoxytrityl-5-(3-cyanopropen-1-yl~-2'-deoxyy-
uridine
14'5'-dimethoxytrityl-5-(4-cyano-2-methylbuten-l-yl)--
2'-deoxyuridine
15'5'-dimethoxytrityl-5-[2-(4-carbmethoxyphenyl)ethenn-
l-yl]-2'-deoxyuridine
16' S'-dimethoxytrityl-5-(4-acetoxybuten-1-yl)-2'-
deoxyuridine
17'5'-dimethoxytrityl-5-(4-acetoxybut-l-yl)-2'-deoxy--
uridine
30 18'5'-dimethoxytrityl-5-[4-(2,~-dinitrophenyl)butyl]--
2'-deoxyuridine
3~.'6~3
I
EXAMPLE VII
Repeating the nucleoside precursor synthesis and
5'-hydroxyl masking procedures of Examples I-VI, but
replacing 5-cnloromercuri-2'-deoxyuridine with sheller-
5 mercuriuridine is productive of the corresponding
5'-dimethoxytrityl-5-substituted uridines.
Examples VIII to XI illustrate the synthesis of
modified citizen nucleosides. Since citizen nucleon
sides, as well as adenosine nucleosides, have reactive
10 groups on their bases unlike the Ursula nucleosides,
such reactive groups are masked to prevent unwanted
reactions therewith. These examples illustrate masking
of reactive groups on the citizen base moiety as in
Reaction 1, as well as masking of the 5'-hydroxyl as in
Reaction 2.
EXAMPLE VIII
5-t3-trifluoroacetylaminopropenyl)-
N4-benzoyl-2'-deoxycytidine
Repeating the nucleoside precursor synthesis pro-
seedier of Example I, but replacing 5-chloromercuri-2'-
deoxyuridine with 5-chloromercuri-2'-deoxycytidine is
productive of 5-(3-trifluoroacetylaminopropenyl)-2'-
deoxycytidine (US Max 287 my). Purified try-
fluoroacetylaminopropenyl~-2'-deoxycytidine (1.3 g,
4.6 Molly) is stirred in 80 ml an hydrous ethanol, bouncily
android (1.5 g, 7 Molly) is added, and the reaction
I 3
-44-
reflexed. Five additional 1.5 g portions of bouncily
android are added hourly. After the reaction is
judged complete by thin layer chromatography [silica
plates eluding n-butanol/methanol/conc NH40H/H~ (60:
inn 6-10 hours, the reaction is cooled and con-
cent rated under reduced pressure to a semisolid. The
solid is ,tritura~ed with ether three times, decanted
and dried. The crude product is crystallized from
water to give chromatographically pure N4-benzoyl-5-
(3-trifluoroacetylaminopropenyl)-2'-deoxycytidine as a
white solid. the product decomposes above 120~C; W Max
311 no.
EXAMPLE IX
5'-Dimethoxytrityl-5-(3-trifluoroacetyl-
aminopropenyl)-N4-benzoyl-2'-deoxycytidine
Repeating the 5'-hydroxyl masking procedure of
Example, but replacing 5-(3-trifluoroacetylamino-
propenyl)-2'-deoxyuridine with 5-(3-trifluoroacetyl-
aminopropenyl)-N4-benzoyl-2'-deoxycytidine is productive
of5'-dime~hoxytrityl-5-(3-trifluoroacetylaminopropennil)-
N4-benzoyl-2'-deoxycytidine.
EXAMPLE X
Repeating the nucleoside precursor synthesis and
S' hydroxyl masking procedures of Examples VIII and
IX, but replacing N-allyltrifluoroacetamide with the
N-alkyl~rifluoroace~amides of Example V is productive of
the corresponding 5'-dimethoxytrityl-5-,(trifluoroacetyl)
aminoalkyl)-N4-benzoyl-2'-deoxycytidines, viz;
S'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten--yule)-
N4-benzoyl-2'-deoxycytidine
5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen--yule)-
N4-benzoyl-2'-deoxycytidine
5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-metthy-
propen-l-yl)-N4-benzoyl-2'-deoxycytidine
-45-
5'-dimethoxytrityl~5-[2-(4-trifluoroacetylaminometthy-
phenyl)ethen-l-yl]-N4-benzoyl-2'-deoxycytidine
5'-dimethoxytrityl-5-(4-trifluoroacetylamino-4-metthy-
buten-l-yl)-N -benzoyl-2'-deoxycytidine
5'-dimethoxytrityl-5-[N-~12 trifluoroacetylaminodo-
decyl)-l-acrylamido]-N4-benzoyl-2'-deoxyeytidine
5'-dimethoxytrityl-5-[N-(pertri~luoroacetylpolylyssol)-
l-acrylamido]-N4-benzoyl-2'-deoxycytidine
EXAMPLE XI
Synthesis of 5'-dimethoxytrityl-N4-benzoyl-5-
(2-carbmethoxyethenyl)-2'-deoxycytidine
5-(2-Carbmethoxyethenyl)-2'-deoxycytidine (0.82 g,
2.6 Molly) is stirred in 50 ml an hydrous ethanol. Ben-
zoic android (500 my, 2.2 Molly) is added, and the
reaction heated to reflex. Five additional 500 my
portions of benzoic android are added hourly. After
the reaction is judged complete by thin layer chrome-
tography (usually 6-8 hours) the reaction is cooled,
and evaporated under reduced pressure to a yellow semi-
solid. Chromatography on silica gel eluding a lunar to 1:3 methanollchloroform mixture followed by
thorough evaporation of appropriately combined fractions
gives N -benzoyl-5-(2-carbmethoxyethenyl)-2'-deoxycyti-
dine as an amorphous white solid. US Max 296 no,
mix 270 no. The solid is dried thoroughly, and
dissolved in 20 ml pardon. Dimethoxytrityl chloride
(1.1 en) is added, and the reaction stirred at ambient
temperature for six hours. Concentration to a solid
followed by column chromatography on silica gel eluding
10% methanol in chloroform yields 5'-dimethoxytrityl-N -
benzoyl-~(2-carbmethoxyethenyl)-2'-deoxycytidine as a
fluffy oft solid.
