Molecular analytical release tags and their use in chemical analysis

Analytical reagents designated "release tags", for labeling molecular species with a highly detectable signal group which can be released in the form of a volatile compound at a desired point in an analytical procedure. In one embodiment, the release tags have the formula EQU (SgCo).sub.x L(Rx).sub.r wherein each Sg is a signal group bearing one or more electronegative substituents, L is any of a wide variety of groups which when attached to a carbonyl group form a readily cleaved linkage, each COL moiety is a release group which upon scission releases signal group Sg in the form of a volative compound, and each Rx is a reactivity group for attaching the release tag compound to a molecular species to be labeled. In a second embodiment, the release tags have the formula EQU SgReRx wherein Sg and Rx are defined as above and Re is a release group which is an olefin, .alpha.-hydroxy ketone or vicinal diol. Conjugates of the release tag compounds and assay methods employing them are also disclosed.

FIELD OF THE INVENTION 
This invention pertains to analytical chemical reagents, and more 
particularly, to cleavable reagents for labeling molecular species in 
analytical procedures and subsequently releasing and detecting 
signal-producing molecules. 
BACKGROUND OF THE INVENTION 
Chemical signal groups are widely used in chemical analysis to label 
substances of interest such as analytes, internal standards, comparison 
substances, and specific binding partners for analytes, so that such 
materials can be followed, detected, or determined in analytical 
procedures. 
Examples of signal groups include radioactive atoms, fluorescent and 
luminescent molecules, metal-containing compounds, electron-absorbing 
groups; enzymes, and light-absorbing compounds. 
Presently-used chemical signal groups suffer from a variety of 
shortcomings. Radioactive atoms in many cases have short half lives, 
present safety and disposal problems, and cause compounds containing them 
to be physically unstable and/or chemically labile. In addition, some 
radioactive materials do not provide high sensitivity, either because they 
do not produce a high level of radioactivity or because beta particles 
produced in the decomposition of the radioactive atoms are quenched by the 
medium to a substantial extent before they can be detected. Also, a 
variety of closely related radioactive tracers which might be employed and 
measured simultaneously in a single system are not available. 
Nonradioactive signal groups suffer from the deficiencies that the signal 
can be dependent on the environment of the label, which necessitates 
careful matching of the matrices of samples and standards if accurate data 
are to be obtained, that the effective signal can be reduced by any 
dilution of the sample during the analytical procedure, and that 
possibilities for using multiple labels simultaneously in a single 
analytical system are limited because of mutual interferences. 
Traditional labels are typically retained on the labeled molecular species, 
and the presence or amount of the labeled material is determined by 
measuring the signal from the label while still attached to the remainder 
of the molecule, and often, in the presence of other constituents of the 
analytical system. As labeled species frequently contain a variety of 
moieties which can interfere with the measurement of the desired signal, 
and in addition, the labeled species cannot always be easily brought into 
a medium which is optimum for the measurement of the signal from the 
label, this can constitute a serious limitation on the utility of labels 
generally in a particular system, or on the use of particular labels which 
an investigator might wish to use. 
An example of such traditional label usage is the common practice of 
labeling molecules with electron-absorbing groups. The molecules are 
inherently volatile or are rendered volatile by the labeling operation. 
They can then be determined in the gas phase by gas chromatography with 
electron capture detection (GC-ECD) or by GC with detection by electron 
capture negative ion mass spectrometry (GC-ECNI-MS). 
The literature contains a few examples of indirect determinations of 
analytes by determination of a molecular species produced by decomposition 
of the analyte or chemical cleavage of a derivative of the analyte. An 
example of the former is the determination of trichloroacetic acid by 
decarboxylation and measurement of the resulting chloroform. See Buchet, 
et al., Arch. Mal. Prof. Med. Tray., 35, 395-402 (1974); and Senft, J. 
Chromatogr., 337, 126-130 (1985). An example of the latter is the analysis 
for T.sub.4 toxin by formation of the labeled derivative 
N-(N-pentafluoro-benzoyl-Met-Gly)-T.sub.4 followed by cyanogen bromide 
cleavage to produce N-pentafluorobenzoyl homoserine lactone. See U.S. Pat. 
Nos. 4,650,750 and 4,709,016 by R. W. Giese. 
Another example of an indirect determination of an analyte is shown in U.S. 
Pat. No. 4,629,689 of Diamond. This reference discloses analytical schemes 
in which at the conclusion of a selective binding assay an enzyme is 
present in a concentration and/or activity which is related to the amount 
of analyte present in the sample, and this enzyme is measured by measuring 
the amount of a readily detectable signal group released from a cleavable 
conjugate of the signal group and another molecular species by the action 
of the enzyme. As an example, the enzyme .beta.-galactosidase was 
determined by measuring the amount of o-nitrophenol released by the 
enzyme-catalyzed cleavage of o-nitrophenyl-.beta.-D-galactopyranoside. 
It is very desirable to have labeling reagents which do not suffer from 
many or most of the above-described disadvantages of traditional reagents, 
and, most importantly, permit multiple species to be labeled and 
determined in a single sample. Such reagents are the subject of the 
present application. 
SUMMARY OF THE INVENTION 
A new class of labeling reagents has recently been conceived, and is 
undergoing continued development. These reagents, called "release tags," 
are basically three-part molecules which can be illustrated by the generic 
formula Sg-Re-Rx in which Sg represents a "signal" group which may be 
determined readily by an analytical detection device, Rx represents a 
"reactivity" group containing a functional group which reacts with a 
substance to be labeled, thereby attaching the release tag, and Re 
represents a "release" group at which cleavage can occur at an appropriate 
time and under appropriate conditions to release the signal group Sg in a 
form suitable for determination. 
The three-part nature of release tags permits a wide variety of such 
materials to be prepared by varying each of the segments. Particularly 
where the signal groups Sg contain electrophilic atoms and thereby are 
electrophoric (electron-absorbing in the gas phase), large numbers of 
closely-related release tag compounds can be prepared by selecting various 
combinations of the electrophilic atoms and substituent groups for 
incorporation into Sg. The reactivity groups Rx can also be varied widely 
to provide release tags capable of bonding specifically and selectively to 
particular substances to be labeled, or to particular classes of such 
substances, as desired. Finally, the release groups Re can be varied 
widely to provide release tags which can be cleaved under particular 
desired conditions to release signal group-containing molecules for 
analytical detection or determination. 
The totality of these features is thus seen to provide the potential for a 
vast multiplicity of release tags, each of which can ultimately release a 
signal group different from those of other release tags. In principle, 
each of a series of many substances can be separately labeled with a 
different release tag. Subsequently the labeled substances can be brought 
together and employed as a combined, analytical reagent. Since the 
"signal" molecules can be released and determined simultaneously, a large 
number of analytes in the sample can be measured simultaneously. Release 
tags are thus seen to be extremely powerful analytical tools. 
Examples of analytical undertakings in which release tags will be of great 
value are the human genome project, infectious disease testing such as 
AIDS, and genetic screening. The need for multiple labels for such 
purposes has been expressed. See e.g., Landegren, U., Kaiser, R., Caskey, 
C. T. and Hood, L., "DNA Diagnostics-Molecular Techniques and Automation", 
Science 242, 229-237, 1988; Rotman, D., "Sequencing the Entire Human 
Genome", Industrial Chemist, Dec. 18-26, 1987; Giese, R. W., 
"Electrophoric Release Tags: Ultrasensitive Molecular Labels Providing 
Multiplicity", Trends in Anal. Chem. 2, 166-168, 1983. 
The present application relates to two classes of release tag compounds. 
The first of these classes includes release tag compounds which are 
cleaved to release signal group-containing molecules by hydrolysis 
followed by decarboxylation. The second class includes release tag 
compounds which release the signal group by oxidation followed by 
decarboxylation. Included in both classes are release tag compounds which 
may also release the signal group thermally, hydrothermally, or by a 
related mechanism. 
In one of its aspects, the present invention relates to release tag 
compounds for labeling substances for analytical purposes, these compounds 
being represented by the formula 
EQU (SgCO).sub.s L(Rx).sub.r (I). 
In formula (I), each Sg is a signal group, each CO is a carbonyl group to 
which an Sg is bonded, each Rx is a reactivity group, L is a linking group 
to which each SgCO group and each Rx group are bonded, each COL portion is 
a release group which is cleavable to release an Sg-containing compound, s 
is an integer of at least one, and r is an integer of at least one. 
Further, each Sg is a C-linked organic moiety containing from 1 to 20 
carbon atoms, the carbon atom of Sg which is bonded to the carbonyl carbon 
adjacent to linking group L being denominated as the .alpha.-position, and 
comprises a radical selected from the group consisting of substituted 
alkyl, substituted keto-alkyl, substituted alkenyl, and substituted 
alkynyl radicals. When Sg comprises a substituted alkyl, substituted 
keto-alkyl, or substituted alkenyl radical, it bears at least two 
electronegative substituents; and when Sg comprises a substituted alkynyl 
radical, it bears at least one electronegative substituent, these 
electronegative substituents being selected from the group consisting of 
halogens, cyano, dihalomethyl, and trihalomethyl. 
When Sg is keto-alkyl, alkenyl, or alkynyl, it comprises at least one 
moiety selected from the group consisting of .beta.-E-alkynyl, 
.alpha.-E-.alpha.-alkynyl, .beta.-E-.alpha.-keto (provided that the 
carbonyl carbon adjacent to linking group L is connected to a nitrogen or 
oxygen atom of L), .alpha.-E-alkenyl, and .alpha.-E-.alpha.-alkenyl, 
wherein E is an electronegative substituent selected from the group 
consisting of halogens, cyano, dihalomethyl, and trihalomethyl. When Sg is 
.beta.-E-alkynyl, it can bear only one electronegative substituent, this 
being clear from the structure of this group as shown in Table I. Sg 
groups which comprise the other moieties listed here will contain at least 
two electronegative substituents. 
When Sg is alkyl, the .alpha.-carbon atom bears at least two of said 
electronegative substituents but no more than one fluorine atom. 
Further, each Sg has properties such that upon release from the release tag 
compound, it forms an electrophoric compound which is sufficiently 
volatile for determination in the gas phase. 
L comprises one of the following groups: oxy, carbonyloxy, amino, 
hydrazino, aminooxy, carbonylamino, carbonylhydrazino, carbonylaminooxy, 
N-pyrrolidino, N-1,4-diaminopiperazino, O-linked 
tris-(hydroxymethyl)-methylamino; an O-linked monosaccharide residue 
derived from a monosaccharide containing only C, H, and C; an O-linked 
monosaccharide residue derived from a monosaccharide possessing at least 
one amino, hydrazino, or hydrazido group; and a polymer residue derived 
from a polymer possessing a plurality of functionalities selected from the 
group consisting of hydroxyl, carboxyl, primary and secondary amines, 
amides, and hydrazides. 
Each Rx is a C-linked or SO.sub.2 -linked organic moiety comprising 1-20 
carbon atoms and at least one reactive functional group compatible with 
each SgCOL portion of the release tag compound and capable of covalently 
reacting the release tag compound via Rx with a labelable substance. 
When Sg is CHCl.sub.2, CCl.sub.3, or CBr.sub.3 and L is an amino moiety, Rx 
comprises a moiety selected from the group consisting of 
carbonylhydrazino, sulfonyl, phenylene, pyridinyl, pyrimidinyl, and vinyl. 
When L is an amino moiety --NH-- directly linked to an alkyl moiety of the 
Rx group, the amino nitrogen may be directly linked to a maximum of one 
--CH.sub.2 -- unit. When L comprises a polymer residue derived from a 
polyamide, the polyamide is a synthetic polyamide. 
The invention also relates to molecular conjugates in which at least one of 
the above-described release tag compounds of formula (I) is covalently 
bound to at least one labelable substance having a reactive site capable 
of reacting with the reactivity group of the release tag compound, and to 
chemical assays which employ release tag compounds or conjugates of such 
compounds with labelable substances. 
In a second aspect, the invention relates to additional release tag 
compounds for labeling substances for analytical purposes, these compounds 
being represented by the formula 
EQU SgReRx (II). 
In formula (II), Sg is a signal group, Re is a release group to which Sg is 
covalently linked and which is cleavable to result in Sg release, and Rx 
is a reactivity group covalently linked to the Re group. 
The group Sg of formula II is an organic moiety comprising at least one 
electronegative substituent and having properties such that upon release 
from the release tag compound, it forms an electrophoric compound which is 
sufficiently volatile for determination in the gas phase. 
The group Re of formula II comprises a functionality selected from the 
group of cleavable linkages consisting of vicinal diols, .alpha.-hydroxy 
ketones, and olefins. 
Finally, the group Rx of formula II is an organic moiety comprising 1-20 
carbon atoms and at least one reactive functional group compatible with 
the release group Re and capable of covalently reacting the release tag 
compound via Rx with a labelable substance. 
The invention further relates to molecular conjugates in which at least one 
of the above-described release tag compounds of formula (II) is covalently 
bound to at least one labelable substance having a reactive site capable 
of reacting with the reactivity group of the release tag compound, and to 
chemical assays which employ the above-described release tag compounds or 
conjugates of such compounds with labelable substances.

DETAILED DESCRIPTION OF THE INVENTION 
As indicated above, the first class of release tag compounds of the present 
application, those which hydrolyze and decarboxylate to release a volatile 
signal molecule, can be represented by the general formula: 
EQU (SgCO).sub.s L(Rx).sub.r (I) 
wherein s and r are each integers of at least one, but may be more than 
one. 
Within Formula I are four subclasses of release tag compounds: 
a) SgCOLRx corresponding to the case where both s and r are one; 
b) (SgCO)L(Rx).sub.r corresponding to the case where s is one and r is 
greater than one; 
c) (SgCO).sub.s LRx corresponding to the case where s is greater than one 
and r is one; 
d) (SgCO).sub.s L(Rx).sub.r corresponding to the case where both s and r 
are greater than one. 
It is thus seen that the release tag compounds can range from moderately 
simple molecules to quite complex materials bearing several signal, 
release, and reactivity groups. 
The signal groups Sg contain from 1 to 20 carbon atoms and are linked to 
carbonyl groups via a carbon atom of Sg. The carbon atom of Sg which is 
adjacent to the carbonyl group is designated as the .alpha. carbon, that 
adjacent to the .alpha. carbon atom is designated the .beta. carbon, and 
that adjacent to the S carbon atom is designated as a .gamma. carbon atom, 
in accordance with normal usage. 
Each Sg group is a substituted alkyl, substituted keto-alkyl, substituted 
alkenyl, or substituted alkynyl group. Where Sg is substituted alkyl, 
substituted keto-alkyl, or substituted alkenyl, it bears at least two 
electronegative substituents selected from the group consisting of 
halogens, cyano, dihalomethyl, and trihalomethyl. When Sg is substituted 
alkynyl, it bears at least one such electronegative substituent, or at 
least two such electronegative substituents if its structure permits. 
Preferably, each Sg group contains three or more such electronegative 
substituents, providing its structure permits this degree of substitution, 
for good sensitivity as a signal group, this being especially true when Sg 
is alkyl. 
More particularly, when Sg is a keto-alkyl, alkenyl, or alkynyl group, it 
is .beta.-E-alkynyl, .alpha.-E-.alpha.-alkynyl, .beta.-E-.alpha.-keto, 
.alpha.-E-alkenyl or .alpha.-E-.alpha.-alkenyl group. In the above-listed 
groups, E stands for an electronegative substituent selected from the 
group consisting of halogens, cyano, dihalomethyl, and trihalomethyl. 
Where the signal group Sg is a .beta.-E-alkynyl group, it necessarily 
bears only a single E substituent. Further, where signal group Sg is a 
.beta.-E-.alpha.-keto group, the carbonyl carbon atom adjacent to linking 
group L is connected to a nitrogen or oxygen atom of L. 
The chemical structures of the above-identified keto-alkyl, alkenyl, and 
alkynyl groups are shown below in Table I. 
TABLE I 
______________________________________ 
Structural Formulae of Preferred Keto-Alkyl, 
Alkenyl, and Alkynyl Groups for Sg 
Name Structure 
______________________________________ 
.beta.-E-alkynyl 
##STR1## 
.alpha.-E-.alpha.-alkynyl 
##STR2## 
.beta.-E-.alpha.-keto 
##STR3## 
.alpha.-E-alkenyl 
##STR4## 
.alpha.-E-.alpha.-alkenyl 
##STR5## 
______________________________________ 
Footnotes for Table I: 
.sup.1 E is halogen, cyano, dihalomethyl, or trihalomethyl. 
.sup.2 In this case, the carbonyl carbon atom to which the .alpha. carbon 
is attached is in turn connected to an oxygen or nitrogen atom of L. 
The Sg groups in Table I were selected for their ease of forming sensitive, 
electrophoric products SgH from corresponding parent compounds SgCO.sub.2 
H, (SgCO).sub.s L(Rx).sub.r, or substances labeled by the release tag 
compounds (SgCO).sub.s L(Rx).sub.r. 
When signal group Sg of formula I is alkyl, the R-carbon atom bears at 
least two of these electrolegative substituents but no more than one 
fluorine atom. Two electronegative substituents are required to be on the 
R-carbon for alkyl Sg, in order to facilitate the subsequent formation of 
SgH, and to make SgH sensitive as an electrophoric species. The presence 
of two or three fluorine atoms on the R-carbon does not adequately achieve 
these properties. 
