Note: Descriptions are shown in the official language in which they were submitted.
ii3.~
A CO~ALENT OLIGONUCLEOTIDE-HORSERADISH
PEROXIDASE CONJUGATE
Description
'rechnical Field
The present invention relates generally to DNA
hybridization probes, and more particularly relates to a
stable, covalent conjugate of an oligonucleotide and
horseradish peroxidase (HRP).
Background Art
Non-isotopically labelled synthetic DNA frag-
ments have found broad application in molecular biology--
e.g., in the areas of DNA sequencing, DNA probe-based
diagnostics, and the like. The conjugate disclosed herein
is prepared using reagents which facilitate the labeling
of oligonucleotides with specific groups by incorporating
one or more modifiable sulfhydryl groups at one or more
~hydroxyl sites within the oligonucleotide.
Methods of introducing a sulfhydryl group at the
5' terminus of synthetic oligonucleotides are known. For
20 example, Connolly, in Nuc. Acids Res. 13(12):4485-4502
(1985) and in Nuc. Acids Res. 15(7):3131-3139 (1987~,
describes a method of incorporating a sulfhydryl moiety
into synthetic DNA using S-trityl-O-methoxy-
morpholinophosphite derivatives of 2-mercaptoethanol, 3
~;;
.~/`'~L'~.
~3~ 3;2
mercaptopropan-l-ol and 6-mercaptohexan-l~ol--i.e., re-
agents given by the formula
~Sk~ o~
where x is 2, 3 or 6. Connolly further describes deriva-
tization o the sulfhydryl-containing oligonucleotides
with thiol-specific probes.
However, this and other prior art methods suffer
from one or more of the following disadvantages:
(1) A short spacer chain linking the 5' terminus
of the oligonucleotide to the sulfhydryl, amino or
hydroxyl group results in destabilization of the
derivatized structure--i.e., proximity of a solid support
or a bulky labeling species to the oligonucleotide chain
causes steric interference and thus hinders use of the
derivatized oligonucleotide in probe-based applications;
lS (2) A hydrophobic spacer chain linking the 5'
terminus of the oligonucleotide to the sulfhydryl, amino
or hydroxyl group provides problems with solubility in the
aqueous solvents commonly used in DNA probe~based methods;
(3) Conventionally used functionalizing reagents
are often incompatible with commonly used D~A synthesis
methodology/ primarily because the functionalizing re-
agents are incompatible with the reagents and sol~ents
typically used therewith;
:
~3~0~i3~
(4) Conventionally used functionalizing reagents
are frequently difficult to synthesize in high yield,
necessitating complex, multi-step reactions;
(5) Certain known reagents require treatment
with multiple activating agents immediately prior to use;
(6) Conventionally used functionalizing reagents
do not allow for "tacking on" of multiple spacer chain to
increase the distance between the terminal sulfhydryl,
amino or hydroxyl moiety and the oligonucleotide chains,
nor, generally, do they allow for multiple functionalization
along an oligonucleotide chain;
(7) Conventionally used functionalizing reagen-ts
do not generally allow for-functionalization at positions
other than at the 5' hydroxyl terminus; and
(8) Conventionally used functionalizing reagents
sometimes require deprotection under harsh conditions, in
such a way that, frequently, the deprotection reaction is
not readily monitorable.
There is thus a need in the art for oligonucleotide
functionalizing reagents and methods which address these
considerations. The present invention involves certain
novel functionalizing reagents which overcome the
aforementioned problems. More specifically, the invention
is directed to a method of "derivatizing" sulfhydryl-function-
alized oligonucleotides which can be prepared using noveloligonucleotide functionalizing reagents as will be described.
Covalent conjugates of oligonucleotides and
labelling enzymes have been described in the literature.
For example, Jablonski et al., in Nuc. Acids Res.
14(15):6115-6128 (1986), describe covalent conjugates of
alkaline phophatase and olignucleotides prepared using
the homobifunctional reagent disuccinimidyl suberate.
Renz and Kurz, in Nuc. ~cids Res. _ (8):3435-3445 (1981),
describe a covalent complex of HRP and oligonucleotides
~3g~3;2
-- 4
using a polyethyleneimine spacer chain having a molecular
weight of about 1400. Also, Ruth and Jablonski, in
N eosides and Nucleotides 6(1&2):541-542 (1981),
disclose conjugates of oligodeoxynucleotides and alkaline
phosphatase having a l9-atom spacer chain between the
oligomer and the enzyme. While these probes have been
used successfully, it would nevertheless be desirable to
provide probes which are more stable and which generate
color faster, thus yielding a more effective and more
readily monitorable means of detection.
Disclosure of the Invention
-
It is accordingly a primary object of the
present invention to provide a stable, readily monitorable,
"derivatized" oligonucleotide which comprises a sulfhydryl
functionalized oligonucleotide covalently conjugated to HRP.
It is a further object of the present invenkion
to provide a method of making such covalent conjugates.
It is another object of the invention to provide
a method of using these conjugates in DNA probe-based ap-
plications.
Additional objects, advantages and novelfeatures of the invention will be set forth in part in the
description which follows, and in part will become appar-
ent to those skilled in the art on examination of the fol-
lowing, or may be learned by practice of the invention.
The objects and advantages of the invention may be real-
ized and attained by means of the instrumentalities and
combinations particularly pointed out in the appended
claims.
In a preferred embodiment of the invention,
oligonucleotide functionalizing reagents are used to
functionalize an oligonucleotide chain at a hydroxyl group
contained therein to introduce a sulfhydryl group~ The
~i
_ 5 _ ~3~ 3~
coupling reaction is effected using standard techniques
for coupling a phosphoramidite to a hydroxyl group of an
oligonucleotide, as described, inter alia, by Beaucage and
Caruthers, Tetrahedron Lett. (1981) 22:1859-1862. After
functionalizatlon, the oligonucleotide is derivatized at
the new sulfhydryl site with E~RP as will be described.