-46- 3
EXAMPLE XII
Repeating the nucleoside precursor synthesis pro-
seedier of Example XI, but replacing 5-(2-carbmethoxy-
ethenyl)-2'-deoxycytidine with the following compounds
5 numbered 19 through 27 below is productive of the
corresponding compounds numbered 19' through 27'
below, respectively, i.e., substituting:
19 5-(2-carbmethoxyethyl)-2'-deoxycytidine
5-(3-carbmethoxyprop-1-yl)-2'-deoxycytidine
10 21 5-(4-oarbmethoxy-2-methylbuten-1-yl)-2'-deoxycyti--
dine
22 5-~3-cyanopropen-1-yl)-2'-deoxycy~idine
23 5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine
24 5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2'-deoxycy-
tiding
5-(4-acetoxybuten-1-yl)-2'-deoxycytidine
26 5-(4-acetoxybut-1-yl)-2'-deoxycytidine
27 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxycytidine
is productive of the following 5'-dimethoxytrityl-N4-
20 benzoyl-5-alkyl-2'-deoxycytidines:
19' 5'-DMT-N4-benzoyl-5 (2-carbmethoxyethen-1-yl)-Z'-
deoxycytidine
20' 5'-DMT-N4-benzoyl-5-(3-carbmethoxyprop-1-yl)-2'-
deoxycytidine
25 21' 5'-~MT-N4-benzoyl-5-(4-carbmethoxy-2-methylbu~en-
l-yl~-2'-deoxycytidine
22' 5'-DMT-N4-benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxy--
cytidine
23' 5'-DMT-N4-benzoyl-5-(4-cyano-2-methylbuten-1-yl)-22'-
deoxycy~idine
_' 5'-DMT-N4-benzoyl-5-[2-(4-carbmethoxyphenyl)ethen--
l-yl]-2'-deoxycytidine
25' 5l-DMT-N4-benzoyl-5-(6-acetoxybuten-l-yl)-2l-de
cytidine
-47-
26' 5'-DMT-N4-benzoyl-5-(4-acetoxybut-1-yl)-2'-deoxy-
cytidine
27' 5'-DMl'-N4-benzoyl-S-[4-(2,4-dinitrophenyl)butyl]--
2'-deoxycytidine
.
S Similarly, the use of the other acid androids, e.g.,
acetic android, anisoyl android, or oilily android,
is productive of the corresponding Nuzzle or No acutely
alkali 2'-deoxycytidines of Examples X and XI wherein
bouncily is replaced by acutely or azalea.
EXAMPLE XIII
Repeating the nucleoside precursor synthesis and
5'-hydroxyl masking procedures of Examples VlII to X,
but replacing 5-chloromercuri-2'-deoxycytidine with
5-chloromercuricytidine is productive of the cores-
15 pounding 5'-dimethoxy~rityl-N4-benzoyl-5-substituted
cytidines.
EXAMPLE XIV
This example typifies the masking of reactive base
moieties and the masking of 5'hydroxyl of adenine
20 nucleosides.
N6-benzoyl-8-(6-aminohexyl)amino-2'-deoxyadenosinee
(4 Molly) is stirred in 60 ml an hydrous ethanol. Trip
fluoroacetic android (6 Molly) is added, and the react
lion stirred at room temperature. Two additional port
I lions of trifluoroacetic android are added hourly.