A further criterion for signal group Sg of formula I is that upon release 
from the release tag compound, the released fragment containing the Sg 
group ultimately forms an electrophoric compound which is sufficiently 
volatile for determination in the gas phase. This is to facilitate 
detection and quantitation of the ultimately formed electrophoric 
compounds by techniques such as gas chromatography and mass spectrometry. 
The most preferred signal groups Sg comprise an alkyl or keto-alkyl moiety. 
Signal groups comprising an alkenyl moiety constitute a second choice, 
while signal groups comprising alkynyl moieties are somewhat less 
preferred. 
The preferred electronegative substituents for inclusion in signal group Sg 
are the cyano group and the halogens fluorine, chlorine, and bromine. A 
signal- group will typically include at least two electronegative 
substituents, which may be the same or different, and where there are two 
or more electronegative substituents in Sg, two of these may be located on 
a single carbon atom or on different carbon atoms of the signal group. As 
the sensitivity of Sg as a signal group increases up to a point, with the 
number of electronegative substituents incorporated therein, preferred 
signal groups contain at preferred signal groups are those which contain 
Particularly two or three carbon atoms and three to five halogen atoms 
selected from the group consisting of chlorine and bromine. 
Linking group L of formula I comprises one of the following groups: oxy, 
carbonyloxy, amino, hydrazino, aminooxy, carbonylamino, carbonylhydrazino, 
carbonylaminooxy, N-pyrrolidino, N-1,4-diaminopiperazino, an O-linked 
tris(hydroxymethyl)methylamino; an O-linked monosaccharide residue derived 
from a monosaccharide containing only C, H, and O; an O-linked 
monosaccharide residue derived from a monosaccharide possessing at least 
one amino, hydrazino, or hydrazido group; a polymer residue derived from a 
polymer possessing a plurality of functionalities selected from the group 
consisting of hydroxyl, carboxyl, primary and secondary amines, amides, 
and hydrazides. Structures of these linking groups are shown in Table II 
below. 
TABLE II 
__________________________________________________________________________ 
Structural Formulae.sup.1 of Linking Groups L 
Description Structure 
__________________________________________________________________________ 
oxy O 
carbonyloxy 
##STR6## 
amino 
##STR7## 
hydrazino 
##STR8## 
aminooxy 
##STR9## 
carbonylamino 
##STR10## 
carbonylhydrazino 
##STR11## 
carbonylaminooxy 
##STR12## 
N-pyrrolidino 
##STR13## 
N-1,4-diaminopiperazino 
##STR14## 
O-linked tris- (hydroxymethyl)-methylamino 
##STR15## 
O-linked monosaccharide residue derived from 
a monosaccharide containing only C, H, and O 
O-linked monosaccharide residue derived from 
a monosaccharide possessing at least one 
amino, hydrazino, or hydrazido group 
polymer residue derived from a polymer 
possessing a plurality of functionalities 
selected from the group consisting of 
hydroxyl, carboxyl, primary and secondary 
amines, amides, and hydrazides 
__________________________________________________________________________ 
Footnote for Table II: 
.sup.1 The partial structural formulae illustrated in this table are 
employed in formula I in the direction shown. 
Where the group L of formula I is --NH-- linked to an alkyl moiety of the 
Rx group, the amino nitrogen may be directly linked to a maximum of one 
--CH.sub.2 -- group in series with the --NH-- group, as otherwise the 
amide may not be readily cleaved. 
Where the group L of formula I is amino, hydrazino, aminooxy, 
carbonylamino, carbonylhydrazino, or carbonylaminooxy, the nitrogen 
atom(s) may bear hydrogen, an alk-G.sub.1 substituent, or an alk-G.sub.2 
substituent, wherein "alk" is an alkyl moiety of 1 to 4 carbon atoms, and 
G.sub.1 and G.sub.2 are defined as follows. G.sub.1 stands for hydrogen 
(--H); carboxymethyl (--CO.sub.2 CH.sub.3); carbonylamino (--CONH.sub.2); 
acetyl (--COCH.sub.3); acetoxy (--OCOCH.sub.3); methoxy (--OCH.sub.3); 
sulfate (--OSO.sub.3 H); formamido (--NHCOH); acetamido (--NHCOCH.sub.3); 
thiomethyl (--SCH.sub.3); sulfonate (--SO.sub.3 H); sulfoxymethyl 
(--SOCH.sub.3); sulfonyl methyl (--SO.sub.2 CH.sub.3); or sulfonamide 
(--SO.sub.2 NH.sub.2). The substituent G.sub.2 may be hydroxy (--OH); 
carboxy (--CO.sub.2 H); or an imidazole (--C.sub.3 H.sub.3 N.sub.2) group. 
Where linking group L of formula I is N-pyrrolidino or 
N-1,4-diaminopiperizino, the ring may contain up to two substituents 
-R.sup.d where -R.sup.d is hydrogen, alk-G.sub.1, -alk-G.sub.2, -G.sub.1, 
or -G.sub.2, these groups having been defined above. 
These substituents were selected for their ability to facilitate the 
formation of SgH. They were also selected for their ability to increase 
the water solubility of the release tag and its conjugates. 
Where linking group L of formula I is tris-(hydroxymethyl)methylamino, each 
oxygen atom may and generally will bear an SgCO- group. 
Where linking group L of formula I is an O-linked monosaccharide residue 
derived from a monosaccharide containing only C, H, and O, the 
monosaccharide from which L is derived may be any of a wide variety of 
monosaccharides, including various trioses, tetroses, pentoses, and 
hexoses. As is well known, these materials can generally exist in 
closed-ring and open-chain forms, both of which are in principle useful in 
the present invention. Where the monosaccharide exists as a closed ring, 
it is linked to SgCO- groups and to -Rx groups through its hydroxyl 
functionalities. Where the monosaccharide exists in the open-chain form, 
it is linked to SgCO- groups through its hydroxyl moieties, but may be 
linked to the -Rx portion of the release tag compound through its hydroxyl 
moieties, and also by means of derivatives of an aldehyde or ketone 
functionality which may be present in the open-chain form of the molecule. 
Some examples of monosaccharides which are useful in the present invention 
are materials such as erythrose, arabinose, xylose, ribose, lyxose, 
glucose, galactose, mannose, gulose, idose, talose, altrose, allose, 
fructose, sorbose, and tagatose. 
Where linking group L of formula I is an O-linked monosaccharide residue 
derived from a monosaccharide possessing at least one amino, hydrazino, or 
hydrazido group, the starting monosaccharide may again be a triose, 
tetrose, pentose, hexose, which contains one or more nitrogen atoms in the 
form of an amino, hydrazino, or hydrazido group. As before, both 
closed-ring and open-chain nitrogen-containing monosaccharides function in 
the invention. In the release tag compounds of the invention including 
linking groups L derived from nitrogen-containing monosaccharides, the 
hydroxyl groups of the monosaccharide carry SgCO- groups while the amino, 
hydrazino, or hydrazido reactive functionality is employed in the 
connection to the reactivity group -Rx. Examples of a number of 
nitrogen-containing monosaccharides useful in the invention are shown in 
Table III below. 
TABLE III 
__________________________________________________________________________ 
Examples of Monosaccharides containing Amino, 
Hydrazino, or Hydrazido Groups 
Name Structure 
__________________________________________________________________________ 
glucosamine 
##STR16## 
2,3,4,5,6-pentahydroxy- 
HOCH.sub.2 (CHOH).sub.4 CH.sub.2 NH.sub.2 
hexylamine 
2,3,4,5,6-pentahydroxy- 
HOCH.sub.2 (CHOH).sub.4 CH.sub.2 NHNH.sub.2 
hexylhydrazine 
2,3,4,5,6-pentahydroxy- 
HOCH.sub.2 (CHOH).sub.4 CH.sub.2 NHNHCONHNH.sub.2 
hexylcarbohydrazide 
2,3,4,5,6-pentahydroxy- 
HOCH.sub.2 (CHOH).sub.5 CONHNH.sub.2 
caproylhydrazide 
1-amino-[1H]-gluconic acid 
HO.sub.2 C(CHOH).sub.4 CH.sub.2 NH.sub.2 
1-hydrazino-[1H]-gluconic acid 
HO.sub.2 C(CHOH).sub.4 CH.sub.2 NHNH.sub.2 
__________________________________________________________________________ 
Where linking group L of formula I is a polymer residue, it is derived from 
a naturally-occurring (except for polyamides) or synthetic polymer having 
multiple, generally repeating, reactive functionalities such as hydroxyl, 
carboxyl, primary and secondary amino, amido, and hydrazido groups. 
Preferred polymers are water soluble. Examples of biopolymers and 
derivatives thereof which can be employed in the invention are: 
polysaccharides and polysaccharide derivatives such as dextran, dextran 
hydrazide, chitosan, and glycol chitosan; natural polynucleotide 
derivatives such as sonicated calf thymus DNA which has been transaminated 
on its cytosine residues with carbohydrazide, an alkyldihydrazide, or an 
alkyl diamine; and synthetic polycytosine DNA oligomers which have 
similarly been transaminated. 
Some examples of synthetic polymers and derivatives thereof which can be 
employed in the invention are: poly(aspartic acid), poly(aspartic acid) 
hydrazide, poly(glutamic acid) hydrazide, polyserine, polyglycine, 
poly(cytidylic acid), poly(asparagine), poly(glutamine), poly(acrylic 
acid), poly(acrylic acid) hydrazide, and poly(acrylamide) hydrazide. In 
the release tag compounds of the invention, some of the reactive 
functional groups on the polymer carry SgCO groups, while at least one of 
the reactive functional groups is connected to reactivity group Rx. By 
thus having a relatively large number of signal groups in the release tag 
compound, the sensitivity of assays employing the release tag is 
significantly increased. Analytical reagents such as specific binding 
proteins (e.g. antibodies) or specific binding polynucleotides (DNA 
probes) can be labeled with polymeric release tags, thereby to attach a 
large number of releasable signal groups. Tiny amounts of these polymeric 
release tag-labeled analytical reagents can then be detected at 
appropriate points in analytical schemes, providing highly sensitive 
assays. Structures of the above-listed polymers and polymer derivatives 
are shown in Table IV below. 
TABLE IV 
__________________________________________________________________________ 
Examples of Polymers Containing Multiple Hydroxyl, Carboxyl, 
Amido, Amino, or Hydrazido Groups 
Name Structure 
__________________________________________________________________________ 
dextran.sup.1 
##STR17## 
dextran hydrazide.sup.2, 9 
##STR18## 
chitosan 
##STR19## 
a glycol chitosan.sup.3, 9 
##STR20## 
a carbohydrazide derivative of a polynucleotide.sup.4, 9 
##STR21## 
polyaspartic acid 
##STR22## 
polyaspartic acid hydrazide.sup.5, 9 
##STR23## 
polyglutamic acid hydrazide.sup.6, 9 
##STR24## 
polyserine 
##STR25## 
polyglycine 
##STR26## 
a carbohydrazide derivative of polycytidylic acid.sup.4, 
##STR27## 
polyasparagine 
##STR28## 
polyglutamine 
##STR29## 
polyacrylic acid 
##STR30## 
polyacrylic acid hydrazide.sup.7, 9 
##STR31## 
polyacrylamide hydrazide.sup.8, 9 
##STR32## 
__________________________________________________________________________ 
Footnotes for Table IV: 
.sup.1 Although the structure of dextran shows .alpha.-1,6 linkages, ther 
are occasional .alpha.-1,2, .alpha.-1,3, and .alpha.-1,4 linkages 
depending on the species. See Stryer, "Biochemistry", W. H. Freeman & Co. 
N.Y., p. 342 (1988). 
.sup.2 Preparation according to Wilchek and Boyer, Meth. Enz., 138E, 
429-442 (1987), by reacting dextran with chloroacetic acid to form 
randomlylocated OCH.sub.2 CO.sub.2 H groups, followed by reaction of thes 
with hydrazine, or with a dihydrazide in the presence of a watersoluble 
carbodiimide. Dextran hydrazides may also be prepared by reacting dextran 
with hydrazine or a dihydrazide in the presence of borohydride or 
cyanoborohydride. 
.sup.3 The hydroxyethyl groups may be located on various OH 
functionalities of the chitosan. Chitosan is reacted with ethylene oxide 
or 2bromoethanol to form glycolchitosan. 
.sup.4 In the example shown, the cytosine residues of a DNA have been 
reacted with carbohydrazide in the presence of sodium bisulfite according 
to Reines and Schulman, Meth, Enz., LIX, 146-156 (1979), resulting in 
transamination. Alkyldiamines can be used similarly, as can 
alkyldihydrazides. 
.sup.5 Preparation according to Wilchek and Boyer, note .sup.2 above, by 
reaction of poly.beta.-benzyl-L- aspartate with hydrazine. Alternatively, 
polyaspartate can be reacted with hydrazine or a dihydrazide in the 
presence of a water soluble carbodiimide. 
.sup.6 Prepared like polyaspartic acid hydrazide but with polyglutamate a 
the starting material. 
.sup.7 Prepared by reacting polyacrylic acid with hydrazine. 
.sup.8 Commercially available. 
.sup.9 Derivatives of polymers having multiple derivatizable functional 
groups are not necessarily fully derivatized, and generally contain some 
underivatized functional groups. 
Carboxylic acid functionalities of the polymer or derivatized polymer can 
be reacted under appropriate conditions with reagents such as hydrazine, a 
dikydrazide such as carbohydrazide or adipic dihydrazide, or an aminoalkyl 
hydrazide, etc., to yield various hydrazide-containing derivatives. 
Similarly, such carboxylic functionalities can be reacted with various 
alkyl diamines or alkyl triamines to yield amino derivatives. Such 
reactions generally employ a reactive ester of the carboxylic acid as the 
starting material or as an intermediate in the reaction. 
Hydrazide-containing compounds can be further derivatized by reaction with 
succinic anhydride, followed by coupling to carbohydrazide or to some 
other dihydrazide, in the presence of a water soluble carbodiimide, to 
yield another form of hydrazide derivative. 
The most preferred linking groups L are the oxy, carbonyloxy, 
carbonylamino, carbonylhydrazino, O-linked 
tris-(hydroxymethyl)methylamino, O-linked glucosamino, and O-linked 
polyserine. Where the reactive functional group of reactivity group Rx is 
a nucleophile, the most preferred linking groups L are the carbonylamino 
and carbonylhydrazino groups. A second preferred set of linking groups L 
includes the amino, hydrazino, N-pyrrolidino, and N-substituted 
polyaspartate hydrazide groups. 
The reactivity group Rx of Formula I may be represented further by the 
general formula L'QRf, where L' is a linking functionality which connects 
the L and Q groups, Rf is the reactive functional group of reactivity 
group Rx, and Q is a spacer moiety bound to Rf and separating this 
reactive functional group from the rest of the molecule. Thus (SgCO).sub.s 
L(Rx).sub.r is (SgCO).sub.s L(L'QRf).sub.r. 
The linking functionality L' is a chemical bond or a multiatom moiety 
having a molecular weight of less than approximately 400 atomic mass 
units. It is bonded to linking group L via a carbon atom or an SO.sub.2 
group of L' and is compatible with each SgCOL portion and each reactive 
functional group Rf of the release tag compound. 
The linking functionalities comprising the L' group are shown in TABLE V 
below. 
TABLE V 
______________________________________ 
Structural Formulae of Linking Functionalities L' 
Description Structure 
______________________________________ 
chemical bond -- 
carbonyl 
##STR33## 
carbonylamino 
##STR34## 
carbonylhydrazino 
##STR35## 
sulfonyl SO.sub.2 
alkyl of 1-10 carbons 
Alk 
phenylene 
##STR36## 
C-pyrrolidinyl 
##STR37## 
C-piperidinyl 
##STR38## 
C-piperazinyl 
##STR39## 
pyridinyl 
##STR40## 
2-oxo-pyrimidinyl 
##STR41## 
vinyl 
##STR42## 
______________________________________ 
The partial formulae shown in this table are employed in Formula I in the 
direction shown. 
Where L' is alkyl, phenylene, C-pyrrolidinyl, C-piperidinyl, C-piperazinyl, 
pyridinyl, or oxo-pyrimidinyl, it may be substituted with up to two 
substituent groups R.sup.d as defined above with respect to linking groups 
L. Where L' is vinyl, it may contain two substituent groups R.sup.c as 
defined above. The groups designated as Y residing on nitrogen atoms in 
the pyrrolidinyl, piperidinyl, or piperazinyl structures may be hydrogen, 
an alkyl group of one to three carbons, or --COCH.sub.3. 
Linking functionality L' is joined to spacer group Q by a chemical bond or, 
in the case where L' is alkyl, phenylene, pyrrolidinyl, piperidinyl, 
piperazinyl, pyridinyl, oxo-pyrimidinyl, or vinyl, L' may also be linked 
to Q by a further linker such as an oxy, amino, hydrazino, aminocarbonyl, 
hydrazinocarbonyl, carbonylamino, carborylhydrazino, or carbonyl group. 