Modes for Carrying Out the Invention
1. Definitions
"Sulfhydryl functionalizing" or simply
"functionalizing" as used herein means incorporating a
protected or unprotected sulfhydryl moiety into an
oligonucleotide chain. The sulfhydryl group introduced by
functionalization is spaced apart from the oligonucleotide
chain by a spacer chain as will be described herein.
"Derivatizing" as used herein means reacting a
functionalized oligonucleotide at the added sulfhydryl
group with a detectable species, i.e.~ one that serves as
a label in probe-based applications. A "derlvatized"
oligonucleotide is thus one that is detectable by virtue
of the "derivatizing" species. As noted above, the
derivatizing species herein is the enzyme horseradish
peroxidase.
An "oligonucleotide" as used herein is a single-
stranded or double-stranded, typically a single-stranded,
chain of nucleotide, typically deoxyribonucleotide,
monomer units. While the reagents and methods of the
present invention may be used in conjunction with a single
nucleotide monomer or with a full-length DNA strand, the
"oligonucleotides" herein are typically single-stranded
and of from about 2 to about 400 monomer units, and, more
-typically for most probe-based applications, from about 2
to about 100 monomer units. Optimal length for use as an
allele-specific oligonucleotide (or "ASO") is about 135-21
base pairs.
Use of the derivatized oligonucleotides in
l~probe-based~' applications is intended to mean use of the
labelled chain to detect or quantify oligonucleotide seg-
ments or sequences in a specimen.
A free sulfhydryl group that is "protected~' is
one that has been reacted with a protecting moiety such
that the resulting protected group will not be susceptible
to any sort of chemical reaction during the synthetic step
or steps during which the protecting group is present.
By 'Istability" of the functionalized or
derivatized oligonucleotide chain is meant substantial
absence of steric interference as well as chemical stabil-
ity under the conditions of most probe-based applications.
By "lower alkyl" and "lower alkoxy" are meant
alkyl and alkoxy substituents, respectively, having from
about l to 6, more typically from about l to 3, carbon
atoms.
2. Structure of the Functionalizinq Reaqents
The sul~hydryl functionalizing reagents used to
prepare the probes of the present invention--i.e., the
covalent oligonucleotide-HRP conjugates--are substantially
linear reagents having a phosphoramidite moiety at one end
linked through a hydrophilic spacer chain to an opposing
end provided with a protected or unprotected sulfhydryl
moiety. These functionalizing reagents are given by the-
s~ructure
R~CH~ Q[CH ~ a~ (CH ~ Q P~OR3 (2
~30~i3~2
--7--
wherein:
R is a protected or unprotected sulfhydryl
moiety,
R is a hydrogen, -CH2OH, or a substituent hav-
ing the formula
X1 X2
--CH2--O--C~ ( 3
Xs x6
in which Xl, X2, X3, X4, X5 and x6 may be the same or dif-
erent and are selected from the group consisting of
hydrogen, lower alkyl and lower alkoxy;
R1 and R2 are independently selected from the
group consisting of hydrogen and lower alkyl;
R3 is ~-cyanoethyl or methyl;
the ~ moieties are selected from the group
consisting of
O O O
-O-, -NH-, -S-, -NH-C-, -NH-C-O-, and -NH-C-NH-
and may be the same or diferent;
n', n'' ~nd n''' are integers in the range of 2
and 10 inclusive; and
n is an integer in the range of 2 and 30
inclusi~e.
~ ~30~?~i3~
Formula (4) represents one example of a
particularly preferred embodiment
1 ~NR1R2
R-CH2-CH2~ 12-CH2~nO-cH2-cH~p~R3 ( )
where R, R , R1, R2, R3 and n are as given above- The
hydrophilic spacer chain in such a case is a polyether
linkaqe, e.g., as shown, formed from polyethylene glycol.
(In other embodiments encompassed by general structure
(2), the spacer chain may also be formed from
polypropylene glycol or the like, or from
poly(oxyalkyleneamines) such as the Jeffamines* sold by
Texaco Chemical Co.)
When it is desired to couple the functionalizing
reagent to an oligonucleotide chain, at any position,
generally, that a nucleoside phosphoramidite could be
coupled to the chain, the R moiety is a protected
sulfhydryl moiety. The protecting group is selected so
that the sulfhydryl moiety remains intact during the
phosphoramidite coupling step--i.e., in which the
phosphoramidite group of the reagent reacts with the
hydroxyl moiety on the oligonucleotide chain. The condi-
tions for this reaction are those used in the conventional
method of synthesizing DNA via the so-called
phosphoramidite route, described, for example, in
Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862
(1981).
3 5 *Trade Mark
~L3~ ii32
g
Examples of particularly preferred sulfhydryl
protecting groups are given by R=
1 X2
\ ~
~ S-C-CH, or -S-C~ (5,6,7)
Xs X~;
It is to be understood that the aforementionecl
exemplary protecting groups are illuskrative only, and
that any number of sulfhydryl protecting groups may be
used so long as the above-described "protecting" criteria
are met.
The opposing, second end of the functionalizing
reagent defined by the phosphoramidite group
~NR1R2 (8)
~oR3
2S
is selected so as to couple, typically, to the terminal S'
hydroxyl of a growing or completed oligonucleotide chain..