-48-
After four hours, the reaction is concentrated to a
solid residue, and lyophilized overnight. The crude
N6-benzoyl-8-(6-~rifluoroacetylaminohexyl)amino-2''-
deoxyadenosine is dried thoroughly and concentrated to
5 a solid residue twice prom pardon. The solid is
stirred in 40 ml of pardon, and dimethoxytrityl
chloride (6.5 Molly) is added. After four hours, the
reaction is concentrated to leave a solid residue.
Purification by column chromatography on silica gel
10 eluding a multi-step gradient of 0 to 15% methanol in
chloroform gives 5'-dimethoxytrityl-N6-benzoyl-8-
(6-trifluoroacetylaminohexyl)amino-2'-deoxyadenosiire
as an off-white solid.
Examples XV to XVII typify the activation of 5'-
masked 5-substituted, and neutral occurring nucleon
sides, to their respective phosphomonochloridites, as
illustrated in Reaction 3 of the diagram.
. EXAMPLE XV
Preparation of 3'-phosphomonochloridite of
5'-DMT-5-(3-t~ifluoroacetylaminoprop-l-yl)-
2'-deoxyuridine
Dry 5-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-
d~oxyuridine (1.54 g, 2.2 Molly) is lyophilized from
20 ml Bunsen three times for more than twelve hours
each to remove residual water and solvents. The result-
in very fluffy white power is transferred to a nitrogen
Jo
-49-
atmosphere while still under vacuum and dissolved in
anhydro~s acetonitrile containing 5 vow % letdown
to a final nucleo.side concentration of 30 my. While
swirling vigorously under nitrogen, one rapid bonus ox
S me~hylphosphodichloridite (1.0 en) is added by syringe.
The reaction is swirled for about one minute under
nitrogen. The resulting crude 5'-DMT-5-(3-trifluoro-
acetylaminoprop-l-yl)-2'-deoxyuridine 3'-methylphospho-
monochloridite reaction solution is then used directly
for deoxyoligonucleotide synthesis (Example XVIII) with
no further purification, [3lP-NMR(CH3CN/CDCl3) generally
indicates 40-70 mow % desired product (167.5 Pam);
remainder is composed of bis-3',~'-[5'DMT-5-(3-trifluoro-
acetylamino~rop-l-yl)-2'-deoxyuridylyl] methylphosphite
(140 Pam) and S'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-
2'-deoxyurLdine 3'-methylphosphonate (9~5 Pam), the
latter product being formed in amounts reflecting the
presence of water in the reaction.
EXAMPLE XVI
Preparation of 3'-phosphombnochloridites
of the naturally-occurring 2'-deoxynucleosides
Repeating the procedure of Example XV, but replace
in 5'~DMT-S-(3-trifluoroacetylaminoprop-1-yl)-2'-
deoxyuridine with:
5'-DMT-2'-deoxythymidinc
5'-DMT-N4-benzoyl-2'-deoxycytidine
S'-DMT-N6-benzoyl-2'-deoxyadenosine
S'-DMT-N2-isobutyryl-2'-deoxyguanosine
is productive of the corresponding phosphomonochlori-
dotes, vows
5' DMT-2'-deoxythymidine 3'-methylphosphomonochloridite
5'-DMT-N4-benzoyl-2'-deoxycytidine 3'-methylphosphomono-
chloridite
-50-
5'-DMT-N6-benzoyl-2'-deoxyadenosine 3'-methylphospho-
monochloridite
5'-DMT-N -isobutyryl-2'-deoxyguanosine 3'-methylphos-
phomonochloridite
.. . ..
EXAMPLE XVII
Repeating the phosphomonochloridite synthesis
procedures of Examples XV and XVI, but replacing methyl-
phosphodichloridite with o-chlorophenylphosphodichlori-
dote is productive of the corresponding 5'-DMT-nucleoside
3l-phosphomonochloridites~ viz.:
5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyurriding
3'-o-chlorophenylphosphomonochloridite
5'-DMT-2'-deoxythymidine 3'-o-chlorophenylphosphomono-
chloridite
5'-DMT-N4-benzoyl-2'-deoxycytidine 3'-o-chlorophenyl-
phosphomonochloridite
5'-DMT-N6-benzoyl-2'-deoxyadenosine 3'-o-chlorophenyl-
phosphomonochloridite
5'-DMT-N -isobutyryl-2'-deoxyguanosine 3'-o-chloro-
phenylphosphomonochloridite
Similarly, the use of ~-chlorophenylphosphodichloridite
is productive ox the analogous 3'-p-chlorophenylphos-
phomonochloridite adduces. [32P]NMR (CHICANO CDCl3) of
o-chlorophenylphosphomonochloridite products 160.7,
160.5 Pam ~diasteriomers).
. . .
Examples XVIII - XXIV typify the chemical synthesis
of oligonucleotides which incorporate modified bases, as
illustrated by Reactions 4 and 5 in the diagram.