Where such linker contains a nitrogen atom, this may in turn bear a 
further substituent of hydrogen, or alkyl or acyl groups of one to three 
carbon atoms. Where the linkage between groups L' and Q involves a 
nitrogen or oxygen atom being bonded to group L', this may not be 
connected to a carbon atom of L' which already bears another hetero atom. 
Linking functionality L' is preferably a chemical bond, a phenylene group, 
or an alkyl group of one to ten carbons. 
The spacer moiety Q contains one to 15 carbon atoms and is linked to 
linking group L via a carbon atom of Q either directly when L' is a 
chemical bond, or indirectly through L' when L' is a multiatom moiety. 
Furthermore, Q is a function of the linkage between L and Q and the 
linkage between Q and reactive functional group Rf such that widen each of 
these linkages involves a hetero atom bonded to Q, such hetero atoms are 
separated from each other by at least two carbon atoms of Q. Preferably, 
when the linkage between L and Q as well as the linkage between Q and the 
reactive functional group Rf each involves a hetero atom bonded to Q, Q 
includes a 2-carbon aliphatic chain or a phenylene group. 
The reactive functional group Rf of reactivity group Rx is an acylating, 
alkylating, electrophilic, or nucleophilic functionality. 
When linking functionality L' is a multiatom moiety, L' includes one of the 
following groups: carbonyl, carbonylamino, carbonylhydrazino, sulfonyl, an 
alkyl group of one to ten carbon atoms, phenylene, C-pyrrolidinyl, 
C-piperidinyl, C-piperazinyl, pyridinyl, pyrimidinyl, and vinyl. 
When linking group L is an oxy, carbonyloxy, aminooxy, or carbonylaminooxy 
moiety, L' is a chemical bend or a multiatom moiety including one of the 
following groups: alkyl of one to ten carbon atoms, phenylene, 
C-pyrrolidinyl, C-piperidinyl, C-piperazinyl, pyridinyl, 2-oxo-pyrimidinyl 
and vinyl. 
When linking group L is oxy or carbonyloxy, reactive functional group Rf is 
an acylating, alkylating, or electrophilic functionality. 
When linking group L is O-linked tris-(hydroxymethyl)methylamino, an 
O-linked monosaccharide residue, or an O-linked polymer residue, L' is a 
chemical bond or a multiatom moiety including one of the following groups: 
carbonyl, carbonylamino, carbonylhydrazino, sulfonyl, alkyl of one to 10 
carbon atoms, phenylene, C-pyrrolidinyl, C-piperidinyl, C-piperazinyl, 
pyridinyl, pyrimidinyl, and vinyl; and Rf is an acylating, alkylating, or 
electrophilic functionality. 
When linking group L is amino, hydrazino, carbonylamino, carbonylhydrazino, 
N-pyrrolidino, or N-(1,4-diaminopiperazino), L' is a chemical bond or a 
multiatom moiety including one of the following groups: carbonyl, 
carbonylamino, carbonylhydrazino, sulfonyl, alkyl of one to ten carbons, 
phenylene, C-pyrrolidinyl, C-piperidinyl, C-piperazinyl, pyridinyl, 
pyrimidinyl, and vinyl; and Rf is an acylating, alkylating, electrophilic, 
or nucleophilic functionality. 
Where L is --NH-- it may be directly linked in turn to a maximum of one 
--CH.sub.2 -- group, alkyl chains (CH.sub.2).sub.n where n is &gt;1 being 
disfavored since amides having the structure --CONH(CH.sub.2).sub.n -- may 
not be readily cleaved. 
Where the reactive functional group Rf is an acylating functionality, it 
may be a carbodiimide-activated carboxyl group, an 
.alpha.-hydroxysuccinimide ester, a 1-hydroxybenzotriazole ester, a 
nitrophenyl ester, an acyl azide, an acyl halide such as the chloride, an 
acyl imidazole, an acyl pyridine such as that resulting from use of 
dimethylaminopyridine, an anhydride, an alkoxyanhydride, a thioester, an 
imidoester, a thioimidoester, phenyl isothiocyanate, an 
oxycarbonylimidazole, or an N-carboxyanhydride. General structures of 
these reactive functional groups are shown in Table VI. 
TABLE VI 
______________________________________ 
Structural Formulae for Acylating Rf Groups 
Description Structure 
______________________________________ 
carbodiimide-activated carboxyl 
##STR43## 
N-hydroxysuccinimide ester 
##STR44## 
1-hydroxybenzotriazole ester 
##STR45## 
nitrophenyl ester 
##STR46## 
acyl azide 
##STR47## 
acyl halide 
##STR48## 
acyl imidazole 
##STR49## 
acyl pyridine 
##STR50## 
anhydride 
##STR51## 
alkoxyanhydride.sup.2 
##STR52## 
thioester.sup.2 
##STR53## 
imidoester.sup.2 
##STR54## 
thioimidoester.sup.2 
##STR55## 
phenylisothiocyanate 
##STR56## 
oxycarbonylimidazole 
##STR57## 
N-carboxyanhydride 
##STR58## 
______________________________________ 
Footnotes for Table VI: 
.sup.1 Exemplary carbodiimides are dicyclohexylcarbodiimide, 
1ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 
1cyclohexyl-3-(2-morpholinyl)-4-ethyl carbodiimidemetho-p-toluene 
sulfonate. Generally, the release tag compound is prepared with one or 
more carboxyl groups as initial reactive functional groups, and these 
groups are caused to react with the carbodiimide to form the 
carbodiimideactivated carboxyl illustrated, which in turn reacts with the 
substance to be labeled. 
.sup.2 R is alkyl 
.sup.3 It will be recognized that many of the groups listed in Table VII 
may bear one or more substituents. 
Where the reactive functional group Rf is an alkylating functionality, it 
may be an .alpha.-haloketo, a primary alkyl bromide or iodide, an epoxide, 
an alkoxypyridinium salt, an imine, a sulfonyloxyalkyl group, or a vinyl 
sulfone. Structures of these groups are shown below in Table VII. 
TABLE VII 
______________________________________ 
Structural Formulae for Alkylating Rf Groups 
Description Structure 
______________________________________ 
.alpha.-haloketo.sup.1 
##STR59## 
primary alkyl bromide or iodide 
##STR60## 
epoxide 
##STR61## 
alkoxypyridinium salt.sup.2 
##STR62## 
imine 
##STR63## 
sulfonyloxyalkyl CH.sub.2OSO.sub.2CH.sub.2 CF.sub.3 
(tresyloxyalkyl illustrated) 
vinyl sulfone SO.sub.2 CHCH.sub.2 
______________________________________ 
Footnotes for Table VII: 
.sup.1 X is halogen 
.sup.2 R is alkyl 
Where the reactive functional group Rf is an electrophilic functionality, 
it may be a nitrophenyl nitrene, an aldehyde, a maleimide, a disulfide, an 
.alpha.-diketone, a .beta.-diketone, or a sulfonyl halide. Structures of 
these groups are shown in Table VIII below. 
TABLE VIII 
______________________________________ 
Structural Formulae for Electrophilic Rf Groups 
Description Structure 
______________________________________ 
nitrophenyl nitrene precursor.sup.1 
##STR64## 
aldehyde 
##STR65## 
maleimide 
##STR66## 
disulfide 
##STR67## 
.alpha.-diketone.sup.2 
##STR68## 
.beta.-diketone.sup.2 
##STR69## 
sulfonyl chloride 
SO.sub.2 Cl 
______________________________________ 
Footnotes for Table VIII: 
.sup.1 The nitrene (..N:) is generated by loss of N.sub.2 from the azide. 
.sup.2 R is H or alkyl 
Where the reactive functional group Rf is a nucleophilic functionality, it 
may be a hydrazine, hydrazide, thiol, 1.degree. or 2.degree. amine, or 
oxyamine. Structures of these groups are shown in Table IX below. 
TABLE IX 
______________________________________ 
Structural Formulae for Nucleophilic Rf Groups 
Description Structure 
______________________________________ 
hydrazine NHNH.sub.2 
hydrazide 
##STR70## 
thiol SH 
amine NH.sub.2 or NHR 
oxyamine ONH.sub.2 
______________________________________ 
The most preferred reactive functional groups Rf are the 
carbodiimide-activated carboxyls, N-hydroxysuccinimide esters, 
1-hydroxybenzotriazole esters, acyl azides, and phenylisothiocyanates. A 
second set of preferred reactive functional groups are the nitrophenyl 
nitrenes, while a third set of reactive functional groups includes amines 
and hydrazides. 
Preferred release tag compounds of Formula I are those which incorporate 
the several preferred Sg, L, L', and Rf groups as discussed above. The 
subsets of release tag compounds derivable by permutations of the 
preferred subsets of the Sg, L, L', and Rf groups are all preferred 
materials. Thus, preferred release tag compounds are those constructed 
employing the Sg, L, L', and Rf groups listed in Table X below, in which 
the numbers 1, 2, and 3 refer to first, second, and third choice subsets 
of the respective groups, and 1' refers to a most preferred but narrow 
subset of linking group L to be employed when reactive functional group Rf 
is a nucleophile. 
TABLE X 
______________________________________ 
Selected Combinations of Preferred Sg, L, 
L' and Rf Groups 
Sg L L' Rf 
______________________________________ 
1 1 1 1 1 
2 1 1 1 2 
3 1 1' 1 3 
4 1 2 1 1 
5 1 2 1 2 
6 1 2 1 3 
7 2 1 1 1 
8 2 1 1 2 
9 2 1' 1 3 
10 2 2 1 1 
11 2 2 1 2 
12 2 2 1 3 
13 3 1 1 1 
14 3 1 1 2 
15 3 1' 1 3 
16 3 2 1 1 
17 3 2 1 2 
18 3 2 1 3 
______________________________________ 
Turning now to the set of release tag compounds having the general formula 
EQU SgReRx (II), 
signal group Sg is a C-linked organic moiety containing from one to twenty 
carbon atoms, the carbon atom of Sg which is bonded to the release group 
Re being denominated as the .alpha.-position since cleavage at Re 
generates initially an SgCO-moiety. Sg includes a substituted alkyl, 
substituted keto-alkyl, substituted alkenyl, or substituted alkynyl group, 
these groups bearing at least one halogen, cyano, dihalomethyl, or 
trihalomethyl electronegative substituent, though where the structure of 
Sg permits, higher numbers of electronegative substituents are preferred, 
as indicated below. 
Further, when signal group Sg is keto-alkyl, alkenyl, or alkynyl, it is a 
.beta.-E-alkynyl, .alpha.-E-.alpha.-alkynyl, .beta.-E-.alpha.-keto, 
.alpha.-E-alkenyl, or .alpha.-E-.alpha.-alkenyl group, where E is a 
halogen, cyano, dihalomethyl, or trihalomethyl group. 
Furthermore, when signal group Sg is alkyl, the .alpha.-carbon atom bears 
at least two of these E moieties but no more than one fluorine atom. 
The electronegative substituents of signal group Sg are preferably selected 
from the group consisting of cyano and halogens. 
The electronegative substituents of signal group Sg may be different, may 
be halogens, and may be located on different carbon atoms of the signal 
group. 
Signal group Sg of formula II most preferably bears at least two 
electronegative substituents, and most preferably bears at least three 
electronegative substituents, which are preferably halogens of at least 
two different varieties. Two of these electronegative substituents are 
preferably located on different carbon atoms, although two of these 
electronegative substituents may be located on a single carbon atom of the 
Sg group. Particularly preferred signal groups are those which contain two 
or three carbon atoms and three to five halogen atoms selected from the 
group consisting of chlorine and bromine. 
As indicated above, the release group Re of the release tag compounds 
represented by Formula II is a vicinal diol, an .alpha.-hydroxy ketone, or 
an olefin. Representative structures of these release groups are presented 
in Table XI below. 
TABLE XI 
______________________________________ 
Release Groups of Release Tag Compounds of Formula II 
Description Structure 
______________________________________ 
vicinal diol 
##STR71## 
.alpha.-hydroxy ketone 
##STR72## 
##STR73## 
olefin 
##STR74## 
______________________________________ 
The reactivity group Rx of the release tags of Formula II is represented by 
the formula QRf, wherein Rf is a reactive functional group which is 
compatible with the release group portion of the release tag compound and 
also capable of forming a covalent bond with a labelable substance, and Q 
is a chemical bond or a C-linked spacer moiety bound to the Rf group and 
including from one to fifteen carbon atoms. Further, Q is a function of 
the Rf group and the linkage between the release group Re and Q such that 
when the release group is an oxirane or an .alpha.-hydroxy ketone and is 
linked via its .alpha.-carbon atom to the Q group, and the reactive 
functional group Rf is linked to Q via a hereto atom, Q comprises at least 
one carbon atom. 
The reactive functional group Rf of Formula II is an acylating, alkylating, 
electrophilic, or nucleophilic functionality, except that where release 
group Re is a vicinal diol or an .alpha.-hydroxy ketone, reactivity group 
Rf may not be a sulfonyl halide. 
Where reactive functional group Rf of release tag Formula II is an 
acylating functionality, it is a carbodiimide-activated carboxyl, an 
.alpha.-hydroxysuccinimide ester, a 1-hydroxybenzotriazole ester, a 
nitrophenyl ester, an acyl imidazole, an acyl pyridine, a thioester, or an 
imidoester. Structures of these reactive functional groups are shown in 
Table VI. 
Where the reactive functional group Rf of release tag Formula II is an 
alkylating functionality, it is an .alpha.-haloketo group, a primary alkyl 
bromide or iodide, an epoxide, an alkoxypyridinium salt, or an imine. 
Structures of these reactive functional groups are shown in Table VII. 
Where the reactive functional group Rf of release tag Formula II is an 
electrophilic functionality, it is a nitrophenyl nitrene precursor, an 
aldehyde, a maleimide, a disulfide, an .alpha.-diketone, a 
.beta.-diketone, or a sulfonyl halide. Structures of these reactive 
functional groups are shown in Table VIII. 
Where the reactive functional group Rf of release tag Formula II is a 
nucleophilic functionality, it is a hydrazine, a hydrazide, a thiol, an 
amine, or an oxyamine. Structures of these reactive functional groups are 
shown above in Table IX. 
In the release tag compounds of formula II, the most preferred signal 
groups Sg are substituted alkyl or keto-alkyl groups, a second choice 
being substituted alkenyl groups. The most preferred release groups Re are 
.alpha.-hydroxyketones, with vicinal diols being a second choice. The most 
preferred reactive functional groups Rf are the carbodiimide-activated 
carboxyls and .alpha.-hydroxysuccinimide esters, with the second choice 
groups being nitrophenyl nitrene precursors, hydrazides, and amines. 
Preferred release tag compounds of Formula II are those which include 
various permutations of the first and second choices of the Sg, Re, and Rf 
groups discussed above. Accordingly, preferred release tag compounds of 
Formula II are indicated in Table XII below, where the number 1 represents 
the most preferred options for the particular portion of the release tag 
compounds, and number 2 represents the second choices for these 
functionalities. 
TABLE XII 
______________________________________ 
Preferred Release Tag Compounds of Formula II 
Sg Re Rf 
______________________________________ 
4 1 1 1 
5 1 1 2 
6 1 2 1 
7 1 2 2 
8 2 1 1 
9 2 1 2 
10 2 2 1 
11 2 2 2 
______________________________________ 
Other preferred release tag compounds of Formula II are those in which the 
release group Re includes a vicinal diol or an .alpha.-hydroxy ketone, and 
the reactive functional group Rf includes a carbodiimide-activated 
carboxyl group or an N-hydroxysuccinimide ester acylating functionality. 
The release tag compounds of the invention are useful for labeling any 
substance, provided that the substance to be labeled possesses at least 
one functional group capable of reacting with the reactive functional 
group Rf of reactivity group Rx of the release tag compound to be 
employed. To put it another way, the release tag compounds of the 
invention make it possible to label a vast number of substances which 
either possess or can be modified to possess a reactive functional group, 
by providing one or more release tags with reactive functional groups 
capable of reaction with the reactive functional groups of the substance 
to be labeled, and causing these materials to react to form a covalent 
linkage. 
Among the many sorts of substances which aloe capable in principle of being 
labeled by the release tag compounds of the invention are materials one 
wishes to analyze for, generally referred to as analytes, and analogs of 
such analytes; materials which constitute primary or secondary binding 
partners for such analytes or analyte analogs; and various substrates for 
enzymes which are used as labels on analyte analogs and on primary and 
secondary binding partners for various analytes. 