As noted above, Rl and R2 are either hydrogen or lower
alkyl, and may be the same or different; in a particularly
preferred embodiment, both Rl and R2 are isopropyl. R3 is
either methyl or ~-cyanoethyl; in a particularly preferred
embodiment, R3 is ~-cyanoethyl. Use of the
phosphoramidite group as a:coupling means is well known in
; 35 the art of DNA synthesis, and reference may be had to
.; ,.~
- 10- ~3~;32
Beaucage and Caruthers (1981), supra, for further descrip-
tion on point.
The spacer chain (9)
~Ct I~QLcH3;ji; Q~L(CH)~ Q-- ( )
is a hydrophilic chain wherein n, n', n'' and ''' are
integers having values as set forth above.
In the preferred embodiment represented by
formula (4), thè spacer chain is the polyether moiety
-CH2-CH2~0-CH2-CH2~nO-cH2-cH- (10)
wherein n is typically 2-30, more typically 2-20 (in some
cases, however, n may be larger than 30, i.e., where
increased distance is desired between the derivatizing
moiety and the oligonucleotide chain). Optimal values for
n provide the spacer chain with a total of at least about
8 carbon atoms along its length. The length of the spacer
chain is quite relevant to the effectiveness of the re-
agents, as providing greater distance between thesulfhydryl group and the oligonucleotide chain: (1)
facilitates coupling of the reagent to DNA; (2) avoids
steric interference which would hinder hybridization and
destabilize the functionalized or derivatized
oligonucleotide chain; ~3) simulates a "solution" type
environment in that freedom of movement of the derivatized
sulfhydryl moiety is enhanced; and (4) avoids interference
with the activity of the derivatizing species, in this
case the enzymatic activity of horseradish peroxidase.
The hydrophilicity of the spacer chain also enhances the
'~
053~2
solubility of the functionalized or derivatized
oligonucleotide chains in aqueous media.
R* is either hydrogen, hydroxyl, or the aromatic
substituent given by (3). Where R* is (3), it is selected
so that the chromogenic cation
X1 x2
X5~,C~[~, ( 11 )
is monitorable upon release. That is, after coupling of
the functionalizing reagent to DNA, deprotection will
yield cation (11) in solution. An example.of a particularly
preferred substituent is dimethoxytrityl (DMT)--i.e.,
R* is -CH2-O-DMT.
While in a preferred embodiment, as illustrated
by structures (2) and (4), R* is bonded to the carbon atom
adjacent to the phosphoramidite group, it is also possible
that R* may be bonded to one or more other carbon atoms
along the spacer chain, as illustrated by formula (2).
3. Use of the Novel Reagents to Functionalize Oligo-
nucleotide Chains
In generalj the coupling reaction between the
functionalizing reagents and a hydroxyl-containing
compound may be represented by the following scheme:
X-OH ~ Reagent (2) --~
(Scheme I)
R ~ ~ Q[CH ~ Q~ (~H ~ \ORI (12)
,.~....
~L3()~
-12-
In Scheme I, X is typically an oligonucleotide chain. The
reaction conditions are the same as those used in the
phosphoramidite route to DNA synthesis, as noted earlier
and as described, inter alia, by Beaucage and Caruthers
(1981), supra.
Compound (12) is deprotected as follows. Where
R is given by formula (3), conversion to an unprotected
hydroxyl group is carried out by treatment with acid. The
protected sulfhydryl moiety at R may be deprotected with,
e.g., silver nitrate.
Multiple functionalization of an oligonucleotide
is possible by making use of multiple R sites where R is
-CH2OH or given by formula (3). After acid deprotection,
further functionalization by reaction at the deprotected
hydroxyl site is enabled. Thus, in the case of
functionalized oligonucleotide (13), for example,
/~-x
R-CH2-CH2~o-cH2-cH23no-cH2-lH-op~o~3 (13)
deprotection of R and further functionaliæation at the
-CH2OH-OH moiety so provided, using a standard
phosphoramidite coupling procedure, gives the compound of
formula (14):
R ~H2-CH2~o-cH2-cH23no-cH2-cH-op--~X
~H~ oR3 (14
f~ CH2-C~12~C)~H2-~H23n~CH2-CH4
R-
-13-
Multiple functionalization at a plurality of hydroxyl
groups along an oligonucleotide chain i~ also possible
using the same chemistry.
4. Synthesis of the Functionalizinq Reaqents
The inventors herein have developed various
routes to the no~el reagents. For the purpose of simplic-
ity, syntheses of the functionalizing reagents will be
discussed in terms of exemplary structure (6) rather than
general structure (4). It is to be understood, however,
that the synthetic methods described apply, in general,
substantially identically to compounds represented by t4).
In a first embodiment, where the functionalizing reagent
to be synthesized is an amine functionalizing reagent,
1'` Scheme II may be followed:
-~ H2-CH2~CH2-CH2~nO CH2-CH2-OH
(15)
Step (1)
~3P ~ R-H
(16) (17) ~ o ~ ~N=N
X_p~NR1R2 ~NRlR2
OR --R CH2-CH2~O-CH2-CH2~nO-CH2-CH2-OP~oR3
Step (2)
(18)
Scheme II
~3~ i3~
-14-
Step (1) represents the Mitsunobu reaction as is well
known in the art. Briefly, the reaction involves
admixture of compounds (15), (16), (17) and (18) in a
polar, organic solvent for a least sev~ral hours, prefer-
ably overnight (see Example 1). Compound (19) is isolated
and coupled to the phosphoramidite (wherein X represents a
halogen~ preferably chlorine) as follows. A molar excess
of the phosphoramidite is added to compound (19) in a
suitable solvent, again, one that is preferably a polar,
organic solvent, under an inert atmosphere. Compound (20)
is isolated--e.g., by column chromatography.