I I I
-51-
EXAMPLE XVIII
Synthesis of deoxyoligonucleotides containing
5-(3-aminopropyl)-uracil and naturally-
occurring nucleated units
The phosphomonochloridite synthesis procedures of
Examples XV and XVI are accomplishes immediately
before deoxyoli~onucleotide synthesis, and the result-
in products are used directly as 30 my crude methyl-
phosphomonochloridites in an hydrous acetonitrile/5
10 vow % letdown.
Solid support (5-DMT-N6-benzoyl-2'-deoxyadenosine
3'-succinamidepropyl silica, 250 my, 20 eke) is put
into a suitable reaction flow vessel (glass or Teflon
column or formula). The solid support is preconditioned
15 by successive treatments with acetonitrile/5 vow % lull-
dine, 2 w/v % iodine in tetrahydrofuran/water/lutidine
for 2 minutes, acetonitrile/5% letdown, chloroform,
4 vow % dichloroacetic acid in chloroform for 2.5 mint
vies, and acetonitrile/5% letdown, where treatments are
total volumes of 5-15 ml in either 2 or 3 portions or
by constant flow, as desired.
The deoxyoligonucleotide is synthesized in accord-
ante with Reaction 4 by sequential addition of the
desired activated 5'-DMT-nucleoside 3'-methyl~hospho-
monochloridite monomer and coupling thereof to the free5'-hydroxyl of the terminal unit of the growing nucleon
tide chain, which unit is initially the only unit of
the chain, i.e., the deoxyadenosine based unit comprise
in the solid support. Additions are by reaction of
10 ml of the crude 30 my monochloridites chosen from
Examples XV and XVI with the now-unprotected 5' hydroxyl
of the chain in either 2 or 3 portions or by constant
flow, for 2 to 6 minutes. The first phosphomonochlori-
dote addition followed by one complete reagent cycle
35 consists of sequential treatments with:
-52~
-5'-DMT-5-(3-trifluoroace~ylaminopropyl)-2'-deoxyuuridine
3'-methylphosphomonochloridite
-acetronitrile/lutidine wash
-capping for S minutes with 0.3 M 4-dimethylaminopyridine
in acetic anhydridellutidine/tetrahydro~uran (1:3:2)
-acetonitrile/5% letdown wash
oxidation with 2% iodine in te~rahydrofuran/water/
letdown (6:2:1) for 2 minutes
-acetronitrile/5% letdown wash
-chloroform wash
removal of DOT by 2.5 minute treatment with 4 vow %
dichloroacetic acid in chloroform
-chloroform wash
-acetonitrile/lutidine wash
The above cycle is repeated thirteen times, each time
replacing 5'-DMT-5-(3-tri1uoroacetylaminopropyl)-2'-
deoxyuridine 3'-methylphosphomonochloridite with a
different one of the following 3'-methylphosphomono-
chloridites:
5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite
5'-DMT-5-(3-tri~luoroacetylaminopropyl)-2'-deoxyurriding
3i-methylphosphomonochloridite .
5'-DMT-N6-benzoyl-2'-deoxyadenosine 3'-methylphosphomono-
chloridite
5'-DMT-N4-benzoyl-~'-deoxycytidine 3'-methylphosphomono-
chloridite
5l-DMT-N2-isobutyryl-2l-deoxyguanosine 3'-methylphospho-
monochloridite
5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyurriding
3'-methylphosphomonochloridite
5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite
S'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyurriding
3'-methylphosphomonochloridite
_53_ I
S'-DMT-deoxythmidine 3'-methylphosphomonochloridite
5'-DMT-5-~3-trifluoroace~ylaminopropyl)-2'-deoxyurriding
3'-methylphosphomonochloridite
5'-DMT-N -isobutyryl-2'-deoxyguanosine 3'-methylphos-
phomonoch`loridite
5'-DMT-N6-benzoyl-2'-deoxyadenosine 3'-methylphospho-
monochloridite
5'-DMT-N4-benzoyl-2'-deoxycytidine 3'-methylphosphomono-
chloridite
in respective order, deleting dichloroacetic acid treat-
mint during the last reagent cycle. The support is
transferred and treated with 2 ml concentrated ammonium
hydroxide for 4 hours at ambient temperature to release
the product prom the support. The supernatant is
removed, the solid washed three times with 0.5 ml con-
cent rated autonomy hydroxide, and the combined super-
Nat ants are sealed and heated at 50C overnight. The
clear yellow supernatant is lyophilized thoroughly
Initial purification is accomplished by reverse phase
20 high pressure liquid chromatography (HPLC) on on RP-8
(C-8) column eluding a 60 minute linear gradient of 0 to
30% vow % acetonitrile in 25 my ammonium acetate, pi 6.8.