Representative examples of analytes which may be labeled by the release tag 
compounds of the invention are materials such as 
a) proteins: for example, protein hormones such as insulin, thyroid 
stimulating hormone (TSH), growth hormone (GH), follicle stimulating 
hormone (FSH), and luteinizing hormone (LH); enzymes such as creatine 
kinase and lactate dehydrogenase (LDH); tumor antigens such as 
carcinoembryonic antigen (CEA); antibodies such as anti human 
immunodeficiency virus (A'HIV), A'hepatitis, IgE, and IgG.sub.1 ; 
receptors such as progesterone receptor and estrogen receptor; and 
transport proteins such as .alpha.-lipoprotein and transferrin; 
b) peptides: for example, hormones such as angiotensin II, glucagon, and 
adrenocorticotrophic hormone (ACTH); 
c) amino acids such as triiodothyronine (T.sub.3), tetraiodothyronine or 
thyroxin (T.sub.4) and .gamma.-aminobutyric acid; 
d) polynucleotides: for example, gene fragments and genes such as the AIDS 
gene and the sickle Hb gene; and RNA such as mRNA, tRNA, rRNA; 
e) nucleotides such as adenosine monophosghate (AMP); 
f) nucleosides such as N.sup.2 -(dG-8-yl)-2-aminofluorene; 
g) nucleobases such as 5-methylcytosine; 
h) lipids: for example, steroids such as cortisol, estradiol, and 
aldosterone; and prostaglandins such as PGE.sub.2 ; 
i) carbohydrates such as blood group antigens; 
j) drugs such as digoxin and theophylline; 
k) cells: for example, lymphocytes such as B lymphocytes and T lymphocytes; 
l) viruses such as the hepatitis, HIV-I, and HIV-II; 
m) vitamins such as Vitamin A, Vitamin D, Vitamin E, Vitamin B.sub.12, and 
folic acid; 
n) coenzymes such as NAD; 
o) bioactive amines such as epinephrine end dopamine; 
p) aflatoxins such as aflatoxin B.sub.1 and aflatoxin G.sub.1 ; 
q) polyaromatic hydrocarbons such as benzo[a]pyrene, and 
7,12-dimethylbenz[a]anthracene; 
r) pesticides such as dieldrin and aldrin. 
A primary binding partner for an analyte is a substance that forms a 
specific noncovalent complex with the analyte. For many types of analytes, 
corresponding antibodies may be obtained as primary bonding partners. Such 
analytes are classified into two broad classes based on their 
sizes--antigens (which are large) and haptens (which are small). Sometimes 
an antibody is the analyte of interest, in which case the corresponding 
antigen or hapten is used as the specific binding partner. 
Other classes of primary binding partners also exist. A certain nucleic 
acid (DNA or RNA) or fragment thereof may be an analyte, in which case the 
complementary nucleic acid (DNA or RNA), generally termed a "DNA probe" 
when it comprises DNA, is the primary binding partner. An enzyme can be a 
binding partner for an inhibitor as an analyte, or vice versa. Similarly, 
lectins bind sugars, avidin and its analogs (e.g., streptavidin and 
succinylavidin) bind biotin, and receptors bind messenger substances such 
as hormones and neurotransmitters. As before, either one of the substances 
in each of these pairs is a primary binding partner for the other. 
A secondary binding partner is a substance that binds to a primary binding 
partner even after the primary binding partner has become bound to its 
analyte. For example, if antibody Ab.sub.1 binds analyte An, forming a 
complex Ab.sub.1 .cndot.An, and a second antibody Ab.sub.2 is available 
which binds in turn to the prior complex onto the Ab.sub.1 part, forming 
Ab.sub.2 .cndot.Ab.sub.1 .cndot.An, then Ab.sub.2 is a secondary binding 
partner for the analyte. The binding of Ab.sub.2 onto Ab.sub.1 is thus 
"piggyback" in nature. The site on Ab.sub.1 that is recognized by Ab.sub.2 
may either be an inherent part of Ab.sub.1, or a hapten or antigen 
recognized by Ab.sub.2 that has been conjugated to Ab.sub.1. 
Since protein A and protein G bind to antibodies at regions remote from the 
antibody binding site, they are often used as secondary binding partners 
in immunoassays. 
Biotin commonly is attached to an antibody for an analyte so that avidin 
(or an avidin analog), which specifically binds to biotin, can function as 
a secondary binding partner relative to the analyte against which the 
antibody was developed. If a conjugate of an antibody Ab.sub.1 and avidin 
(i.e., Av-Ab.sub.1) binds to an analyte An forming the complex Av-Ab.sub.1 
.cndot.An and this complex in turn can bind to biotin forming 
biotin.cndot.Av-Ab.sub.1 .cndot.An, then biotin is a secondary binding 
partner for An. Similarly, biotin conjugated to a substance X (i.e., 
X-biotin) is a secondary binding partner for An if 
X-biotin.cndot.Av-Ab.sub.1 .cndot.An can form. 
A hapten can function as a secondary binding partner. For example, a hybrid 
antibody can be prepared which binds the analyte An in one binding site 
and a hapten H to another. Thus, hapten H is then a secondary binding 
partner for An. If hapten H is first conjugated to some other substance X 
forming H-X, then H-X is a secondary binding partner for An if the complex 
An.cndot.Ab.cndot.H-X forms. 
Similarly, if a nucleic acid analyte NA.sub.A is recognized by (hybridizes 
to) nucleic acid NA.sub.1, forming the complex NA.sub.1 .cndot.NA.sub.A, 
and nucleic acid NA.sub.2 can further bind to this complex by binding to 
an unused part of NA.sub.1, forming NA.sub.2 .cndot.NA.sub.1 
.cndot.NA.sub.A, then NA.sub.2 is a secondary binding partner relative to 
NA.sub.A. 
Proteins such as antibodies, avidin, streptavidin, lectins, protein A, and 
protein G are commonly used as primary or secondary specific binding 
proteins. Related forms of these and other proteins are also used, e.g. 
the Fab, and F(ab').sub.2 parts of antibodies. Succinylavidin is another 
example. 
Polymer-modified proteins may also be used as primary or secondary binding 
partners. Examples of the polymers employed in producing such 
polymer-modified proteins are other proteins, polypeptides, 
polysaccharides, polynucleotides, and synthetic polymers such as 
polyacrylic acid or polyacrcylylhydrazide. In use, the polymer on the 
polymer-modified protein carries many copies of a given release tag or of 
releasable SgCO groups, thus allowing the polymer-modified protein to be 
detected with high sensitivity. Similarly, polymer-modified 
polynucleotides can be prepared for detection with very high sensitivity. 
Examples of primary and secondary binding partners which are conveniently 
labeled by the release tag compounds of the invention are 
a) proteins: for example, antibodies, avidin, streptavidin, lectins, 
protein A, and protein G; 
b) polymer-modified proteins: for example, antibody-poly asp hydrazide, 
antibody-dextran, antibody-polyethyleneimine, antibody-dextran, 
avidin-dextran, and avidin-polyglu-hydrazide; 
c) peptides: for example, angiotensin II; 
d) polynucleotides: for example, complementary DNA and RNA; 
e) polymer-modified polynucleotides: for example, 3'-tailed DNA and RNA, 
DNA-polyglu hydrazide, and DNA-dextran hydrazide; 
f) carbohydrates: for example, glucose; 
g) haptens: for example, digoxin, digoxigenin, and fluorescein; and 
h) biotin. 
Many of the above-listed materials can function either as primary or 
secondary binding partners, depending on the assay being conducted. 
Examples of enzyme substrates which may be labeled by the release tag 
compounds of the invention are: carbohydrates such as chitin and 
glycolchitin, dextran, glucose-6-phosphate, and galactose glycosides; 
lipids such as cholesterol esters; nucleotides such as ATP and AMP; 
polynucleotides such as DNA and RNA; peptides such as dipeptides and 
dipeptide esters; proteins such as albumin; and esters suck as 
p-nitrophenyl esters, phosphate esters, and carboxylic acid esters. 
As explained above, the release tag compounds of the invention are capable 
of forming conjugates with a wide variety of other substances. Such 
conjugates are fully covalent materials in which at least one release tag 
compound is covalently linked to at least one other molecular moiety. 
Conjugates may thus be symbolized as (substance).sub.u (tag).sub.t where 
the tag is a residue of a release tag which is covalently bound to the 
substance. The subscripts u and t indicate that depending on the 
particular release tags and substances chosen for the conjugate, 
conjugates may contain one release tag and one other substance to be 
labeled, one release tag and multiple other substances, one substance 
labeled by multiple release tags, and multiple substances labeled with 
multiple release tags. Where the release tag employed originally contained 
multiple reactive functional groups Rf, in the resulting conjugate not all 
of these are necessarily reacted with substance to be labeled. The 
substance being labeled may also possess multiple reactive functional 
groups initially, not all of which are necessarily reacted with release 
tag compounds in forming the conjugate. 
The release tag compounds employed in forming conjugates of the invention 
must each have at least one signal group-containing unit SgCO-, and in 
release tag compounds of formula I will frequently have multiple such 
units. In release tag compounds bearing multiple signal groups and 
multiple reactive functional groups, the remaining portion of the molecule 
linking these together is generally relatively large, and may or may not 
be precisely definable. 
In conjugates containing multiple release tag residues, these conjugates 
can be derived from the same or different release tag molecules. 
Similarly, where the conjugate contains multiple subunits, as in a protein 
possessing quaternary structure, these may also be the same or different. 
The release tag compounds of the invention are synthesized using principles 
and reactions which are well known to those skilled in the art. They are 
prepared basically in three stages. In the first stage (stage one), 
molecular species containing the signal group Sg, the release group Re, 
and the reactivity group Rx in final or precursor form (i.e., carrying a 
protecting group) are obtained either commercially or via synthesis. The 
second stage (stage two) involves carrying out appropriate chemical 
reactions to join these materials into the release tag compound SgReRx 
which may, however, still contain certain functionalities in precursor or 
protected form. In the third stage (stage three), any such functionalities 
are converted to the desired final form. 
Obtaining the signal groups Sg in stage one of the synthetic process 
generally involves preparation of electrophoric carboxylic acids such as 
trichloroacetic acid, which correspond to Sg--CO.sub.2 H. Many such 
carboxylic acids are known, and others may be prepared conveniently by 
reactions such as halogenation of precursor unsaturated carboxylic acids, 
and quenching with carbon dioxide of Grignard reagents prepared from 
halogenated hydrocarbons. When mixtures of halogenated carboxylic acids of 
varying halogen content or substitution pattern are generated in synthetic 
procedures, these can usually be fractionated chromatographically, to 
yield multiple signal group precursors from a given reaction. 
For synthesis of the release tag compounds of formula I, many of the 
release groups are obtained in stage one in a precursor form. It is their 
coupling to the signal group-containing moiety Sg--CO.sub.2 H which yields 
the final form of the release group. The release group precursors are 
typically such materials as simple amino acids, hydroxy acids, diamines, 
and similar difunctional molecules, many of which are commercially 
available. The second functional group in these molecules is required for 
the attachment of the reactivity group Rx. 
The reactivity groups Rx or their precursors for stage three are generally 
commercially available because of the widespread usefulness of such 
reactivity groups in bio-organic chemistry. 
For synthesis of the release tag compounds of formula II, where Re involves 
a diol, R-hydroxy ketone, or olefin, this functionality is established 
either before or after incorporation of the release group precursor into a 
release tag, by reactions such as oxidation, hydrolysis, elimination, the 
Wittig reaction, or combinations of these. More particularly, for the 
synthesis of the release tag compounds of formula II, one can make use of 
many of the reagents and reactions which allowed the first class to be 
prepared. For the formation of olefin and glycol release tags, the general 
strategy is to start with SgCOCl, and form the corresponding aldehyde by a 
reduction reaction, several of which have been described (March, J., 
Advanced Organic Chemistry, J. Wiley, New York, 3rd Edn., 1985, p. 396). 
The aldehyde can be converted into an olefin by a Wittig reaction ((a) 
House, H. O., Jones, V. K., Frank, G. A. J. Org. Chem. 1964 29, 3327; (b) 
House, H. O., Rasmusson, G. H. Ibid, 1961, 26, 4278; (c) Maercker, A., 
Org. React., New York, 1965, 14, 270; (d) House, H. O., Modern Synthetic 
Reactions, 2nd Edn., W. A. Benjamin, Inc., Menlo Park, Calif., 1972, pp. 
682-709.) or a Horner-Emmon's reaction (Reviews: (a) Boutagy, J. and 
Thomas, R., Chem. Rev. 1974, 74, 87; (b) Wadsworth, W. S., Org, React., 
New York, 1977, 25, 73.) This establishes an olefin release group. In 
turn, a glycol release group can be formed by oxidizing the olefin with 
alkaline potassium permanganate, or osmium tetroxide in pyridine, or a 
peracid as described (House, H. O., Ibid., pp. 275, 298). A reactivity 
group (Rx) is then incorporated as in the preparation of the first class 
of release tags, taking advantage of an appropriate functional group 
introduced in the Wittig reaction. 
For hydroxyketo release tags, SgCOCl is reacted with an organocadmium 
compound, to form a corresponding ketone as has been reviewed (Cason, 
Chem. Revs., 40, 1947, 15). The ketone in turn is brominated as described 
(House, Ibid., 529) and hydrolyzed as described (Wagner, R. B. and Zook, 
H. D., Synthetic Organic Chemistry, John Wiley and Sons, New York, 1953, 
p. 170) to form the corresponding .alpha.-hydroxyketone. As desired, the 
.alpha.-hydroxy and keto groups can be reversed under acidic conditions 
(the .alpha.-ketol rearrangement) as has been described (March, Ibid., p. 
967). A reactivity group Rx is then incorporated as in the preparation of 
the first class of release tags. 
Related procedures for synthesizing olefinic, glycol, and 
.alpha.-hydroxyketone release tags can be developed from standard 
reactions in organic chemistry by one skilled in the art. 
The general literature on peptide synthesis is quite relevant to the 
preparation of release tag compounds. Release tags commonly utilize amide 
linkages, the formation of which is the heart of peptide synthesis. 
Protecting groups are also important in peptide synthesis and the same 
ones can be used as necessary for most if not all of the protections 
needed in release tag synthesis. 
In Table XIII below are listed a number of representative electrophoric 
carboxylic acids Sg--CO.sub.2 H, as well as representative chemical 
reactions by which they may be formed from commercially available starting 
materials. Such materials serve as precursors of the Sg-containing portion 
of the release tags, the halogenated portions of the molecules being the 
ultimate signal groups Sg. Those skilled in the art will recognize that 
other starting materials can be subjected to the illustrated reaction 
conditions, and the illustrated starting materials can be subjected to 
reaction conditions other than those particularly shown, to yield yet 
additional electrophoric carboxylic acid products. 
TABLE XIII 
__________________________________________________________________________ 
Preparation of SgCO.sub.2 H 
No. 