An alternative method of synthesizing the amine
functionalizing reagents herein, and one which may also be
used to give the sulfhydryl functionalizing reagents, is
given by Scheme III:
S3~
(III ~Ula~lOS) ~;
O_u~=O
O
T --1-- 0 ~
U~ o - D. 5z
Q,:: _
C ~
h u~ O Z
..
I :C" I o I/O~ I
: X I I: 3
O ~ ~
_ U ~ ~1 5 1
ct~ O O
r~ I I T
o T 1
~ C - C O O I
T 1,~ o ~ _ ~ _
-- V
S
V ~ V ~ V -- V
,a ~ T T ~ ~ T ~
a
3L3~ 3;~
-16-
~H2-ci~12~o-cH~-cH~no-cH2-cH2~-c ~2/c~-c\
(22) \ C /
Acid Step (3) ~H3~H3
R-CH2-CH2~-CH2-CH2~n\O-CH2-CH2-O-CH2-CH-CH `
(23) -- OH OH
1) R-~X Ste~ (4)
2) X p~NR1R2 R~
R-C~12-CH2~0-CH2-CH23ncH2-c~-OP~oR3R
In Scheme III, steps la-lc and la'-lb' represent
alternative routes to intermediate (21). In steps la-lc,
the protected diol (21) is formed Dy: reaction of the
polyethylene glycol with allyl bromide (reaction carried
out at room temperature for at least about a few hours,
preferably overnight) to give (19); reaction of (19) with
osmium tetroxide to give diol (20) under conventional,
known conditions; and protection of the diol by reaction
with 2,2-dimethoxypropane. Steps la'-lb' give (21) via
reaction of the tosylated glycol with the solketal anion.
Step 2 represents the Mitsunobu reaction as shown in
Scheme II, where R is as defined earlier, while the acid
treatment of Step 3 deprotects the diol. Step 4-1
introduces a chromogenic moiety where R is given by (5)
(and may thus be omitted where R is hydrogen) and Step 4-
2 introduces the phosphoramidite. "X~' in both Steps 4-l
and 4-2 is a halogen leaving group, preferably chlorine.
~130U~3~
-17-
A third synthetic method, ~pecific for the
production of sulfhydryl functionalizing reagents, is
gi~en by Scheme IV.
Ho-CH2-CH2*-CH2-~H~3n~ CH2-C~2-OH (18)
e~ 3 P, ¢iJ~ -SHIt
O O Step (l)
~ O J ~N-N ~ ~
~ + S-CH2-CH2~-CH2-CH2~nO-CH2-cH2-OH ~24)
/NR1R2
X~P~o 3 Step (2)
NR1 R2
~+s-CH2-CH~ CH2-CH23no
(25)
Scheme IV
In Scheme IV, Step l is carried out at a low temperature,
preferably about 0C or less, and the triphenylphosphine,
diisopropylazodicarboxylate and S-tritylmercaptan are al-
lowed to react overnight. The phosphoramidite is added in
Step 2, and ~25) is o~tained in good yield. Here, R of
structure (4) is shown as -S-C03 (0=phenyl throughout) but
~OQ53Z
-18-
may in fact be any number of protected sulfhydryl
moieties.
5. Derivati~ation with HRP
The functionalized oligonucleotide chains
prepared using the above-described reagents are primarily
useful in probe-based applications. That is, the primary
purpose of introducing a sulfhydryl group into an
oligonucleotide chain is to enable derivatization at that
site with a laheled species. The present application is
directed to derivatization with the enzyme horseradish
peroxidase.
The derivatized oligonucleotides of the present
invention are conjugates comprising an oli~onucleotide
chain covalently coupled to HRP, the conjugates given by
the structure (26)
HRP-NH-C-(CH~ Q[CH ~ ~ (CH ~ Q-P-O-X
O n OH (26)
wherein
R , Q, n, n', n" and n'" are as defined above
for compound (2), and X is an oligonucleotide chain.
The length of the oligonucleotide chain is
typically in the range of about 2 and 100 monomer units.
Where the conjugate is to be used as an ASO, as noted
earlier, the number of monomer units in the chain is
preferably about 13-21.
~3Q~S32
--19--
In an exemplary embodiment, the conjugates of
the invention may be represented by the structure (27)
o
HRP-NH-C-(CH ) -N/U--I I
2 5 ~ S-CH2-CH2~0-CH2-CH2~nCI-CH2-CH-OP--O--X
O OH
( 2 7 )
where R , X and n are as given above. The conjugates of
formula (27) result from coupling of exemplary sulfhydryl
functionalizing reagent (4) to oligomer X.
The covalent con~ugates represented by Formula
(26) are prepared by the procedure illustrated in Scheme
V:
HRP-NH-C-(cH2)s- ~ + HS ~ H~ Q @ H ~ Q ~ H ~ Q-P-O-X
mal-sac HRP complex
(28) (2g)
HRP-NH-C-(cH2)s-N ~ R ~ O
S 4CH3~Q I(CH3;;~Q _(Ctl)~Q--P--O--X
O _ _ n OH
~26)
Scheme V
~L3~53~
-20-
Preparation of mal-sac-HNS~, i.e., the (N-
maleimido-6-aminocaproyl [mal-sac] derivatlve of 4-
hydroxyl-3-nitrobenzene sulfonic acid sodium salt ~HNSA])
and the corresponding mal-sac-HNSA HRP complex (28) is
described in Examples 6 and 7 below.
Thiolated oligonucleotide (29) is prepared as
described in Example 8. Typically, the tritylthio
oligonucleotides are detritylated just prior to use in the
reaction of Scheme V.