The 5'-DMT-terminated product, eluding as a sharp peak
at about 40 minutes J is collected; all shorter chains,
25 both capped and uncapped, elude before 25 minutes. The
collected product is evaporated to a solid residue,
treated with 80% acetic acid at ambient temperature
for 20 minutes (to remove DOT), lyophilized to a solid
residue, and dissolved in a small amount of aqueous
buffer. The product, generally greater than OWE home-
generous after HPLC, is further purified by conventional
electrophoresis on owe polyacrylamide gels (1 to 6 mm
thick) by excision and extraction of the appropriate
product band (product generally migrates slower than
-54~
unmodified deoxyoligonucleodites of similar length).
The purified 5-aminopropyl-uracil-containing pentadeca-
deoxyoligonucleotide product illustrated diagrammatic
gaily below, wherein Us = 5-(3 aminopropyl)uracil,is
thereby produced.
CA G U T U T Us G
O O' O O O O O O
HOPE O-,P-O~--S:~-P-O~ r P top o, JO p O\r~P~-o~--o Pow
-
ox owe P-o¦ o P ox I I¦-- I¦-
Note the conventional deprotection of the oligo-
nucleated with ammonia has also removed the trifler-
acutely masking group on the substituent.
The length and sequence of this oligonucleotide may
then be determined by P-kinasing and sequencing using
suitable protocols, for example the protocols hereto-
fore used to determine the length and sequence of the
prior art oligonucleotides in which the bases of the
nucleated units therein are unmodified.
15Similarly,intentional variation of the order and
number of methylphosphomonochloridite additions employed
here is productive of other 5-(modified)uracil-containing
deoxyoligonucleotides which vary in selected length and
base sequence. In addition, replacement of the nucleon
side 3'-methylphosphomonochloridite adduces of
Examples XV and XVI with the corresponding owe- or
~-chlorophenylphosphomonochloridite adduces of Example
XVII and inclusion of pyridinium oximate treatment to
remove chlorophenyl blocking groups (at the end of the
deoxyoligonucleotlde synthesis and before concentrated
-55-
ammonium hydroxide treatment) is produc~ivè of the
same deoxyoligonucleotide products.
EXAMPLE XIX
Repeating the phosphomonochloridite and deoxyoligo-
5 nucleated synthesis procedures of Examples XV to XVIII,
but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-
deoxyuridine with the 5'-DMT-5-alkyl-2'-deoxyuridines
numbered 28 through 36 below is productive of the
corresponding oligonucleotides having Ursula bases Us
lo numbered 28' through 36' below, respectively, i.e.,
substituting:
285'-dimethoxytrityl-5-(3-trifluoroacetylaminopropenn-
l-yl)-deoxyuridine
29 5'-dimethoxytrityl-5-(4-tri~luoroacetylaminobut-
1-yl)-2'-deoxyuridine
305'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten--
l-yl)-2'-deoxyuridine
315'-dimethoxytrityl-5-(6-trifluoroacetylaminohex-1--
yl)-2'-deoxyuridine
20 32S'-dimethoxytrityl-5-(5-tri~luoroacetylaminohexen--1-
yl)-2'-deoxyuridine
335'-dimethoxytrityl-5-(2-trifluoroacetylaminoprop-22-
yl)-2'-deoxyuridine
- 34 5'-dimethoxytrityl-5-(3-trlfluoroace~ylamino-2-
methyl-propen-1-yl)-2'-deoxyuridine
35 5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-
methyl-prop-l-yl)-2'-deoxyuridine
36 5'-dimethoxytrityl-5-[2- (~-trichluoroacetylamino-
methylphenyl~ethen-l-yl]-2'-deoxyuridine
_ 5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpoly-
lysyl)-l-acrylamido]-2'~deoxyuridine
_ 5'-DMT-5-[N-(7-trifluoroacetylaminoheptyl)-l-
acrylamido]-2'-deoxyuridine
-56~
is productive of the deoxynucleotides corresponding to
the product of Example XVIII, wherein Us is:
28' 5-(3-aminopropen-1-yl) Ursula
29' 5-(4-aminobut-1-yl) Ursula
30' 5-(4-aminobuten-1-yl) Ursula
31' 5-(6-aminohex-1-yl) Ursula
32' 5-(6-aminohexen-1-yl) Ursula
33' 5-(3-aminoprop-2-yl) Ursula
34' 5-(3-amino-2-methylpropen-1-yl) Ursula
35' 5-(3-amino-2-methylprop-1-yl) Ursula
36' 5-[2-(4-aminoethylphenyl)ethen-1-yl] Ursula
37' 5-[N-(polylysyl)-l-acrylamido] Ursula
38' 5-[N-(7-aminoheptyl)-1-acrylamido] Ursula
Similarly, by employing other 5'-DMT-5-(acylaminoalkyl)-
2'-~eoxyuridine~, the analogous ~eoxyoligonucleoti~es
are produced.