Starting Material 
Reactants 
Product(SgCO.sub.2 H) 
__________________________________________________________________________ 
1 CHCl.sub.2COOH.sup.(1a) 
Br.sub.2 /Red P 
CBrCl.sub.2COOH 
2 CH.sub.2 ICOOH.sup.(1a) 
Cl.sub.2 /Red P 
CCl.sub.2 ICOOH 
3 CH.sub.2 ICOOH.sup.(1a) 
Br.sub.2 /Red P 
CBr.sub.2 ICOOH 
4 CFH.sub.2COOH.sup.(1a) 
Br.sub.2 /Red P 
CBr.sub.2 FCOOH 
5 CFH.sub.2COOH.sup.(1a) 
Cl.sub.2 /Red P 
CCl.sub.2 FCOOH 
6 CH.sub.2 ClCOOH.sup.(1a) 
Br.sub.2 /Red P 
CBr.sub.2 ClCOOH 
7 CH.sub.3 COCO.sub.2 H.sup.(1a) 
PBr.sub.5 
CH.sub.3 CBr.sub.2 CO.sub.2 H 
8 CH.sub.3 COCO.sub.2 H.sup.(1c) 
PCl.sub.5 
CH.sub.3 CCl.sub.2 CO.sub.2 H 
9 CCl.sub.2CClCOOH.sup.(1d) 
Cl.sub.2 /CCl.sub.4 
CCl.sub.3CCl.sub.2COOH 
10 CCl.sub.3COCOOH.sup.(1g) 
1) NaBH.sub.4 
CCl.sub.3CBrClCOOH 
2) PCl.sub.5 
3) Br.sub.2 /Red P 
11 CCl.sub.2CClCOOH.sup.(1d) 
BrCl CCl.sub.3CClBrCOOH 
12 CCl.sub.2CClCOOH.sup.(1d) 
Br.sub.2 /CCl.sub.4 
CBrCl.sub.2CBrClCOOH 
13 CCl.sub.3COCOOH.sup.(1d) 
PBr.sub.5 
CCl.sub.3CBr.sub.2COOH 
14 CH.sub.2 ClCOCOOH.sup.(1c,h) 
1) Br.sub.2 Na.sub.2 CO.sub.3 
CClBr.sub.2CCl.sub.2COOH 
2) PCl.sub.5 
15 CH.sub.3COCOOH.sup.(1a) 
1) Br.sub.2 /Na.sub.2 CO.sub.3 
CBr.sub.3CCl.sub.2COOH 
2) PCl.sub.5 
16 HC CCOOH.sup.(1l) 
1) Br.sub.2 /CCl.sub.4 
CClBr.sub.2CClBrCOOH 
2) alc KOH 
3) Cl.sub.2 /CCl.sub.4 
17 CH.sub.2 BrCOCOOH.sup.(1b,g) 
1) Cl.sub.2 /Na.sub.2 CO.sub.3 
CBrCl.sub.2CBr.sub.2COOH 
2) PBr.sub.5 
18 CH.sub.3COCOOH.sup.(1a) 
1) Br.sub.2 /Na.sub.2 CO.sub.3 
CBr.sub.3CBrClCOOH 
2) NaBH.sub.4 
3) SOCl.sub.2 
4) Br.sub.2 /Red P 
19 CCl.sub.2CClCOOH.sup.(1d) 
1) Zn dust 
CClBr.sub.2CBr.sub.2COOH 
2) Br.sub.2 excess 
20 CH.sub.3COCOOH.sup.(1a) 
1) Br.sub.2 /Na.sub.2 CO.sub.3 
CBr.sub.3CBr.sub.2COOH 
2) PBr.sub.5 
21 HC CCOOH.sup.(1l) 
Cl.sub.2 /CCl.sub.4 
HCl.sub.2 CCCl.sub.2COOH 
22 CCl.sub.3COCOOH.sup.(1g) 
1) NaBH.sub.4 
CCl.sub.3CHClCOOH 
2) PCl.sub.5 
23 CH.sub.2 ClCOCOOH.sup.(1c,h) 
1) Cl.sub.2 /Red P 
CHCl.sub.2CClBrCOOH 
2) NaBH.sub.4 
3) PCl.sub.5 
4) Br.sub.2 /Red P 
24 CH.sub.2 BrCOCO.sub.2 H.sup.(1b) 
1) Cl.sub.2 /Red P 
CCl.sub.2 BrCHClCOOH 
2) NaBH.sub.4 
3) PCl.sub.5 
25 CCl.sub.3COCOOH.sup.(1g) 
1) NaBH.sub.4 
CCl.sub.3CHBrCOOH 
2) PBr.sub.5 
26 CH.sub.2 ClCOCOOH.sup.(1c,h) 
1) Br.sub.2 /Na.sub.2 CO.sub.3 
CHBrClCCl.sub.2COOH 
2) PCl.sub.5 
27 HC CCOOH.sup.(1l) 
1) 1 eq. Br.sub.2 /CCl.sub.4 
CHBrClCBrClCOOH 
2) Cl.sub.2 
28 CH.sub.2 BrCOCOOH.sup.(1b) 
1) 1 eq. Br.sub.2 /Na.sub.2 CO.sub.3 
CHBr.sub.2CCl.sub.2COOH 
2) PCl.sub.5 
29 CH.sub.2 ClCOCOOH.sup.(1c,h) 
1) 1 eq. Cl.sub.2 /Na.sub.2 CO.sub.3 
CHCl.sub.2CBr.sub.2COOH 
2) PBr.sub.5 
30 CH.sub.3COCOOH.sup.(1a) 
1) Br.sub.2 /Na.sub.2 CO.sub.3 
CBr.sub.3CHClCOOH 
2) NaBH.sub.4 
3) PCl.sub.5 
31 CH.sub.2 ClCOCOOH.sup.(1a) 
1) Br.sub.2 /Na.sub.2 CO.sub.3 
CBr.sub.2 ClCHBrCOOH 
2) NaBH.sub.4 
3) PBr.sub.5 
32 CH.sub.2 BrCOCOOH.sup.(1b,g) 
1) 1 eq. Br.sub.2 /Na.sub.2 CO.sub.3 
CHBr.sub.2CBrClCOOH 
2) NaBH.sub.4 
3) PCl.sub.5 
4) Br.sub.2 /Red P 
33 CH.sub.2 ClCOCOOH.sup.(1b,g) 
1) 1 eq. Br.sub.2 /Na.sub.2 CO.sub.3 
CHBrClCBr.sub.2COOH 
2) PBr.sub.5 
34 HC CCOOH.sup.(1l) 
Br.sub.2 /CCl.sub.4 
Br.sub.2 CHCBr.sub.2COOH 
35 CH.sub.3COCOOH.sup.(1a) 
1) Br.sub.2 /Na.sub.2 CO.sub.3 
CBr.sub.3CHBrCOOH 
2) NaBH.sub.4 
3) PBr.sub.5 
36 CH.sub.2 BrCOCOOH.sup.(1b) 
1) Cl.sub.2 /Na.sub.2 CO.sub.3 
CCl.sub.2 BrCCl.sub.2COOH 
2) PCl.sub.5 
37 CH.sub.2 BrCOCOOH.sup.(1b) 
PCl.sub.5 
CH.sub.2 BrCCl.sub.2COOH 
38 CH.sub.2 BrCOCOOH.sup.(1b) 
1) Cl.sub.2 /Na.sub.2 CO.sub.3 
CHClBrCHClCOOH 
2) NaBH.sub.4 
3) PCl.sub.5 
39 CH.sub.2 ClCOCOOH.sup.(1b) 
1) NaBH.sub.4 
CH.sub.2 ClCClBrCOOH 
2) PBr.sub.5 
3) Cl.sub.2 /Red P 
40 CH.sub.2 ClCOCOOH.sup.(1b) 
1) 1 eq. Cl.sub.2 /Na.sub.2 CO.sub.3 
CHCl.sub.2CHBrCOOH 
2) NaBH.sub.4 
3) PBr.sub.5 
41 CH.sub.2 BrCOCOOH.sup.(1b) 
1) Br.sub.2 /Red P 
CHBr.sub.2CHClCOOH 
2) NaBH.sub.4 
3) PCl.sub.5 
42 C.sub.6 H.sub.5 CH.sub.2 CO.sub.2 H.sup.(1a) 
Br.sub.2 /Na.sub.2 CO.sub.3 
C.sub.6 H.sub.5 CBr.sub.2 CO.sub.2 H 
43 CH.sub.2 ClCOCOOH.sup.(1b) 
1) NaBH.sub.4 
CHClBrCHBrCOOH 
2) H.sup.+ (H.sub.2 O) 
3) Br.sub.2 /CCl.sub.4 
44 CH.sub.2 ClCOCOOH.sup.(1b) 
PBr.sub.5 
CH.sub.2 ClCBr.sub.2COOH 
45 C.sub.6 H.sub.5 CH.sub.2 CO.sub.2 H.sup.(1a) 
Cl.sub.2 /Na.sub.2 CO.sub.3 
C.sub.6 H.sub.5 CCl.sub.2 CO.sub.2 H 
46 CH.sub.2 BrCOCOOH.sup.(1b) 
1) NaBH.sub.4 
CHBr.sub.2CHBrCOOH 
2) H.sup.+ (H.sub.2 O) 
3) Br.sub.2 /CCl.sub.4 
47 CH.sub.2 BrCOCOOH.sup.(1b) 
PBr.sub.5 
CH.sub.2 BrCBr.sub.2COOH 
48 CH.sub.2 ClCOCOOH.sup.(1b) 
PCl.sub.5 
CH.sub.2 ClCCl.sub.2COOH 
49 CHCl.sub.2CHO.sup.(1a) 
1) HCN CHCl.sub.2CHClCOOH 
2) H.sub.3 0.sup.+ .DELTA. 
3) PCl.sub.5 
50 C.sub.10 H.sub.7 CH.sub.2 CO.sub.2 H.sup.(1a) 
Cl.sub.2 /Red P 
C.sub.10 H.sub.7 CCl.sub.2 CO.sub.2 H 
51 CCl.sub.2CClCOOH.sup.(1a) 
ICl CCl.sub.3CIClCOOH + 
CCl.sub.2 ICCl.sub.2COOH 
52 CF.sub.2CFCOOCH.sub.3.sup.(1g) 
1) Cl.sub.2 
CF.sub.2 ClCFClCOOH 
2) NaOH/H.sup.+ 
53 CF.sub.2CFCOOCH.sub.3.sup.(1g) 
1) Br2 CF.sub.2 BrCFBrCOOH 
2) NaOH/H.sup.+ 
54 CF.sub.2CFCOOCH.sub.3.sup.(1g) 
ICl CF.sub.2 ICFClCOOH + 
CF.sub.2 ClCFICOOH 
55 C.sub.10 H.sub.7 CH.sub.2 CO.sub.2 H.sup.(1a) 
Br.sub.2 /Red P 
C.sub.10 H.sub.7 CBr.sub.2 CO.sub.2 H 
56 CH.sub.2 FCOCOOH.sup.(1k) 
1) Cl.sub.2 /Red P 
CCl.sub.2 FCCl.sub.2COOH 
2) PCl.sub.5 
57 CH.sub.2 FCOCOOH.sup.(1k) 
1) Cl.sub.2 /Red P 
CCl.sub.2 FCHClCOOH 
2) NaBH.sub.4 
3) PCl.sub.5 
58 CH.sub.2 FCOCOOH.sup.(1k) 
1) Cl.sub.2 /Red P 
CCl.sub.2 FCHFCOOH 
2) NaBH.sub.4 
3) SF.sub.4 
59 CF.sub.3CH.sub.2CH.sub.2 OH.sup.(1f) 
1) K.sub.2 Cr.sub.2 O.sub.7 /H.sup.+ 
CF.sub.3CCl.sub.2COOH 
2) Cl.sub.2 /Red P 
60 CF.sub.3CH.sub.2CH.sub.2 OH.sup.(1f) 
1) K.sub.2 Cr.sub.2 O.sub.7 /H.sup.+ 
CF.sub.3CHClCOOH 
2) Cl.sub.2 leq./Red P 
61 CF.sub.3CH.sub.2CH.sub.2 OH.sup.(1f) 
1) K.sub.2 Cr.sub.2 O.sub.7 /H.sup.+ 
CF.sub.3CBr.sub.2COOH 
2) Br.sub.2 /Red P 
62 CH.sub.2 FCOCOOH.sup.(1f) 
1) Cl.sub.2 /Red P 
CCl.sub.2 FCBr.sub.2COOH 
2) PBr.sub.5 
63 CH.sub.2 FCOCOOH.sup.(1f) 
1) Br.sub.2 /Red P 
CBr.sub.2 FCBr.sub.2COOH 
2) PBr.sub.5 
64 CH.sub.2 FCOCOOH.sup.(1f) 
1) Br.sub.2 /Red P 
CBr.sub.2 FCHFCOOH 
2) NaBH.sub.4 
3) SF.sub.4 
65 CH.sub.2 FCOCOOH.sup.(1f) 
1) Br.sub.2 /Red P 
CBr.sub.2 FCCl(CN)COOH 
2) HCN 
3) PCl.sub.5 
66 NCCH.sub.2COOH.sup.(1b,a) 
Cl.sub.2 /Red P 
NCCCl.sub.2COOH 
67 NCCH.sub.2COOH.sup.(1b,a) 
Br.sub.2 /Red P 
NCCBr.sub.2COOH 
68 NCCH.sub.2CH.sub.2COOCH.sub.3.sup.(1e) 
1) Cl.sub.2 /Red P 
NCCH.sub.2CCl.sub.2COOH 
2) NaOH/H.sup.+ 
69 NCCH.sub.2CH.sub.2COOCH.sub.3.sup.(1e) 
1) Br.sub.2 /Red P 
NCCH.sub.2CBr.sub.2COOH 
2) NaOH/H.sup.+ 
70 CCl.sub.3COCOOH.sup.(1e) 
1) KCN/H.sup.+ 
CCl.sub.3CCl(CN)COOH 
2) PCl.sub.5 
71 CCl.sub.3COCOOH.sup.(1e) 
1) KCN/H.sup.+ 
CCl.sub.3CBr(CN)COOH 
2) PBr.sub.5 
72 CCl.sub.3COCOOH.sup.(1e) 
1) KCN/H.sup.+ 
CCl.sub.3CF(CN)COOH 
2) SF.sub.4 
73 CH.sub.3COCOOH.sup.(1e) 
1) Br.sub.2 /Na.sub.2 CO.sub.3 
CBr.sub.3CCl(CN)COOH 
2) KCN/H.sup.+ 
3) PCl.sub.5 
74 HC CCOOH.sup.(1l) 
1 mole Br.sub.2 
BrCHCBrCOOH 
75 HC CCOOH.sup.(1l) 
1 mole Cl.sub.2 
CHClCClCOOH 
76 HC CCOOH.sup.(1l) 
1) Br.sub.2 /CCl.sub.4 (excess) 
Br.sub.2 CCBrCOOH 
2) NaNH.sub.2 
77 HC CCOOH.sup.(1l) 
1) 1 mole Cl.sub.2 
ClBrCCClCOOH + 
2) Br.sub.2 
ClBrCCBrCOOH 
3) alc KOH 
78 
##STR75## 1) K.sub.2 Cr.sub.2 O.sub.7 /H.sup.+ 2) KCN/H.sup.+ 3) 
H.sub.3 O.sup.+ 4) PCl.sub.5 
##STR76## 
79 CCl.sub.2CClCOOH.sup.(1a) 
CHCl.sub.3 /KtOBu 
##STR77## 
80 
##STR78## 1) K.sub.2 Cr.sub.2 O.sub.7 /H.sup.+ 2) MeOH/H.sup.+ 3) 
NaOEt/Cl.sub.2 4) NaOH/H.sup.+ 
##STR79## 
81 
##STR80## 1) K.sub.2 Cr.sub.2 O.sub.7 /H.sup.+ 2) HCN 3) H.sub.3 
O.sup.+ 4) PCl.sub.5 
##STR81## 
82 Cl.sub.2 CCClCO.sub.2 H.sup.(1d) 
Zn dust/heat 
ClC CCO.sub.2 H 
83 HC CH, HCOCO.sub.2 CH.sub.2 CH.sub.3.sup.(1a) 
1. NaNH.sub.2 
HC CCHClCO.sub.2 H 
2. H.sub.3 O.sup.+ 
3. PCl.sub.5 
4. NaOH 
5. H.sub.3 O.sup.+ 
CF.sub.3 CH.sub.2 Br.sup.(1i) 
1. CdCl.sub.2 
CF.sub.3 CCl.sub.2 COCO.sub.2 H 
2. ClCOCO.sub.2 Et 
3. NaOH 
4. Cl.sub.2 /Red P 
N CCH.sub.2 OH.sup.(1m) 
1. PI.sub.2 
N CCCl.sub.2 COCO.sub.2 H 
2. CdCl.sub.2 
3. ClCOCO.sub.2 Et 
4. NaOH 
5. Cl.sub.2 /Red P 
HC CCHClCO.sub.2 Et.sup.(1m) 
1. Br.sub.2 
BrHCCBrCHClCO.sub.2 H 
2. NaOH 
__________________________________________________________________________ 
Footnotes to TABLE XIII: 
Starting material sources: 
.sup.(1a) Aldrich Chemical Company 
.sup.(1b) Morton Thiokol, Alfa Products 
.sup.(1c) Biochemical Laboratories, Inc. 
.sup.(1d) Columbia Organic Chemicals 
.sup.(1e) CTC Organics 
.sup.(1f) Interchemical Corporation 
.sup.(1g) K&K Laboratories 
.sup.(1h) Mide Chemical Corporation 
.sup.(1i) Pfaltz and Bauer, Inc. 
.sup.(1j) Reliable Chemical Company 
.sup.(1k) United State Biochemical Corporation 
.sup.(1l) Wiley Organic 
.sup.(1m) Alfa 
2)Many other electrophoric carboxylic acids are listed in Beilstein. 
the initially prepared electrophoric carboxylic acid Sg--CO.sub.2 H is 
frequently further converted into another generally more reactive 
derivative for reaction with the precursor of the release group. Thus, for 
example, the carboxylic acid can be reacted with thionyl chloride, 
phosphorus pentachloride, or oxalyl chloride to yield the corresponding 
acid chloride. The anhydride of the carboxylic acid may be obtained by 
reacting the acid chloride with trichlorotrifluoro acetone hydrate in dry 
benzene in the presence of pyridine. Alternatively, the anhydride may be 
prepared by treating the carboxylic acid directly with P.sub.2 O.sub.5 or 
acetic anhydride in refluxing benzene. The carboxylic acid may be 
converted to the corresponding aldehyde as described by Harrison, I. T., 
and Harrison, S., Compendium of Organic Synthetic Methods, 
Wiley-Interscience, New York, 1971, pp. 132-137. Additional methods are 
provided in Volumes 1 through 5 of this series. See for example Wade, L. 
G., Volume 5, pp. 93-96, 1984. The aldehyde Sg--CHO may be converted to 
the corresponding acetal Sg--CH(OR).sub.2 by an acid catalyzed reaction 
with an alcohol ROH. The .alpha.-keto acid Sg--CO--CO.sub.2 H may be 
prepared by reacting the acetal Sg--CH(OR).sub.2 with HCN, followed by 
reaction with sulfuric acid in water, and finally with chromium oxide. 
This .alpha.-keto acid may in turn be converted to the corresponding acid 
chloride by reaction with thionyl chloride or other reagents as discussed 
above. The above-described carboxylic acid Sg--CO.sub.2 H or one of its 
reactive derivatives as discussed above is employed in a subsequent 
reaction with the precursor of the release group Re in the synthesis of 
the desired release tag compounds. 
The volatile compound derived from the S.sub.g moiety in a release tag is 
detected preferably by an electron capture detector (ECD) or by electron 
capture negative ion mass spectrometry (ECNI-MS). Examples of other gas 
phase detectors that may be used are as follows: flame ionization 
detector, electron impact MS, positive ion chemical ionization MS, 
thermospray MS, fast atom bombardment MS, fast ion bombardment MS, 
atmospheric pressure ionization MS, particle beam MS, electrospray MS, 
plasma desorption MS, laser ionization MS, laser desorption MS, thermal 
conductivity detector, nitrogen-phosphorous detector, photoionization 
detector, flame photometric detector, and ion mobility detector. 