The mal-sac HRP complex (28) is coupled to
thiolated oligonucleotide (29) by simple admixture,
preferably at room temperature or lower. The reaction
mixture is allowed to remain at low temperature--e.g.,
about 0C--at least overnight and preferably at least
about several days, at which point the covalent HRP
conjugate (26) is isolated and purified, preferably
chromatographically.
Prior to use in probe-based applications, the
conjugates are stored in a phosphate buffer (added salts
optional) maintained at a pH of from about 5.5 to about
7.5, preferably about 6.0, at a temperature of from about
-10C to about 30C (with the proviso that the solution
not be frozen), optimally about 4C.
For use in hybridization, the conjugate solu-
tions are normally diluted (the final concentrationvarying depending on use) with hybridization buffer and
used according to standard hybridization techniques (see,
e.g., Maniatis, et al.j Molecular Cloninq, New York: Cold
Spring Harbor Laboratory, 1982). The general procedure
followed is well known in the art, and typically involves:
(1) providing a covalent conjugate according to the inven
tion, which conjugate includes an oligomer having a
nucleotide sequence substantially complementary to that of
an analyte of interest, i.e., sufficiently complementary
to enable hybridization; ~2) contacting, in solution, the
S3~
-21-
analyte of interest with the covalent conjugate; and (3)
detecting the presence of nucleic acid complexes which
form by assaying for HRP activity.
Generally, the covalent conjugate hybridizes to
an analyte that is attached to a solid support and is then
detected thereon.
In sum, the advantages of the novel HRP
conjugates in probe-based applications are many. A
primary advantage is the relatively long, hydrophilic
spacer chain which provides an optimum distance between
the HRP and the oligonucleotide, ensuring that full bio-
logical activity of the HRP is retained and enhancing the
effectiveness of hybridization. The novel conjugates, by
virtue of the "R " moiety, also allow multiple
derivatization of one oligonucleotide, i.e., attachment of
two or more "spacer-HRP" chains either linked end-to-end,
bound at various points within an oligonucleotide chain,
or both. Finally, in contrast to other enzyme/oligomer
conjugates, e.g., alkaline phosphatase systems, ease of
detection is enhanced by the rapid generation of color.
-
It is to be understood that while the invention25 has been described in conjunction with the preferred
specific embodiments thereof, that the foregoing descrip-
tion as well as the examples which follow are intended to
illustrate and not limit the ~cope of the invention.
Other aspects, advantages and modifications within the
scope of the invention will be apparent to those skilled
in the art to which the invention pertains.
~ 3~?S3~
- 22 -
Example 1
(a) Reaction of tetraethylene glycol with
phthalimide (see Step(l), Scheme II): Tetraethylene
glycol (38.85 g, 200 mmole) and triphenyl phosphine ~52.46
g, 200 mmole) were dissolved in 200 mL of dry TEIF, and
phthalimide (29.43 g~ 200 mmole) added. A solution of
diethylazo dicarboxylate (DEAD) (34.83 g; 200 m~lole) in
100 mL of dry THF was added dropwise to the reaction
mixture, with cooling and stirring. The reaction mixture
was stirred overnight at room temperature. Solvent was
then removed under reduced pressure, and the residue
partitioned between 250 mL of ~2 and 250 mL of diethyl
ether. The aqueous layer was washed five times with 200
mL of diethyl ether and,concentrated under vacuum. The
residue was dried by azeotropic distillation of toluene (3
x 100 mL) and weighed. The 25.89 g obtained was then
purified on an 5iO2 column using ethyl acetate as an
eluant. The product fractions were collected and
concentrated to a syrup (11.75 g; 36.3 mmole; 18.2%) which
was allowed to crystalli~e overnight.
The structure of the product obtained in (a) was
confirmed by lH NMR as:
o
~ O ~ O o ~ OH
~ b) Synthesis of the allyl derivative (see Step
lb, Scheme III): To a solution of the alcohol obtained in
step (a) (4.67 g; 14.4 mmole) in 100 mL of dry T~F was
added Na~ (520 mg; 21.67 mmole). The mixture was stirred
for one hour, and then allyl bromide (1.9 mL; 2.61 g;
21.67 mmole) was added. The suspension was stirred
overnight, at which point it was filtered and the solvent
removed under reduced pressure. The residue was purified
~,
~IL300S32
-23-
on an SiO2 column using a mixture of ethyl acetate and
hexane (70:30) as eluant. Fractions containing the
desired product were pooled and concentrated to a syrup
weighing 2.84 g (7.82 mmole; 54.3~). Elemental analysis
was as follows. Calc.: C, 62.80; H, 6.93; N, 3.85.
Found: C, 62.49; H, 6.99; N, 3.82.
Proposed structure of the product obtained:
o
10 ~1 ~ --~ ,
(c) Synthesis of the corresponding diol (see
Step lb, Scheme III): To a solution of the allyl ether
prepared in step (b) (2.84 g; 7.82 mmole) and N-methyl
morpholine N-oxide tl.83 g; 15.63 mmole) in 180 mL of DMF/
H2O (8:1) was added osmium tetroxide (8.13 mL of a solu-
tion 25 mg/mL in t-butanol; 800 umole). The resulting
amber solution was stirred at room temperature. After 48
hours, a solution of sodium hydrosulfite (2.13 g) in water
(10 mL) was added to the reaction mixture. A black
precipitate formed and the suspension was stirred for 1
hour. The mixture was filtered and concentrated under
reduced pressure. The residue was purified on an SiO2
column using a mixture of methylene chloride and methanol
as the eluant. Elemental analysis was as follows. Calc.:
C, 62.80; H, 6.93; N, 3.85. Found: C, 62.49; H, 6.99; N,
3.82.