. .
EXAMPLE XX
Repeating the phosphomonochloridite and deoxyoligo-
20 nucleated synthesis procedures of Examples XV to XVIII,
buy replacing 5'-DMT-5-(3-acetylaminopropyl)-2'-deoxy-
Rodney with 5-substituted-2'-deoxyuridines numbered
37 through 46 below is productive of the corresponding
oligonucleotides having the Us Ursula bases numbered
37' through 46', below respectively; i.e., substituting;
37 5'-DMT-5-~propen-1-yl)-2'-deoxyuridine
38 5'-DMT-5-(2-earbmethoxyethyl)-2'-deoxyuridine
39 5'-DMT-5-(3-carbmethoxyprop-1-yl)-2'-deoxyuridine
5'-DMT-5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-
deoxyuridine
41 5'-DMT-5-(3-cyanopropen-1-yl)-2'-deoxyuridine
42 5'-DMT-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxy-
uridine
~57~
43 5'DMT-5-12-(4-carbmethoxyphenyl)ethen-l-yl)-2'-
deoxyuridine.
44 5'-DMT-5-(4-acetoxybuten-l-yl)-2'-deoxyuridine
. 45 5'-DMT-S-(4-ace~oxybut-l-yl~-2'-deoxyridine
46 5'-DMT 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxy-
uridine
is productive of the products wherein, Us is:
37' 5-(propen-l-yl)uracil
38' 5 (2~carboxyethyl)uracil
lo 39' 5-(3-carboxyprop-l-yl)uracil
40' 5-(4-carboxy-2-methylbuten-l-yl)uracil
Al' 5-(3-cyanopropen-l-yl~uracil
42' 5-(4-cyano-2-methylbuten-l-yl)uracil
43' 5-12-(4-carboxyphenyl)ethen-l-yl]uracil
44' 5-(4-hydroxybuten-l-yl)uracil
45' 5-(4-hydroxbut-l-yl)uracil
46' 5-[4-(2,4-dinitrophenyl)butyl~uracil
Note In 46' the dinitrophenyl is a ligand recognition
type reporter group, i.e. use of antidinitrophenyl
antibody as the ligand. Similarly, by employing other
appropriate 5'-DMT-5-alkyl-2'-deoxyuridines, the analog
gout deoxyoligonucleotides are produced.
EXAMPLE XXI
Repeating the procedures of Examples XV to XVIII,
but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)-
2'-deoxyuridine with 5'-DMT-N4-benzoyl-5-(3-trichloro-
acetylaminoproply)-2'-deoxycytidine is productive of
deoxyoligonueleotides as in Example TV ereLn Us
[5-(3-aminopropyl)-uracil] is replace by Amman-
propyl)cytosines. For example,
; : ,
-58~
C A G Cm T Cm T em I;
O O C) O O O O O
HO OPT OPT -0-1~-0\ -POW\ -0\ -O-PI-O\ r I- \ POW I
Jo ~o~g-oio o owe
where Cm = 5-(3-aminopropyl)cytosine.
EXAMPLE XXII
Repeating the deoxyoligonucleotide synthesis pro-
seedier of Example XXI but replacing 5'-DMT-N4-benzoyl-
5-(3-trichloroacetylaminopropyl)-2~-deoxycytidine with
compounds numbered 47 through 57 below is productive of
the corresponding oligonucleotides having the Cm cry-
cosine bases numbered 47' through 57' below, respect
lively, i.e., substituting:
47 5-D~T-N -benzoyl-5-~3-trifluoroacetylaminopropen-
1-yl)-2'-deoxycytidine
48 5'-DMT-N -benzoyl-5- (4-tri~luoroacetylaminobut-1-
yl~-2'-deocycytidine
49 5'-DMT-N4-benzoyl-5-~4-trifiuoroacetylaminobuten-
l-yl)-2'-deoxycytidine
5'-DMT-N4-benzoyl-5-(6-trifluoroacetylaminohex-1-
yl)-2'-deoxycytidine
51 5'D~r-N4-benzoyl-5-(6-trifluoroacetylaminohexen-1--
yl)-2'-deoxycytidine
52 5'-DMT~N4-benzoyl-5-(3-trifluoroace~ylaminoprop-2--
ye)- 2'-deoxycytidine
53 5'-DMT-N4-benzoyl-5-(3-trifluoroacetylamino-2-
methylpropen-l-yl)-2'-deoxycytidine
54 5'-DMT-N4-~enzoyl-5-(3-trifluoroacetylamino~2-
methylprop-l-yl)-2'-deoxycytidine
55 5'-DMT-N4-benzoyl-5[2-(4-trifluoroacetylaminomethyye-
phenyl)e~hen-l-yl]-2'-deoxycytidine
_59_ I 3
56 5'-DMT-N4-benzoyl-5-[N-(pertrifluoroacetylpoly-
lysyl)-l-acrylamido)-2'-debxycytidine
57 5'-DMT-N4-benzoyl~-[N-~trifluoroacetylaminoheptyl))-
acrylamido]-2'-deoxycytidine
is productive of the products wherein Cm is:
47' 5-(3-aminopropen-l-yl)cytosine
48' 5-(4-aminobu~ yl~cytosine
49' 5-;4-aminobuten-l-yl)cytosine
50' 5-(6-aminohex-l-yl)cytosine
lo Al' 5-(6-aminohexen-l-yl)cytosine
52' 5-(3-aminoprop-2-yl)cytosine
53' 5-(3-amino-2-methylpropen-l-yl~cytosine
54' 5-(3-amino-2-methylprop-l-yl)cytosine
55' 5-~2-(4-aminomethylphenyl)ethen-l-yl]cytosine
I 5-[N-(polylysyl)-l-acrylamido]cytosine
57' 5-[N-(7-aminoheptyl)-l-acrylamido]cytosine
Similarly, by employing other N4-acyl-5-(acylaminoalkyl)-
2'-deoxycytidines the analogous deoxyoligonucleotides
are produced.