EXPERIMENTAL 
Rotary--flash injector 
An injector assembly was prepared by connecting a Varian rotary valve 
assembly (part No. 03-908719-00) to the front end of a Varian flash 
injector body (part No. 01-001014-00) by means of a nut bored with 
opposite threads at each end to accept and connect the valve assembly and 
the injector body, respectively. This nut was provided with a gas inlet 
line for introduction of carrier gas. 
Within the flash injector body was placed a glass insert tube which 
extended from the top end of the injector body and out the bottom end. 
This glass insert was 130 mm in length and 6 mm OD, and fit closely in the 
bore of the injector body. The first 2.6 cm of the glass insert had an 
inside diameter of 4 mm, and the remainder of the insert possessed a 2 mm 
bore. At the juncture of the 4 mm and 2 mm ID portions of the glass insert 
was placed a plug of clean glass wool. 
The injector assembly was mounted on a Varian Model 6000 GC equipped with 
an electron capture detector, the injector body being housed in a heated 
injector block (Varian part No. 62-000203-00). The lower end of the glass 
insert was connected to a capillary GC column, the front end of which was 
placed within the glass liner approximately 80 mm -from its outlet end. 
The outlet end of the flash injector body was located at the top of the 
column oven of the gas chromatograph. 
Chromatography 
The chromatographic column was a 0.32 mm ID.times.7 m Quadrex 007 column 
(5% phenyl methyl silicone; Quadrex, Inc.), 5 .mu.m film thickness. 1 
.mu.l injections of sample solutions were made onto the glass wool in the 
glass insert, with a 5 .mu.l syringe fitted with an 11.5 cm stainless 
steel needle, the injector body being maintained at 300.degree. C. and the 
column being at a low temperature such as 50.degree. C. After sample 
injection, the column was held at its initial temperature for about three 
minutes, then programmed quickly at 50.degree. C./min to 150.degree. C., 
and held at this temperature for five minutes. Nitrogen flow through the 
column was 3 ml/min, measured at RT, with the column at 50.degree. C. The 
detector was maintained at 310.degree. C. 
WORKING EXAMPLES 
N-Trichloroacetyl-p-aminobenzoic acid (CCl.sub.3 CO-ABA) (W1), 
p-Aminobenzoic acid (1 g, 7.3 mmol) and 7 mL (32.8 mmol) of trichloroacetic 
anhydride were refluxed for 0.5 hr. More anhydride (3 mL) was added and 
heating was continued for 16 hr. Water (15 mL) and ethyl acetate (30 mL) 
were added, and, after shaking, the separated organic layer was dried 
(anhyd. Na.sub.2 SO.sub.4) and evaporated (rotary evaporator) to give the 
product as yellowish white crystals. The product was a single spot by TLC 
(silica; ethyl acetate/hexane, 2/3), and its structure was confirmed by 
its spectral characteristics. 
N-Trichloroacetyl-p-aminobenzoic acid N-hydroxysuccinimide ester (CCl.sub.3 
CO-AB-NHS) (W2). 
Compound W1 (290 mg, 1.03 mmol) was dissolved in 5 mL of dimethylformamide 
(DMF) and the temperature was raised to 70.degree. C. 
N,N-(carbonyldiimidazole (144 mg,, 0.90 mmol) was added, and 70.degree. C. 
was continued until CO.sub.2 evolution ceased (30 min). 
N-Hydroxysuccinimide (102 mg, 0.09 mmol) was added, heating was 
discontinued, and the reaction mixture was stirred for 17.5 hr. The 
solvent was removed under high vacuum, and the addition of 15 mL of 
isopropanol gave a white precipitate (304 mg, 81%) which was a single spot 
by silica TLC (2:3 ethyl acetate:hexane, v/v), and which melted at 
258.degree. C. After recrystallization from isopropanol, the structure of 
the product was confirmed by MS, IR, and .sup.1 H NMR. 
N-Trichloroacetyl-N-methyl-p-aminobenzoic acid (CCl.sub.3 CO-MABA)(W3). 
4-(Methylamino)benzoic acid (1.00 g, 6.62 mmoL) and trichloroacetic 
anhydride (3.07 g, 9.94 mmoL) were refluxed in 20 mL of dry benzene for 30 
min. After evaporation and flash column chromatography (ethyl 
acetate/hexane 3/7 v/v), the product was obtained as a white solid (1.1 g, 
56%), the structure of which was confirmed by its spectral 
characteristics. 
N-Trichloroacetyl-N-methyl-p-aminobenzoic acid N-hydroxysuccinimide ester 
(CCl.sub.3 CO-MAB-NHS) 
Compound W3 (285.5 mg, 0.96 mmol) and N,N-carbonyldiimidazole (156 mg, 0.96 
mmol) were heated with stirring at 70.degree. C. in 5 mL of 
dimethylformamide until no more CO.sub.2 evolved (10 min). After the heat 
was removed, stirring was continued for 30 min, N-hydroxysuccinimide (111 
mg, 0.96 mmol) was added and the reaction mixture was stirred at RT for 17 
hr. The solid residue was purified by recrystallization from isopropanol, 
yielding a white solid (250 mg, 66%), m.p. 183.degree. C., the structure 
of which was confirmed by its spectral characteristics. 
Trichloroacetylmethylaminobenzoyl-Albumin (CCl.sub.3 CO-MAB-Albumin) (W5). 
Bovine Serum Albumin (BSA, 1 mg; Sigma Chemical Co.) was dissolved in 1 mL 
of potassium phosphate buffer, pH 8 and 1.74 mg of compound W4 dissolved 
in 100 .mu.L of dimethylsulfoxide was added, followed by stirring at RT 
for 17 hr. The resulting solution was dialyzed against 4.times.1 L of 0.01 
M ammonium bicarbonate at 4.degree. C. over a period of 3 d. Based on the 
TNBS test, 74% of the amino groups in BSA had been modified with 4. 
Diaminooctyl-DNA(DAO-DNA) (W6). 
Sodium bisulfite was prepared by adding 3.15 g of sodium sulfite and 7.15 g 
of sodium metabisulfite to 25 ml of water. 1,8-Diaminooctane (7.2 g) was 
added and the pH was adjusted to 7 with concentrated hydrochloric acid. 
Calf thymus DNA (Sigma, 139 mg) was dissolved in 20 mL of water and 
denatured by heating to 100.degree. C. for 30 min followed by rapid 
cooling in an ice bath. The single stranded DNA was then sonicated for 40 
min at 0.degree. C. and added to the sodium bisulfite-diaminooctene 
solution. This gave a final concentration of 2 M bisulfite and 1 M 
diaminooctane. The mixture was clarified by centrifugation (4000.times.g) 
and then stirred at 60.degree. C. (oil bath) for 42 hrs. The reaction 
mixture was cooled and filtered (0.2 .mu.m filter) to remove a small 
amount of particulate material. The product was desalted in two 25 mL 
portions over a BioRad P-4 column (340.times.2.6 cm) equilibrated in 0.02 
M sodium chloride, 1 mM EDTA, pH 8. After the void volume (60 mL), the 
product was collected in the next 50 mL fraction and each of these two 
fractions was separately dialyzed overnight against 4 L of water and 
lyphilized, yielding together 99.5 mg (72%). Seventy-five percent of the 
cytosine residues were substituted with DAO, based on alkaline 
hydrolysis-HPLC. 
N-Trichloroacetyl-p-aminobenzoyl-BSA(CCl.sub.3 CO-AB-BSA) (W7) 
N-Trichloroacetyl-p-aminobenzoic acid NSH ester (16.6 mg, 0.04 mmol, 
Compound W2) was added in small portions over 15 min to a solution of 2 mg 
(0.02 .mu.mol) of bovine serum albumin (BSA) in a 1.8 mL of a 0.1 M 
potassium phosphate buffer, pH 8/DMSO, 1:1, followed by stirring for 18 h 
at room temperature. This solution was centrifuged and the clear 
supernatant was passed through a PD10 column (Pharmacia) 2 times using 
0.01 M KPB, pH 8.0, followed by lyophilization. Protein analysis (BCA 
test; Pierce Chemical Co.) and amino group analysis by a trinitrobenzene 
sulfonic acid (TNBS) test indicated that 82% of the primary amino groups 
on BSA were modified. 
N-Trichloroacetyl-p-aminobenzoyl-DAO-/DNA(CCl.sub.3 CO-AB-DAO-DNA) (W8) 
Formamide/DMSO (700 .mu.L, 4:3) containing 15 mg (0.038 mmol) of 
N-trichloroacetyl-p-aminobenzoic acid NSH ester (W2) was added dropwise to 
a solution of DAO-DNA in 800 .mu.L of 0.1 M KPB pH 8. The resultant cloudy 
solution (precipitation of the DAO-DNA by the organic solvent) was stirred 
at room temperature for 18 h. The reaction mixture was centrifuged and the 
clear supernatant was passed through a PD10 column twice using 0.01 M KPB 
pH 8 followed by lyophilization. 
Enzymatic digestion and HPLC analysis of modified DAO-DNA, 
A sample of lyophilized CCl.sub.3 CO-AB-DAO-DNA (W8) was dissolved in 2.5 
mL of Tris buffer pH 8.8 (0.025 M, 1 mM EDTA) and passed through PD10 
column using the same buffer. To 1.5 mL of this solution, containing 
approximately 37 .mu.g of CCl.sub.3 CO-AB-DAO-DNA based on A.sub.260 was 
added 30 .mu.L of 1 M CaCl.sub.2 solution and 50 .mu.g of staphylococcal 
nuclease (Sigma), followed by incubation at 37.degree. C. for 3 h. HPLC 
analysis (C18-silica) showed a 66% disappearance of DAO-cytidylic acid and 
related peaks when compared against standard DAO-DNA treated similarly. 
N-Trichloroacryloyl-p-aminobenzoic acid (CCl.sub.2 CClCO-ABA) (W9) 
Trichloroacrylic acid (500 mg, 2.85 mmol; Alpha Chem. Co.) was added to 7 
mL of SOCl.sub.2 and the mixture was refluxed for 6 h. After cooling, 5 mL 
of benzene was added and the solvent was concentrated on the rotary 
evaporator to 1/3 of its original volume. This step was repeated 4 times, 
until most of SOCl.sub.2 was evaporated. Three mL of acetonitrile were 
added followed by a suspension of 383 mg (2.80 mmol) of p-amino-benzoic 
acid in 3 mL of acetonitrile. After 30 min of stirring, TLC showed the 
disappearance of most starting material and the presence of a product with 
higher R.sub.f value. The product was purified by preparative silica TLC 
(EtOAC/Hexane/acetic acid, 2:3:0.05) which gave a pure white powder (89% 
yield); MS (EI) m/z 293 (M.sup.+). 
Aqueous stability test of N-Trichloroacryloyl-p-aminobenzoic acid (W9) 
N-Trichloroacryloyl-p-aminobenzoic acid (440 .mu.g) was dissolved in 1 mL 
of methanol, and 100 .mu.L of this solution was added to 300 .mu.L of 
potassium phosphate buffer (0.1 M, pH 8). In the same way a solution of 
p-aminobenzoic acid was prepared. Both solutions were analyzed by HPLC 
(C18-silica column, 0.01 M potassium phosphate buffer pH 4.5 initially, 
then 0 to 38% acetonitrile over 29 minutes. This gave a retention time of 
6.8 min for p-aminobenzoic acid and 26 min for the product. The solutions 
were kept at room temperature. Analysis by HPLC demonstrated that the 
N-trichloroacryloyl-p-aminobenzoic acid was stable at least for four days. 
By this last day the solution of the p-aminobenzoic acid had discolored 
and additional peaks were seen. 
N-Trichloroacryloyl-p-aminobenzoic acid N-hydroxysuccinimide ester 
(CCl.sub.2 CClCO-AB-NHS) (W10) 
Dicyclohexylcarbodiimide (10 mg, 0.05 mmol) was added as a solid to a 
stirred suspension of N-trichloroacryloyl-p-aminobenzoic acid (14 mg, 
0.047 mmol) and N-hydroxysuccinimide (5.98 mg, 0.05 mmol) in 3 mL of dry 
CH.sub.2 Cl.sub.2 at 0.degree. C. The reaction mixture was stirred under 
N.sub.2 for 3 h and allowed to come to room temperature. The insoluble 
dicyclohexylurea was filtered, and rotary evaporation of the filtrate 
yielded a white solid. The product was purified by TLC using 
ETOAc:hexane:acetic acid (2:3:0.05). NMR indicated the presence of 4 
methylene hydrogens at .delta. 3.0. MS (EI) 390 (M.sup.+) and 276 (base 
peak). 
N-Trichloroacryloyl-p-aminobenzoyl-BSA (CCl.sub.2 CClCO-AB-BSA) 
NSH ester W10 (22 mg) was added as a solid to 5 mg of bovine serum albumin 
(BSA) dissolved in 2.5 mL of 0.1 M potassium phosphate buffer, pH 
8/dimethylsulfoxide, 60:40. The immediate white suspension was stirred 
overnight at room temperature. After centrifugation the clear supernatant 
was passed through a PD10 column twice, then lyophilized. A protein assay 
(Pierce BCA) and TNBS test indicated that 52% of the BSA amino groups had 
been modified. 
N-Trichloroacryloyl-p-aminobenzoyl-DAO-DNA (CCl.sub.2 CClCO-AB-DAO-DNA) 
NSH ester W10 (19 mg, 48 .mu.mol) in 300 .mu.L of DMF was added dropwise to 
1 mL of a cold, stirring solution of DAO-DNA (2.7 mg) in 0.1 M KPB, pH 8. 
The reaction mixture was allowed to come to room temperature, 1.6 mL of 
DMF was added to give a clear solution, and stirring was continued for 18 
h. After centrifugation, the clear supernatant was passed through a PD10 
column and lyophilized. Digestion and HPLC analysis showed the 
disappearance of the DAO-cytidylic acid peaks and the presence of new 
peaks derived from the modification. 
Detection of CCl.sub.3 CO-AB-BSA by GC-ECD 
The conjugate CCl.sub.3 CO-AB-BSA (W6) was dissolved in water and dilutions 
were made giving concentrations of this conjugate ranging from 
2.4.times.10.sup.-16 to 6.times.10.sup.-13 mole/.mu.L. Injection of 1 
.mu.L volumes of these solutions into the GC-ECD each gave a peak for 
chloroform, the released electrophore. Injection of water or a solution of 
albumin in water gave no peak for chloroform. The resulting standard curve 
is shown in FIG. 1A, including an inset presenting a chromatogram for the 
smallest amount of CCl.sub.3 CO-AB-BSA injected. In FIG. 1B is shown a 
chromatogram from the injection of 2.7.times.10.sup.-13 mole of CCl.sub.3 
CO-AB-BSA. Each injection was made over a 10 sec interval into the 
rotary-flash injector described above. 
CCl.sub.3 CO-AB-BSA may also be detected by headspace injection GC-ECD in 
which an aqueous sample of CCl.sub.3 CO-AB-BSA is heated and the vapor, 
containing chloroform, is injected into a GC-ECD. The released chloroform 
may also be determined by electron capture negative ion mass spectrometry 
or by ion mobility spectrometer detector. CCl.sub.3 CO-AB-BSA may also be 
detected by heating an aqueous sample of CCl.sub.3 CO-AB-BSA and 
extracting the released chloroform in isooctane, toluene or a related 
organic solvent for injection into a GC-ECD. 
Detection of CCl.sub.2 CClCO-AB-BSA by GC-ECD 
An aqueous solution of CCl.sub.2 CClCO-AB-BSA was heated at 60.degree. C. 
for 1 hr to enhance the aggregation of the CCl.sub.2 CClCO-AB-BSA. 
Injection as above of 1 .mu.L of this solution into a GC-ECD gave a peak 
for CCl.sub.2 CClH. 
Detection of CCl.sub.3 CO-AB-DAO-DNA by GC-ECD 
To an aqueous solution of CCl.sub.3 CO-AB-DAO-DNA was added polylysine 
(Sigma) for the purpose of developing aggregates of these two substances. 
Injection as above of 1 .mu.L of this solution into a GC-ECD gave a peak 
for CHCl.sub.3. 
Detection of a mixture Of CCl.sub.2 CClCO-AB-BSA and CCl.sub.3 
CO-AB-DAO-DNA by GC-ECD 
One .mu.L of an aqueous solution containing 331 ng of CCl.sub.3 
CO-AB-DAO-DNA, 334 ng of polylysine, 147 ng of CCl.sub.2 CClCO-AB-BSA, and 
19.3 ng of albumin was injected as above into a GC-ECD. This gave the 
chromatogram shown in FIG. 2, displaying peaks for CCl.sub.3 H and 
CCl.sub.2 CClH. 
GC-ECD Of 2,2-Dichloropropionic Acid 
Injection as above of one .mu.L of water containing 2.78 ng of 
2,2-dichloropropionic acid (Aldrich Chem. Co.) into a GC-ECD gave a peak 
for 1,1-dichloroethane. 
Determination Of Ethanol by Thermal Release of an Electrophore 
Ethanol is reacted with trichloroacetic anhydride to yield 
ethyltrichloroacetate. 