Proposed structure of the product:
~ O ~ O ~ O ~ ~ OH
O
- 24 - ~3~532
(d) Labelling with DMT: The diol obtained in
part (G) (1.0 g; 2.50 mmole) was coevaporated with
anhydrous pyridine (2 x 15 mL). The dry residue was then
dissolved in 25 mL of the same. DMT-Cl (0.92 g; 2.75
mmole) was added to the solution. The reaction was car-
ried out at room temperature and monitored by TLC
(CH3Cl:MeOH approximately 97.3) until appearance of the
product.
After one hour, 10 mL of methanol was added and
the reaction mixture was stirred for ten additional
minutes. Next, the reaction was quenched with 10 mL of
ice water and extracted with ethyl acetate (2 x 75 mL).
The organic laye-r-was washed once with 5% NaHCO3
(50 mL), twice with saturated NaCl solution and dried over
Na2SO4. The product was evaporated down to an oily
residue under reduced pressure.
This residue was chromatographed using the above
solvent system. The final product was used without
further purification in step (e). Yield: 86.3% of
theoretical (1.51 g actual / 1.75 g theoretical).
Proposed structure of the product obtained in this step:
0~'~/--ODNIT
o
(e) Preparation of the phosphoramidite: The
product obtained in step (d) (1.0 g; 1.4 mmole) was dis-
solved in 10 mL of acid-free chloroform and placed in a
250 mL round bottom flask preflushed.with dry argon. To
..{, ....
~3~C~S32
- 25 -
this solution (.72 g, 5.6 mmole) of C(CH3) 2-CH~2-N-E~ was
added. Then, the phosphoramidite
Cl
NC-CH2-CH2-O-P~ (CH
CH (CH3) 2
(O . 66 g; 2.8 INmole) was added with a syringe over a two-
minute period. The reaction was carried out at ro~m
temperature and under argon. After ons hour, the mixture
was transferred with 50 mL of ethyl acetate in a 250 mL
separatory funnel and extracted with saturated NaCl solu-
tion four times. The organic layer was dried over Na2SO4
and evaporated down to an oily residue under vacuum. This
residue was chromatographed with 1~ Et3N in ethyl acetate.
Yield: 48.4% of theoretical (0.610 g actual / 1.26 g
theoretical).
Example 2
Essentially the same procedure was followed as
set forth in Example 1, but the tetraethylene glycol
starting material was not in this case initially reacted
with phthalimide.
(a) Synthesis of the allyl derivative of
pentaethylene glycol: To a solution of pentaethylene
20 glycol (5.65 g; 20 mmole) in 100 mL of dry THF was added
the potassium salt of t-butanol (2.24 g; 20 mmole). The
mixture was stirred for 30 minutes and 18-crown-6 (53 mg;
0.2 mmole) was added. The mixture was stirred for an ad-
ditional 30 minutes and then allyl bromide (2.42 g; 1.73
mL; 20 mmole) was added. A white precipitate, presumably
potassium bromide, was noted to form and stirring was
continued overnight. The reaction mixture was filtered
through a Whatman GFB* filter, adsorbed onto 8 g of SiO2,
and fractionated on an SiO2 column using a mixture of
methylene chloride and acetone (1:1) as eluant. The
*Trade Mark
~,
., L,
- 26 - ~30~S32
pooled fractions yielded 4.28 g (13.28 mmole; 66.4~)
product. Elemental analysis was as follows. Calc.: C,
55.88; H, 9.38. Found: C, 55.56; H, 9.76.
Proposed structure of the product:
~o ~0 ~0 0 0 ,~0~ 0
(b) Synthesis of the corresponding diol: To a
solution of the allyl ether prepared in step (a) (4.28g;
13.28 mmole) in 270 mL of a mixture of acetone and water
(8:1) was added N-methyl morpholine (3.11 g; 4.6 mL; 26.55
mmole; 2 eq.) followed by osmium tetroxide (25 mg/mL in t-
butanol; 338 mg; 13.5 mL; 1.33 mmole [0.1 e~.~}. The re-
action mixture was stirred overnight. The next morning, a
solution of sodium hydrosulfite ~3.62 g) in 15 mL water
was added. After 45 minutes of stirring, the suspension
was filtered through a Whatmarl GFB filter. The solvent
was evaporated, the residue taken up in methanol, and the
suspension filtered. The filtrate was concentrated to an
amber syrup, which was then purified on SiO2 using a
mixture of methylene chloride, methanol, and acetic acid
(80:20:5) [.~3 as eluant. The fractions containing product
were pooled and concentrated to yield 3.3 g (9.26 mmole;
69.7~ yield) product.
Proposed structure of the product:
~o ~ ~ ~ O ~ O ~ ~ H
; (c) The triol prepared in step (b) (3.3 g; 9.26
mmole) was taken up in 60 mL acetone and cupric sulfate
(45 g; 28.20 mmole) was added. To the resulting bluish
suspension was added 60 mL H2SO4, at which point the solu-
',~
~3~ S32
-27-
tion turned yellow. The flask was stoppered and stirred
over a weekend. The suspension was then filtered through
a Whatman GFB filter and the filtrate treated in 2.5 g
Ca(OH)2 for one hour. The suspension was filtered again
and the filtrate concentrated and purified on an SiO2
column. The column was run in 97:3 chloroform: methanol
and then again using 8:1 chloroform: methanol. The column
fractions were pooled, yielding 3.03 ~ (7.64 mmole; 82.5%)
product.
Proposed structure of the product:
HO ~ ~ ~ ~ ~ ~
0--~
lS
Example 3
Synthesis of
/ OCH2CH~CN
DMT~--~o~o----o~ ~ \N--CH(CH3)2
CH(CH3)2
was carried out as follows.