EXAMPLE XXIII
: Repeating the deoxyoligonucleotide synthesis pro-
seedier of Example XXI, but replacing 5'-DMT-N4-benzoyl-
5-(3-trifluoroacetylaminopropyl)-2'-deoxycytidine with
the compounds numbered 58 through 68 below is productive
of the corresponding oligonucleo~ides having the Cm
citizen bases numbered 58' through 68' below, respect
lively, i.e., substituting:
-60-
58 5'-DMT-N4-benzoyl-5-(propen-1-yl)-2'-deoxycytidinee
59 5'-DMT-N4-benzoyl-5-(2-carbmethoxyethyl)-2' Dixie-
cytidine
60 5'-DMT--N4-benzoyl-5-(2-carbmethoxyethen-1-yl)-2'--
deoxycytidine
61 5'-DMT-N4-benzoyl-5-(3-carbmethoxyprop-1-yl)-2'-
deoxycytidine
62 5'-DMT-N4-benzoyl-5-(4-carbmethoxy-2-methylbuten-
l-yl)-2'-deoxycytidine
63 5'-DMT-N4-benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxy--
cytidine
64 5'-DMT-N4-benzoyl-5-(4-cyano-2-methylbuten-1-yl)-
2'-deoxycytîdine
65 5l-DMT-N4-benzoyl-5-[2-(4-carbmethoxyphenyl)ethen--
1-yl]-2'-deoxycytidine
66 5'-DMT-N4-benzoyl-5-(4-acetoxybuten-1-yl)-2'-
deoxycytidine
67 5'-DMT-N4-benzoyl-5-(4-acetoxybut-1-y1)-2'-
deoxycytidine
68 5'-DMT-N4-benzoyl-5-~4-(2,4-~initrophenyl)butyl]-
2-deoxycytidine
.
is productive of the products wherein Cm is;
58' 5-(propen-1-yl)cytosine
59' 5-(2-carboxyethyl)cytosine
60' 5-(2-carboxyethen-1-yl)cytosine
61' 5-(3-carboxyprop-1-yl)cytosine
62' 5-(4-carboxy-2-methylbuten-1-yl)cytosine
63' 5-(3-cyanopropen-1-yl)cytosine
64' 5 (4-cyano-2-methylbuten-1-yl)cytosine
9.~3~6~11
-61-
65'. 5-[2-(4-carboxyphenyl)ethen-1-yl]cytosine
66' 5-~4-hydroxybuten-1-yl)cytosine
67' 5-(4-hydroxybut-1-yl)cytosine
68' 5-[4-(2,4-dinitrophenyl)butyl]cytosine
Similarly, by employing other appropriate 5'-DMT-N4-
acyl-5-alkyl-2'-deoxycy~idines the analogous Dixie-
oligonucleotides are produced.
EXAMPLE XXIV
Repeating the phosphomonochloridite and deoxyoligo-
nucleated synthesis procedures of Examples XV-XXIII,
but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)-
2'-deoxyuridine with 5'-DMT-N5-benzoyl-8-(6-trifluoro-
acetylaminohexyl)amino-2'-deoxyadenosine is productive
of deoxyoligonucleotides as in Example XVIII, except
that the Us is replaced by Am, and Am = 8-(6-aminohexyl)
amino-2'-deoxyadenosine.
Examples XXV to XXVIII typify the binding of report
ton groups to oligonucleotides containing appropriately
modified bases, as illustrated in Reaction 6.