The ethyltrichloroacetate was dissolved in water (2.76 ng/.mu.l) and 1 
.mu.L was injected into a GC-ECD as above. A peak for chloroform was 
obtained. 
PREDICTIVE EXAMPLES 
The following examples illustrate signal groups Sg such as those shown in 
Table XIII, release groups, reactivity groups, and connecting moieties in 
a variety of release tag compounds, and provide suggested synthetic 
procedures for preparation of these compounds. Release tags which are 
illustrated in the form of carboxylic acids would generally be employed in 
practice as more reactive carboxylic acid derivatives such as N-hydroxy 
succinimide or benzotriazole esters, anhydrides, or acid chlorides, etc. 
##STR82## 
Carboxymethoxylamine-hydrochloride (Aldrich) is reacted with an anhydride 
of an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in 
Table XIII to afford the product. 
##STR83## 
An anhydride of an electrophoric carboxylic acid SgCO.sub.2 H such as 
those shown in Table XIII is reacted with hydrazine to form an 
SgCO-substituted hydrazine, which in turn is reacted with succinic 
anhydride to afford the product. 
##STR84## 
2-Hydroxyisobutyric acid (Aldrich) is combined with an acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII in the presence of pyridine to afford the product. 
##STR85## 
The diethyl acetal of an aldehyde corresponding to an electrophoric 
carbexylic acid SgCO.sub.2 H such as those shown in Table XIII is reacted 
with hydrogen cyanide (generated from sodium cyanide plus sulfuric acid), 
followed by heating in aqueous sulfuric acid to afford the carboxylic acid 
corresponding to the intermediate .alpha.-hydroxy nitrile. This product is 
oxidized with chromic oxide in pyridine or acetic acid to give the 
2-oxo-carboxylic acid, which is in turn reacted with thionyl chloride to 
afford the 2-oxo-carboxylic acid chloride. This is finally reacted with 
2-hydroxyisobutyric acid in pyridine to afford the desired product. 
##STR86## 
An acid chloride of an electrophoric 2-oxo-carboxylic acid SgCOCO.sub.2 H 
(prepared as above in connection with (4)) is reacted with p-aminobenzoic 
acid to give the product. 
##STR87## 
An acid chloride of an electrophoric 2-oxo-carboxylic acid SgCOCO.sub.2 H 
(prepared as above in connection with (4)) is reacted with hydrazine to 
give the corresponding substituted hydrazine, which is reacted in turn 
with succinic anhydride to give the product. 
##STR88## 
Carboxymethoxylamine hydrochloride is reacted with an acid chloride of an 
electrophoric 2-oxo-carboxylic acid SgCOCO.sub.2 H (prepared as above in 
connection with (4)) to give the product. 
##STR89## 
N-Acetylhydroxyproline (Sigma) is reacted with an acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII in the presence of pyridine to give the product. 
##STR90## 
Hydroxyproline (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
product. 
##STR91## 
1,4-Diaminopiperazine is reacted with one equivalent of an anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII to yield the monosubstituted SgCO-1,4-diaminopiperazine. 
##STR92## 
1,4-Diaminopiperazine is reacted with one equivalent of an anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII to yield the monosubstituted SgCO-1,4-diaminopiperazine. This 
compound is reacted in turn with succinic anhydride to give the product. 
##STR93## 
Tris-(Hydroxymethyl)aminomethane (Aldrich) is reacted with succinic 
anhydride to give N-succinyl-tris-(hydroxymethyl)-aminomethane, which in 
turn is reacted with an acid chloride of an electrophoric carboxylic acid 
SgCO.sub.2 H such as those shown in Table XIII in pyridine to give the 
product. 
##STR94## 
Gluconic acid lactone (Sigma) is reacted with hydrazine to yield 
gluconylhydrazide, which is reacted in turn with succinic anhydride to 
yield N-gluconyl-N'-succinylhydrazine. This latter compound is reacted 
with an acid chloride of an electrophoric carboxylic acid SgCO.sub.2 H 
such as those shown in Table XIII in pyridine to yield the product. 
##STR95## 
p-Hydroxybenzoic acid is reacted with an acid chloride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII in pyridine 
to give the product. 
##STR96## 
6-Aminonicotinic acid (Aldrich) is reacted with an anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII to give the product. 
##STR97## 
3-Amino-4-hydroxybenzoic acid (Aldrich) is reacted with an anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII to give the product. 
##STR98## 
Serine (Sigma) is reacted with an anhydride of an electrophoric carboxylic 
acid SgCO.sub.2 H such as those shown in Table XIII to give the product. 
##STR99## 
Cysteic acid (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
product. 
##STR100## 
Histidine (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
product. 
##STR101## 
Histidylglycine (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
product. 
##STR102## 
Asparagine (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
product. 
##STR103## 
4-Amino-2-nitrophenol (Aldrich) is reacted with an anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII to form the corresponding N-SgCO-substituted-4-amino-2-nitrophenol, 
which is reacted in turn with iodoacetate to give the product. 
##STR104## 
Sulfanilamide (Aldrich) is reacted with succinic anhydride to form 
4-(succinamido)-sulfanilamide, which in turn is reacted with an acid 
chloride of an electrophoric carboxylic acid SgCO.sub.2 H such as those 
shown in Table XIII to give the product. 
##STR105## 
N.sup..alpha. -Acetylasparagine (Sigma) is reacted with an acid chloride 
of an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in 
Table XIII to give the product. 
##STR106## 
Glycylglycine is reacted with an anhydride of an electrophoric carboxylic 
acid SgCO.sub.2 H such as those shown in Table XIII to give the product. 
##STR107## 
Cytosine (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
corresponding N-SgCO-substituted cytosine, which is reacted in turn with 
iodoacetate to give the product. 
##STR108## 
p-Aminobenzoic acid is reacted with an acid chloride or anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII to yield the N-SgCO-substituted carboxylic acid, which is in turn 
activated with N,N-carbonyldiimidazole then reacted with 
N-hydroxysuccinimide to produce the N-hydroxysuccinimide ester of the 
carboxylic acid. This is finally reacted with hydrazine to give the 
product. 
##STR109## 
The N-SgCO-substituted-p-aminobenzoic acid prepared as an intermediate in 
the synthesis of 27 above is reacted with ethylene diamine to give the 
product. 
##STR110## 
The N-SgCO-substituted-p-aminobenzoic acid prepared as an intermediate in 
the synthesis of 27 above is reacted with thionyl chloride to give the 
product. 
##STR111## 
The N-SgCO-substituted-p-aminobenzoic acid prepared as an intermediate in 
the synthesis of 27 above is reacted with dicyclohexylcarbodiimide and 
p-nitrophenol to give the product. 
##STR112## 
The compound shown as 27 above is reacted with sodium nitrite in aqueous 
acid to give the product. The product can also be obtained by reacting the 
N-SgCO-substituted-p-aminobenzoic acid prepared as an intermediate in the 
synthesis of 27 above with diphenylphosphorazidate. 
##STR113## 
The N-SgCO-substituted-p-aminobenzoic acid prepared as an intermediate in 
the synthesis of 27 above is reacted with dicyclohexylcarbodiimide and 
N-hydroxybenzotriazole to give the product. 
##STR114## 
The N-SgCO-substituted-p-aminobenzoic acid prepared as an intermediate in 
the synthesis of 27 above is reacted with isobutylchloroformate to give 
the product. 
##STR115## 
The compound shown as 27 is reacted with 1,4-phenylenediisothiocyanate 
(Aldrich) to give the product. 
##STR116## 
The compound shown as 29 is reacted with methanethiol (Aldrich) to give 
the product. 
##STR117## 
The compound shown as 27 is reacted with dimethylsuberimidate (Aldrich) to 
give the product. 
##STR118## 
Iodoacetic acid is reacted with dicyclohexylcarbodiimide and 
N-hydroxysuccinimide to give iodoacetyl-N-hydroxysuccinimide, which is 
reacted in turn with compound 27 above to give the product. 
##STR119## 
The N-hydroxysuccinimide ester of the N-SgCO-substituted-p-aminobenzoic 
acid prepared as an intermediate in the synthesis of 27 above is reacted 
with 3-aminopropanol (Aldrich) to give the corresponding 3-aminopropanol, 
which is reacted in turn with tresyl chloride to give the product. 
##STR120## 
The compound shown as 27 is reacted with succinimidyl-3-(2-pyridyldithio) 
propionate (SPDP, Sigma) to give the product. 
##STR121## 
The compound shown as 39 above is reacted with dithiothreitol to give the 
product. 
##STR122## 
The compound shown as 27 is reacted with .gamma.-maleimidobutyric acid 
N-hydroxysuccinimide to give the product. 
##STR123## 
The N-hydroxysuccinimide ester of the N-SgCO-substituted-p-aminobenzoic 
acid prepared as an intermediate in the synthesis of 27 above is reacted 
with 1,4-diaminopiperazine to give this product. 
##STR124## 
The N-hydroxysuccinimide ester of the N-SgCO-substituted-p-aminobenzoic 
acid prepared as an intermediate in the synthesis of 27 above is reacted 
with sulfanilic acid to give the corresponding sulfanilic acid derivative, 
which is reacted in turn with thionyl chloride to give the product. 
##STR125## 
Carboxymethoxylamine is reacted with trifluoroacetic anhydride to give 
N-(trifluoroacetyl)carboxylamine. This latter compound is reacted with 
dicyclohexylcarbodiimide and the hydrazide shown as 27, followed by 
removal of the trifluoroacetyl groups at alkaline pH to give the product. 
##STR126## 
The compound shown as 27 is reacted with 1,2,3,4-diepoxybutane (Aldrich) 
to give the product. 
##STR127## 
4-(Oxyacetyl)phenoxyacetic acid is prepared as described (Duerksen, P. J. 
and Wilkinson, K. D., Anal. Biochem. 160, 1987, pp. 444-454). This 
compound is then activated with dicyclohexylcarbodiimide and reacted with 
the compound shown as 27 to give the product. 
##STR128## 
4-Fluoro-3-nitrophenyl azide is synthesized as described (Forster, A. C., 
McInnes, J. L., Skingle, D. C., and Symons, R. H., Nucl. Acids Res. 13, 
1985, pp. 745-761) and reacted with the compound shown as 28 to form the 
product. Photolysis of the product as described (Forster, Ibid.) forms the 
corresponding release tag nitrophenyl nitrene. 
##STR129## 
.alpha.-D-Glucopyranosylphenylisothiocyanate (Sigma) is reacted with an 
acid chloride of an electrophoric carboxylic acid SgCO.sub.2 H such as 
those shown in Table XIII in pyridine to give the product. 
##STR130## 
Carboxymethoxylamine hydrochloride (Aldrich) is reacted with the anhydride 
of an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in 
Table XIII, followed by treatment of the initial product with 
N,N'-carbonyldiimidazole and N-hydroxysuccinimide to give the product. 
##STR131## 
Hydrazine is reacted with the anhydride of an electrophoric carboxylic 
acid SgCO.sub.2 H such as those shown in Table XIII. The initially-formed 
product is then reacted successively with succinic anhydride, acidic 
methanol, and hydrazine, followed by NaNO.sub.2 in aqueous acid to give 
the product. 
##STR132## 
2-Hydroxyisobutyric acid (Aldrich) is reacted with the acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, in the presence of pyridine, and the resulting substituted 
carboxylic acid is then reacted with the water-soluble carbodiimide 
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to give the product. 
##STR133## 
An acid chloride of an electrophoric 2-oxo-carboxylic acid SgCOCO.sub.2 H 
(prepared as above in connection with compound 4) is reacted in the 
presence of pyridine with 2-hydroxyisobutyric acid, and the resulting 
substituted carboxylic acid is subsequently reacted with 
N,N'-carbonyldiimidazole and N-hydroxysuccinimide to give the product. 
##STR134## 
An acid chloride of an electrophoric 2-oxo-carboxylic acid SgCOCO.sub.2 H 
(prepared as above in connection with compound 4) is reacted with 
p-aminobenzoic acid, and the intermediate substituted carboxylic acid is 
then further reacted with N,N'-carbonyldiimidazole and 
N-hydroxysuccinimide to give the product. 
##STR135## 
An acid chloride of an electrophoric 2-oxo-carboxylic acid SgCOCO.sub.2 H 
(prepared as above in connection with compound 4) is reacted with 
hydrazine, and the resulting substituted hydrazine is further reacted with 
succinic anhydride and then with N,N'-dicyclohexylcarbodiimide and 
N-hydroxybenzotriazole to give the product. 
##STR136## 
An acid chloride of an electrophoric 2-oxo-carboxylic acid SgCOCO.sub.2 H 
(prepared as above in connection with compound 4) is reacted with 
carboxymethoxylamine, and the intermediate substituted carboxylic acid is 
further reacted with p-nitrophenol in the presence of 
N,N'-dicyclohexylcarbodiimide to give the product. 
##STR137## 
An acid chloride of an electrophoric carboxylic acid SgCO.sub.2 H such as 
those shown in Table XIII is reacted in the presence of pyridine with 
N-acetylhydroxyproline (Sigma), then the intermediate substituted 
carboxylic acid is further reacted with N,N'-dicyclohexylcarbodiimide and 
N-hydroxysuccinimide to give the product. 
##STR138## 
An anhydride of an electrophoric carboxylic acid SgCO.sub.2 H such as 
those shown in Table XIII is reacted with hydroxyproline (Sigma), and the 
intermediate substituted carboxylic acid is then further reacted with 
N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR139## 
One equivalent of an anhydride of an electrophoric carboxylic acid 
SgCO.sub.2 H such as those shown in Table XIII is reacted with hydrazine, 
then the intermediate substituted hydrazine is further reacted with 
succinic anhydride, and the intermediate substituted carboxylic acid is 
finally reacted with isobutyl chloroformate to give the product. 
##STR140## 
One equivalent of an anhydride of an electrophoric carboxylic acid 
SgCO.sub.2 H such as those shown in Table XIII is reacted with 
1,4-diaminopiperazine, then the substituted aminopiperazine is further 
reacted with succinic anhydride, and finally the substituted carboxylic 
acid reacted with isobutyl chloroformate to give the product. 
##STR141## 
Tris-(hydroxymethyl)aminomethane (Aldrich) is reacted with succinic 
anhydride, the intermediate polyol is reacted with an acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, and finally, the carboxylic acid is reacted with 
N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR142## 
Gluconic acid lactone (Sigma) is reacted with hydrazine, the intermediate 
product is reacted with succinic anhydride, the hydroxyl functionalities 
are reacted in the presence of pyridine with an acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, and finally, the substituted carboxylic acid is reacted with 
p-nitrophenol and N,N'-dicyclohexylcarbodiimide to produce the product. 
##STR143## 
p-Hydroxybenzoic acid (Aldrich) is reacted in the presence of pyridine 
with an acid chloride of an electrophoric carboxylic acid SgCO.sub.2 H 
such as those shown in Table XIII, then the substituted carboxylic acid is 
further reacted with N,N'-dicyclohexylcarbodiimide and 
N-hydroxysuccinimide to give the product. 
##STR144## 
6-Aminonicotinic acid (Aldrich) is reacted with an anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, then the resulting substituted carboxylic acid is further reacted 
with N,N'-dicyclohexylcarbodiimide and N-hydroxybenzotriazole to yield the 
product. 
##STR145## 
4-Hydroxyphenylacetic acid is reacted in the presence of pyridine with an 
acid chloride of an electrophoric carboxylic acid SgCO.sub.2 H such as 
those shown in Table XIII, then the intermediate substituted carboxylic 
acid is further reacted with N,N'-dicyclohexylcarbodiimide and 
N-hydroxybenzotriazole to yield the product. 
##STR146## 
4-Hydroxyphenyl propionic acid (Aldrich) is treated as described above for 
the preparation of compound 64, to produce the product. 
##STR147## 
3-Amino-4-hydroxybenzoic acid (Aldrich) is reacted with an anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, then the intermediate substituted carboxylic acid is further reacted 
with N,N'-carbonyldiimidazole and N-hydroxysuccinimide to give the 
product. 
##STR148## 
Serine (Sigma) is reacted with an anhydride of an electrophoric carboxylic 
acid SgCO.sub.2 H such as those shown in Table XIII, then the intermediate 
substituted carboxylic acid is further reacted with 
N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR149## 
Cysteic acid (Sigma) is reacted as described above for the preparation of 
compound 67, to yield the product. 
##STR150## 
Histidine (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII, then the 
intermediate substituted carboxylic acid is further reacted with the 
water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
to give the product. 
##STR151## 
Histidylglycine (Sigma) is reacted with the reagents employed in the 
preparation of compound 69 above, to give the product. 
##STR152## 
Asparagine (Sigma) is reacted with an anhydride of an electrophoric 
carbozylic acid SgCO.sub.2 H such as those shown in Table XIII, and the 
intermediate substituted carboxylic acid is further reacted with 
N,N'-carbonyldiimidazole and N-hydroxysuccinimide to give the product. An 
analog may be made using glutamine instead of asparagine. 