(a) Hexaethylene glycol (10.0 g; 35.40 mmole)
was coevaporated with anhydrous pyxidine (3 x 25 mL) and
then dissolved in 100 mL of the same. DMT-Cl (13.17 g;
38.94 mmole) was added to the solution. The reaction was
carried out at room temperature and monitored by TLC
(CHCl3:MeOH approximately 8:1) until the appearance of
product. After two hours, 25 mL of methanol was added and
the reaction mixture was stirred for 15 additional
minutes. Next, the reaction was quenched with 50 mL ice
~3(~S~2
-28-
water and extracted with ethyl acetate (3 x 150 mL). The
organic layer was washed with 5% NaHCO3 (2 x 100 mL),
saturated NaCl (2 x 100 mL), dried o~er Na2SO4 and then
evaporated down to an oily residue (yellowish color).
This oily residue was chromatographed on a silica gel
column (400 g). The column was eluted first with
CHC13:MeOH (approximately 97:3), then with CHC13:NeOH (ap-
proximately 90:10). The fractions were combined and
evaporated to dryness to give an oily residue. The
material obtained was presumed to be of the structure
HO ~ ~'^\~'' ~ ~ ~ ~ O-DMT
and was used without further purification in the synthesis
of the corresponding phosphoramidite.
(b) The procedure of Example l(e) was followed
using 2.0 g (3.40 mmole) of the compound obtained in (a),
1.6 g (6.80 mmole) of the phosphoramidite
NC-CH2-cH2 P\ ,cH(cH3)2
CH(CH3)2
and 1.76 g (13.60 mmole) of [(CH3)2-CH]-N-Et. Elemental
analysis of the product was as expected for C42H61N2OloP
xH20. Calc.: C, 63.49; ~, 7.93; N, 3.52. Found: C,
63.36; H, 7.95; N, 4.11. Yield: 85.4% of theoretical
(2.28 g / 2.67 g).
~3~S;32
-29-
Example 4
(a~ Synthesis of
~ ~ 5 ~ ~ O''~_,Or'^\~OH
(compound ~26~; see Step l, Scheme IV) was carried out as
follows. To a 0C solution of triphenylphosphine (7.87 g;
mmole) in 75 mL dry THF was added the
azodicarboxylate(NCOOCH(CH3))2 (6.07 g; 30 mmole) with
stirring. After one hour, a solution of tetraethylene
glycol (S.83 g; 30 mmole) in 10 mL dry THF was added. All
material dissolved to give a pale yellow solution. After
one hour, a solution of the mercaptan 03C-SH in 20 mL dry
THF was added dropwise with cooling and stirring. The
reaction mixture was stirred overnight and the solvent
removed under reduced pressure. The residue was applied
to an SiO2 column and frac~ionated using methylene
chloride followed by a mixture of mixture of methylene
chloride and CH3CN (2:1). The material was
rechromatographed on SiO2 using CH3CN as eluant, and the
product was removed from ~3P=O by ~aking small (ap-
proximately 15 mL) fractions. The fractions were pooled,
yielding 5.22 g (11.53 mmole; 38.4~ overall; 77% of
theoretical). Elemental analysis was as follows. Calc.:
Cr 71.65; H, 7.12; S, 7.08. Found: C, 71.32; H, 7.21; S,
7.15.
.
~3~C~S32
-30-
(b) Synthesis of thecorresponding
phosphoramidite
~CH(CH,)2
0 N
~+5--~-- ~p/ \CH~CÇ i3)2
~ O ~ N
was then carrisd out according to the method described in
Example l(e), using the reaction product of step (a) (4.22
g; 9.30 mmole), the phosphoramidite
,CI
NC--CH2-cH2-o P\ ,CH(CH3)2
1H(CH3)2
(4.40 g; 18.60 mmole) and [(CH3)2-CH]2-N-Et (4.81 g; 37.20
mmole). Yield: 75.3~ of theoretical (4.57 g / 6.07 g).
Example 5
(a) Synthesis of
0
~ + 5 ~ O ~ O ~ ~ H
was carried out as follows. To a O C solution of
triphenylphosphine (7.87 g; 30 mmole) in 75 mL dry THF was
added the diisopropyl azodicarboxylate (NCOOCH(CH3))2
(6.07 g; 30 mmole) with stirring. After one hour, a solu-
tion of tetraethylene glycol (5.83 g; 30 mmole) in 10 mL
dry THF was added. All material dissolved to give a pale
yellow solution. After one hour, a solution of the
mercap~an ~3C-SH in 20 mL dry THF was added dropwise with
~3~S~
cooling and stirring. The reaction mixture was stirred
overnight and the solvent removed under reduced pressure.
Th~ residue was applied to an SiO2 column and fractionated
using methylene chloride followed by a mixture of mixture
of methylene chloride and C~3CN (2-1). The material was
rechromatographed on SiO2 using CH3CN as eluant, and the
product was removed from ~3P=O by taking small (ap-
proximately 15 mL) fractions. The fractions were pooled,
yielding 5.22 g (1~.53 mmole; 38.4% overall; 77~ of
theoretical). Elemental analysis was as follows. Calc.:
C, 71.65; H, 7.12; S, 7.08. Found: C, 71.32; H, 7.21; S,
7.15.
(b) Preparation of the phosphoramidite:
/N[CH(CH~)~
S 0-- 0-- ~ 0 P~
cb C H2C H2C N
The product obtained in step ~a) (4.22 g; 9.30 mmole) was
dissolved in 10 mL of acid-free chloroform and placed in a
250 mL round bottom flask preflushed with dry argon. To
this solution (.72 g, 5.6 mmole) of [(CH3)2-CH]2-N-Et was
added. Then, the phosphoramidite
~CI
Nc-cH2-cH2-o P\ ,cH(cH3)2
CH(CH3)2
(0.66 g; 2.8 mmole) was added with a syringe over a two-
minute period. The reaction was carried out at roomtemperature and under argon. After one hour, the mixture
was transferred with 50 mL of ethyl acetate in a 250 ml
separatory funnel and extracted with saturated NaCl solu-
tion four times. The organic layer was dried over Na2SO4
~3~S3~
~ 32 -
and evaporated down to an oily residue under vacuum. This
res~due was chromatographed with 1% Et3N in ethyl acetate.