EXAMPLE XXV
Fluoresceinated deoxyoligonucleotides
A purified pentadeca-nucleotide (from Example XVIII)
of the structure
C A G Us T us T us G
O O O O O O O
~10\ -O-P O\ OPT -0-~-0\ -O-P-O\ -POW\ OPT 'I\ POW
r Jo I_
PUP P ~1'
-62- 3
[where Us is 5~[N-(7-aminoheptyl)-l-zcrylamido]uracil
is dissolved await Aye units per ml in aqueous 300mM
sodium borate or sodium carbonate buffer, pi 9.5, con-
twining 30 sodium chloride. Solid fluoresce in is-
thiocyanate (OHS my per ml) is added, and the mixture sealed and shaken gently at 4 C to 25 C overnight. The
reaction is chromatographed directly on a column of
G-50 Seafood to separate unbound fluoresce in adduces
which are retained; the fluoresceinated deoxyoligo-
nucleated adduces elude near the void volume. Early fractions containing significant Aye units are combined
and lyophilized to solid product of structure similar
to the starting pentadecadeoxyoligonucleotide where
us is now either
O o S
HNJ~N~CH2~NHCNH-Fluorescein
OWN
or unrequited 5-[N-(7-aminoheptyl)-l-acrylamido]uracil.
I Max (H20) 262 no, 498 no.
Repeating the procedure on compounds recited in
Examples XIX, XXI, XXII, and XXIV is productive of the
corresponding fluoresceinated or polyfluoresceinated
I deoxyoligonucleotides in like manner.
EXAMPLE XXVI
Attachment of reporter groups other than fluoresce in
can be accomplished by repeating the procedure of Example
I 3
-63-
XXVD but replacing fluore5cein isothiocyanate with,
for example:
2,4-dinitrophenyl ~sothiocyanate
l-fluoro-2,4-dinitrobenzene
amino ethyl isoluminol lsothiocyanate
aminoethylaminonaphthalene-1,2-carboxylic hydrazide
isothiocyanate
N,N'-bi~ talkyl~ulfonyl)-N~aryl-N'-isothiocyanatoaryl-
dioxamide
. 10 m-sulfonyl aniline isothiocyanate
N-hydroxysuccinimidyl button
9- (N-hydroxysuccinimidyl carboxy)-N-methylacridine,
or cyanogenbromide-activated Suffer
is productive no the corresponding.adducts wherein the
attached group is other than fluoresce in.
EXAMPLE XXVII
Attachment of isoluminol and free primary
amine-containing reporter group
purified pentadecanucleotide from Example XVIII
20 of the structure: .
C A G U T Us T em G
O O O O ' O O ' O O
. 1~0~,~ OX I I O\ -O-P-O\ I I I\ -UP Ox -Ox
"I
O O- ox ox O-
where TV it 5-(2-carboxyeth~nyl)urac~l] it dissolved
in water await Aye units per my and diluted with one
volume pardon. Aminobutyl ethyl lsoluminol is added
I
to a final concentration of 1 mg/ml,followed by add-
lion of a five-fold molar excess of loathly-
dimethylaminopropyl) carbodiimide. The reaction is
sealed and shaken gently in the dark for 12 to 48
hours. The reaction mixture is concentrated under
reduced pressure to a solid residue, and chroma~ographed
directly on a column of G-50 Seafood I; the isoluminol-
deoxyoligonucleotide conjugates elude near the void
volume. Early fractions containing significant Aye
lo units are combined and lyophilized to solid product
of structure similar to the starting deoxyoligonucleotide
where Us is now either
HO N-isoluminol
or unworked 5-(2-carboxyethenyl)uracil.
Repeating the procedure on compounds from Examples
XV and XXIII wherein R2 contains car boxy is productive
of the corresponding deoxyoligonucleotide-isoluminol
adduces in like manner.
Repeating the procedure, but replacing aminobutyl
isGluminol with other reporter groups containing a free
primary amine is productive of the corresponding Dixie-
oligonucleotide-reporter adduces in like manner.
-65~
EXAMPLE XXVIII
Attachment of dinitrophenyl reporter groups
A purifiefl nonanucleotide of the structure:
HO i O-P-O~O-P-O~¦--o_P-O¦--o-P-O~o-P~O O-P-Oi 0- too
where Am = 8-(6aminohexyl)aminoadenine is dissolved at
20 Aye units per ml in 250 my sodium carbonate buffer,
pi 9, and 1-fluoro~2,4-dinitrobenzene is added. The
reaction solution is shaken at ambient temperature
overnight, then chromatographed directly on a column
of Seafood G-50. Early fractions containing signify-
cant Aye units are combined and concentrated to given oligonucleotide product similar to the starting
decanucleotide wherein Am is now either
NH2
NH ~N2
,.1 N2
or unrequited 8-(6-aminohexyl)aminoadenine.
Repeating the procedure, but replacing Amman-
hexyl)~minoadenine with other modified bases containing
a free primary amine is similarly productive of the
corresponding dinitrophenylated oligonucleotide adduces.