##STR153## 
4-Amino-2-nitrophenol (Aldrich) is reacted with an anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, the intermediate substituted phenol is further reacted with 
iodoacetate, then the substituted carboxylic acid is finally reacted with 
N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR154## 
Sulfanilamide (Aldrich) is reacted with succinic anhydride, the 
intermediate carboxylic acid is then reacted at the sulfanilamide nitrogen 
with an acid chloride of an electrophoric carboxylic acid SgCO.sub.2 H 
such as those shown in Table XIII, and the resulting substituted 
carboxylic acid is finally reacted with N,N'-dicyclohexylcarbodiimide and 
N-hydroxysuccinimide to give the product. 
##STR155## 
N.sup..alpha. -acetylasparagine (Sigma) is reacted with an acid chloride 
of an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in 
Table XIII, then the substituted carboxylic acid is further reacted with 
N,N'-carbonyldiimidazole and N-hydroxysuccinimide to give the product. An 
analog may be made using N.sup..alpha. -acetylglutamine instead of 
N.sup..alpha. -acetylasparagine. 
##STR156## 
Glycylglycine (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII, then the 
intermediate substituted carboxylic acid is further reacted with 
N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR157## 
Cytosine (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII, the 
resulting derivative is reacted with iodoacetate, and the substituted 
carboxylic acid produced in that reaction is further treated with 
N,N'-carbonyldiimidazole and N-hydroxysuccinimide to give the product. 
##STR158## 
The material shown as compound 57 above is reacted with ethylenediamine to 
give the product. 
##STR159## 
The compound shown as number 71 above is reacted with hydrazine and the 
resulting intermediate material is further reacted with 
1,4-phenylenediisothiocyanate to give the product. 
##STR160## 
The material shown as compound 55 above is reacted with methane thiol 
(Aldrich) to give the product. 
##STR161## 
The material shown as compound 58 above is reacted with hydrazine and the 
resulting product is further reacted with dimethylsuberimidate (Aldrich) 
to give the product. 
##STR162## 
The material shown as compound 50 above is reacted with 3-aminopropanol 
(Aldrich) and the resulting alcohol is further reacted with tresyl 
chloride to give the product. 
##STR163## 
The material shown as compound 68 above is reacted with hydrazine, then 
the intermediate substituted hydrazine is further reacted with 
succinimidyl-3-(2-pyridyldithio)propionate (Sigma) to give the product. 
##STR164## 
The material shown as compound 82 above is reacted with dithiothreitol to 
give the product. 
##STR165## 
The material shown as compound 53 above is reacted with sulfanilic acid 
and the resulting intermediate product is further with thionyl chloride to 
give the product. 
##STR166## 
Carboxymethoxylamine (Aldrich) is reacted with trifluoroacetic anhydride 
to give N-(trifluoroacetyl)carboxylamine. The material shown as compound 
75 above is reacted with hydrazine, then with the 
N-(trifluoroacetyl)carboxylamine in the presence of the water-soluble 
carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide followed by 
hydrolysis with aqueous sodium hydroxide to remove the trifluoroacetyl 
group and give the product. 
##STR167## 
The material shown as compound 67 above is reacted with hydrazine, then 
the resulting substituted hydrazine is further reacted with epibromohydrin 
(Aldrich) to give the product. 
##STR168## 
4-Fluoro-3-nitrophenylazide is synthesized as described (Forster, Ibid.), 
and is then reacted with the material shown as compound 27 above to form 
the product. Subsequent photolysis as described (Forster, Ibid.) yields 
the corresponding nitrophenyl nitrene. 
##STR169## 
One equivalent of an anhydride of an electrophoric carboxylic acid 
SgCO.sub.2 H such as those shown in Table XIII is reacted with hydrazine, 
then the resulting substituted hydrazine is further reacted with succinic 
anhydride and the resulting substituted carboxylic acid is finally reacted 
with N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR170## 
2-Hydroxyisobutyric acid (Aldrich) is reacted with an acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, then the resulting substituted carboxylic acid is further reacted 
with N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR171## 
The anhydride of an electrophoric carboxylic acid SgCO.sub.2 H such as 
those shown in Table XIII is reacted with ethanolamine and the resulting 
substituted alcohol is further reacted with N,N'-carbonyldiimidazole to 
give the product. 
##STR172## 
The anhydride of an electrophoric carboxylic acid SgCO.sub.2 H such as 
those shown in Table XIII is reacted with hydrazine and the resulting 
substituted hydrazine is further reacted with N,N'-carbonyldiimidazole to 
give the product. 
##STR173## 
The anhydride of an electrophoric carboxylic acid SGCO.sub.2 H such as 
those shown in Table XIII is reacted with hydrazine, and the resulting 
substituted hydrazine is further reacted with disuccinimidyl carbonate to 
give the product. 
##STR174## 
2,3-Diaminopropionic acid (Aldrich) is reacted with one equivalent of an 
anhydride of an electrophoric carboxylic acid SgCO.sub.2 H such as those 
shown in Table XIII, yielding a mixture of N2-SgCO-2,3-diaminopropionic 
acid and N3-SgCO-2,3-diaminopropionic acid. The latter compound is 
isolated and reacted with phosgene to give the product. 
##STR175## 
An anhydride of an electrophoric carboxylic acid SgCO.sub.2 H such as 
those shown in Table XIII is reacted with carbohydrazide to give the 
product. 
##STR176## 
Homocysteine thiolactone is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII, the 
resulting carboxylic acid is further reacted with ammonia, and the 
resulting thiol-containing amide is finally reacted with 
2,2'-dipyridyldisulfide to give the product. 
##STR177## 
Glucosamine (Sigma) is reacted with sodium borohydride and the resulting 
intermediate product is further reacted with succinic anhydride. The 
substituted carboxylic acid resulting from this reaction is reacted in 
turn, in the presence of pyridine, with an acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, and then with N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide 
to give the desired product. 
##STR178## 
Glucose (Sigma) is reacted with ammonia and sodium borohydride and the 
resulting amine is then reacted with succinic anhydride to form a 
substituted cayboxylic acid. This is reacted in turn, in the presence of 
pyridine, with an acid chloride of an electrophoric carboxylic acid 
SgCO.sub.2 H such as those shown in Table XIII, and then further reacted 
with N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. For this reaction glucamine can also be obtained from Hiils 
America. 
##STR179## 
Glucosamine (Sigma) is reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
product. 
EQU (SgCO).sub.s Dextran hydrazide (99) 
Dextran hydrazide is partly reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
product. 
EQU (SgCO).sub.s Dextran hydrazide-succinyl-EDAC (100) 
The material shown as compound 99 above is reacted with succinic anhydride 
and the resulting succinyl derivative is further reacted with the 
water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
to give the product. 
EQU (SgCO).sub.s Dextran-carbonyl imidazole (101) 
Dextran is reacted in the presence of pyridine with an acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, then the resulting dextran derivative is further reacted with 
N,N'-carbonyldiimidazole to give the product. 
EQU (SgCO).sub.s Dextran aldehyde (102) 
Dextran is reacted in the presence of pyridine with an acid chloride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, then the resulting dextran derivative is treated with aqueous 
periodate to give the product. 
EQU (SgCO).sub.s Poly(ser)-EDAC (103) 
Poly(ser) is reacted in the presence of pyridine with an acid chloride of 
an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, then the intermediate polymer derivative is further reacted with the 
water-soluble carbodiimide 1-ethyl-3-(3-dimethylamino- propyl)carbodiimide 
to give the product. 
EQU N.sup..alpha. -acetyl-(SgCO).sub.s Poly(ser)-DAO-succinyl-EDAC(104) 
Poly(ser) is reacted with acetic anhydride followed by reaction in the 
presence of pyridine with an acid chloride of an electrophoric carboxylic 
acid SgCO.sub.2 H such as those shown in Table XIII. The resulting polymer 
derivative is then treated with 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and 
1,8-diaminooctane. The resulting product is further treated with succinic 
anhydride followed by EDAC to give the product. 
EQU (SgCO).sub.s Poly(C)-hydrazide (105) 
Poly(C)-hydrazide is partly reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII to give the 
product. 
EQU (SgCO).sub.s Poly(C)-hydrazide-succinyl-EDAC (106) 
The material shown as compound 105 above is reacted further with acetic 
anhydride and then with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 
give the product. 
EQU (SgCO).sub.s poly(gly)-EDAC (107) 
Poly(gly) is reacted in the presence of pyridine with an acid chloride of 
an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, then further reacted with 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to give the product. 
EQU N.sup..alpha. -Acetyl-(SgCO).sub.s Poly(gly)-DAO (108) 
Poly(gly) is reacted first with acetic anhydride and then further reacted 
in the presence of pyridine with an acid chloride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII, and 
finally reacted further with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
and 1,8-diaminooctane to give the product. 
EQU (SgCO).sub.s Poly(asp)-hydrazide (109) 
Poly(asp)-hydrazide is partly reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII, to give 
the product. 
EQU (SgCO).sub.s Poly(asp)-hydrazide-succinyl-EDAC (110) 
The material shown as compound 109 above is further reacted with succinic 
anhydride and then with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
(EDAC), to give the product. 
EQU (SgCO).sub.s Glycol chitosan (111) 
Glycol chitosan is partially reacted with the anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII, to give 
the product. 
EQU (SgCO).sub.s Glycol chitosan-succinyl-EDAC (112) 
The material identified as compound 111 above is further reacted with 
succinic anhydride and then with 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), to give the product. 
EQU (SgCO).sub.s Poly(acrylamide)-ED-succinyl-EDAC (113) 
Poly(acrylamide)-ED is partly reacted with an anhydride of an electrophoric 
carboxylic acid SgCO.sub.2 H such as those shown in Table XIII, and then 
further reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), 
to give the product. 
EQU (SgCO).sub.s Poly(asn)-EDAC (114) 
Poly(asn) is reacted in the presence of pyridine with an acid chloride of 
an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, then the product of this reaction is further treated with 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), to give the product. 
EQU (SgCO).sub.s Poly(asn)-DAO (115) 
Poly(asn) is reacted in the presence of pyridine with an acid chloride of 
an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, and then reacted further with 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and 1,8-diaminooctane 
(DAO), to give the product. 
EQU (SgCO.sub.s Poly(asn)-DAO-succinyl-EDAC (116) 
The material shown as compound 115 above is reacted further with succinic 
anhydride and then finally with 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) to give the product. 
##STR180## 
Tris-(hydroxymethyl)aminomethane (Sigma) is reacted with an anhydride of 
an electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, and then treated with a solution of barium hydroxide. The product of 
this reaction is further treated with methylchloroacetate and sodium 
hydride, then treated with aqueous sodium hydroxide, and finally reacted 
with N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR181## 
3,5-Diaminobenzoic acid (Aldrich) is reacted with the anhydride of an 
electrophoric carboxylic acid SgCO.sub.2 H such as those shown in Table 
XIII, and the resulting material is further reacted with 
N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the 
product. 
##STR182## 
This material is prepared in the same way as compound 118 above except 
that 3,4-diaminobenzoic acid (Aldrich) is employed as a starting material. 
A release tag-labeled antibody is prepared by reacting an antibody with a 
release tag reagent having a reactive functional group capable of 
covalently attaching to the antibody. Examples of release tags which are 
suitable for conjugation with an antibody are: CCl.sub.3 CO-AB-NHS, 
CCl.sub.3 CO-MAB-NHS, CCl.sub.2 CClCO-AB-NHS, CCl.sub.3 -diol-gly-NHS, and 
SgCO-CHOH-NHS, where AB stands for p-aminobenzoic acid, MAB stands for 
N-methyl-p-aminobenzoic acid, and NHS stands for N-hydroxysuccinimide. 
Also included among the exemplary release tags are the compounds, numbered 
as 46, 48-76, 78-80, 86-93, 96, 97, 100, 101, 103, 104, 106, 107, 110, 
112, 113, 114, 116, and 117, where the numbers refer to the release tags 
shown in the above listing of exemplary release tag compounds. This 
grouping of release tag compounds which are suitable for labeling 
antibodies is referred to as Group I in the discussion below. 
A release tag-labeled antibody can also be prepared by reacting an antibody 
in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) 
with one of the following release tags: 27, 77, 85, 94, 99, 105, 108, 109, 
111, and 115, where these numbers refer to the above-listed exemplary 
release tag compounds. The above-identified set of release tag compounds 
which are suitable for the reaction with an antibody in the presence of 
EDAC are defined as Group II for purposes of the discussion to follow. In 
addition, a release tag-labeled antibody can be prepared by reacting an 
antibody with exemplary release tag compounds 98 or 102, defined as Group 
III, in the presence of NaCNBH.sub.3 or NaBH.sub.4. 
A release tag-labeled antibody can also be prepared by reacting the 
antibody with aqueous periodate and then with a release tag from Group II 
in the presence of NaCNBH.sub.3 or NaBH.sub.4. 
A release tag-labeled DNA can be prepared by preparing an aminoalkyl DNA as 
described (Ehrat, M., Cecchini, D. and Giese, R. W., J. Chromatogr. 326, 
1985, pp. 311-320) and subsequently reacting this with a release tag from 
Groups I, II, or III as defined above. 
A release tag-labeled DNA can also be prepared by reacting the DNA in the 
presence of NaHSO.sub.3 as described (Ehrat, Ibid.) with a release tag 
from Group II. 
A release tag-labeled avidin, streptavidin, protein A, protein G, or lectin 
can be prepared by reacting it as above with a release tag compound from 
Group I. 
##STR183## 
An .alpha.,.beta.-unsaturated carboxylic acid bearing a .beta.-Sg 
substituent is prepared in any of the ways known to the art, then the 
double bond is oxidized to the diol by reaction with osmium tetroxide in 
the presence of pyridine and THF. This oxidation can also be accomplished 
by reacting the olefinic carboxylic acid with performic acid as described 
by Wagner and Zook, Synthetic Organic Chemistry, Wiley Interscience, N.Y., 
pp. 179-180, 1953. The .alpha.,.beta.-dihydroxy carboxylic acid is then 
reacted with glycine methyl ester in the presence of 
N,N'-dicyclohexylcarbodiimide, the ester functionality is saponified by 
treatment with base, and finally, the resulting carboxylate is reacted 
with N-hydroxysuccinimide in the presence of DCC to yield the desired 
product. For the particular case in which the group Sg is Cl.sub.3 --, the 
starting Sg-substituted .alpha.,.beta.-unsaturated carboxylic acid can be 
produced from 3-hydroxy-4,4,4-trichlorobutyric-.beta.-lactone (Aldrich) by 
acid hydrolysis to effect dehydration as described by Wagner and Zook, 
Ibid., p. 50. Alternatively, one can start with this same lactone, and 
brominate it to yield the .alpha.-bromolactone as described by House in 
"Modern Synthetic Reactions" 2nd Ed., W. A. Benjamin, Menlo Park, Calif., 
pp. 476-478 or 459 and 473, 1972. Upon hydrolysis as described by Wagner 
and Zook, Ibid., pp. 170-171, the .alpha.,.beta.-dihydroxy carboxylic acid 
is produced, and this is in turn reacted further with glycine methyl ester 
and N-hydroxysuccinimide to produce the product as described above. 
EQU Sg-diol-CO-gly-BSA (121) 
BSA is reacted with the material shown above as compound 120 to form the 
product. Upon oxidation with aqueous permanganate-periodate as described 
by House, Ibid., p. 278, the diol is cleaved and SgH is released. 
##STR184## 
This .alpha.-hydroxy ketone is prepared by any of the following three 
procedures: 
(a) Acetic acid is reacted with two equivalents of NaH, the product is 
treated with an anhydride of an electrophoric carboxylic acid SgCO.sub.2 H 
such as those shown in Table XIII, the product of this step is further 
treated with bromine and sodium carbonate, then treated with sodium 
hydroxide, reacidified, and finally subjected to N-hydroxysuccinimide in 
the presence of N,N'-dicyclohexylcarbodiimide to give the desired product. 
(b) The acid chloride of an electrophoric carboxylic acid SgCO.sub.2 H such 
as those shown above in Table XIII is reacted with methanol to give 
SgCO.sub.2 CH.sub.3. Methyl acetate is reacted with sodium methoxide and 
the resulting carbanion is reacted with the SgCO.sub.2 CH.sub.3 to yield 
SgCOCH.sub.2 CO.sub.2 CH.sub.3. This is brominated in the presence of 
sodium bicarbonate, the product is saponified, then reacidified, and 
finally, the resulting acid is treated with N-hydroxysuccinimide in the 
presence of N,N'-dicyclohexylcarbodiimide to yield the product. 
(c) Sg-CO-CHO is reacted with HCN, the mixture is acidified, and the 
resulting carboxylic acid is treated with N-hydroxysuccinimide in the 
presence of N,N'-dicyclohexylcarbodiimide to give the product. 
##STR185## 
The material shown above as compound 7 is reacted with 
dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the product. 
##STR186## 
The material shown above as compound 13 is reacted with 
dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the product. 
##STR187## 
The material shown above as compound 15 is reacted with 
dicyclohexylcarbodiimide and N-hydroxysuccinimide to give the product. 
Other embodiments of the invention will be apparent to those skilled in the 
art from a consideration of this specification or practice of the 
invention disclosed herein. It is intended that the specification and 
examples be considered as exemplary only, with the true scope and spirit 
of the invention being indicated by the following claims.