Example 6
Preparation of mal-sac-HNSA Ester
One molar equivalent (2.24 g) of 4-hydroxy-3-
nitrobenzene sulfonic acid sodium salt (HNSA) was mixed
together with one molar equivalent (2.0~ g) of
dicyclohexylcarbodiimide and one molar equivalent (2.10 g)
of N-maleimido-6-aminocaproic acid in 25 m~ of
dimethylformamide (DMF) at room temperature overnight. A
white precipitate of dicyclohexylurea was formed. The
precipitate was filtered and 300 mL diethyl ether was
added to the mother liquor. After about 10 minutes to 4
hours a gummy solid precipitated from the mother liquor.
This solid was found to contain 58~ of active HNSA ester
and 42% of free ~INSA.
The analysis consisted of dissolving a small
amount of the precipitate in 10 mM phosphate buffer at pH
7.0 and measuring absorbance at ~06 nm; this reading
provides the amount of unreacted free HNSA which is the
contaminating material in the crude HNSA ester. ~ddition
of very small amounts of concentrated strong base (5N
NaOH) hydrolyzed the ester. A second reading was taken.
Subtraction of the first reading from the second yielded
the amount of ester in the original material. For
purification purposes, the solid was dissolved in DMF,
placed on a LH20 Sephadex* column and eluted with DMF so
that the ester was separated from the contaminating free
HNSA. The progress of purification was monitored by thin
layer chromatography using chloroform, acetone and acetic
acid (6:3:1 v:v:v) as eluting solvent. The product was
positively identified as mal-sac HNSA ester by its re-
activity with amines. The yield of crude ester produced
*Trade Mark
. , I .
~3~5~
- 33 -
was estimated to be approximately 30% of theore-tical; the
purified material consisted of 99~ ester.
The ester thus obtained was found to dissolve
fully in water and was found to be stable in water for
several hours, provided no nucleophiles were added. The
purified ester was found to be stable for extended periods
when stored desiccated.
Example 7
Preparation of Conjugate of mal-sac
HNSA Ester and~Horseradish Peroxidase (HRP)
An amide of mal~sac HNSA ester and HRP was
prepared as follows: -
A total of 40 mg (1.0 moles) of HRP (SigmaChemical Co.) was dissolved in 0.5 mL of 0.1 M phosphate
15 buffer at pH 7.0 to yield a total am ne concentration of
3.7 x 10 M. Then, 5 mg (1.1 x 10 moles) of the mal-
sac HNSA ester of Example 5A, calculated from the data in
Example 6A, was dissolved in 0.5 mL of the HRP solution.
The mixture was stirred at room temperature, and the ~RP
20 fraction (2.8 mL) was collected on a Pharmacia G-25 column
using 0.1 M phosphate buffer, pH 6.0, as eluant.
Example 8
Preparation of HRP-Oilgonucleotide Conjugates
A thiol-functionalized oligomer was prepared
25 using the following l9-mer which had been synthesized on a
Biosearch 8630* DNA Synthesizer: d(TGTTTGCCTGTTCTCAGAC).
The sulfhydryl functionalizing reagent obtained
in Example l(b) was mixed with a solution of the oligomer
and coupled thereto under standard phosphoramidite
30 coupling conditions (see, e.g., Reaucageand Caruthers
(1981), supra).
The tritylthio oligomer was purified by a
standard chromatographic technique using a preparative
*Trade Mark
~t
~.
~L3~S32
- 34 -
~RP-1 column and the following solvent gradient (wherein solvent
"A" designates C113CN and "B" designates 5~ CH3CN in O.lM TEAA,
pH 7.3): (1) A, 10~ --> 40%, 15 min.; (2) A, 40% --> 100%, 15
min.; and (3) A, 100%, 5 min. The tritylthio oligomers eluted
after about 20 minutes.
The purified tritylthio oligomer so obtained was
detritylated using silver nitrate and dithiothreitol (0.1 M and
0.15 M, respectively, in 0.1 M TEAA, pH 6.5). The ditritylated
oligomer was then passed through a G-25 (NAP-10) column,
concentrated under vacuum to approximately 100 vl, and used
right away in the following conjugation reaction.
The mal-sac HRP complex prepared in Example 7 (700 vl)
was aliquoted into the thiooligomer to give a final volume of
800 vl. The individual reaction vessels were allowed to remain
at room temperature for approximately one hour, and then at
about 4C for two days, at which point the four conjugates were
removed and purified on a DEAE* Nucleogen weak anion exchange
column using the following solvent gradient ("B" designates
20 mM Na2P04, pH 6; "C" designates 20 mM Na2P04 ~- lM NaCl,
pH 6): (1) B, 0 --> 100~, 30 min.; (2) C, 100%, 10 min.; and
2S (3) C, 100 --> 0%, 5 min. Remaining unconiugated HRP and
oligomer eluted after about 2 and about 15-40 min (depending on
the size of the oligomer), respectively, while the conjugate
eluted after about 15-40 min as well (also depending on the size
of the oligomer). The identity of the product was confirmed by
ultraviolet spectroscopy, monitoring peak absorbances of the
oligomer (at 260 nm) and of the heme group of HRP ~at 402 nm).
*Trade Mark
