Title: Nucleic acid sequence identification
Abstract: Disclosed are methods for determining the presence or absence of a target nucleic acid (e.g. DNA) sequence in a sample nucleic acid, the method comprising: (a) exposing the sample to a detection agent comprising a colloid metal surface associated with a SER(R)S active species (SAS) such as an azo dye and with a target binding species (TBS) which may be PNA which is complementary to the target, and (b) observing the sample agent mixture using SER(R)S to detect any surface enhancement of the label wherein the binding of the TBS to the target sequence causes surface enhancement SAS. The detection agent may be exposed to the sample in step (a) as two or more separate components and will generally comprise a first agent and a second agent each having a different TBS, each TBS being capable of binding to the target sequence, and wherein the binding of the first and second TBS to the target sequence brings a metal surface associated with each TBS into proximity thereby causing surface enhancement of an SAS associated with one or both of the metal surfaces.
Patent Number: 7,001,721 Issued on 02/21/2006 to Whitcombe, et al.
| Inventors:
|
Whitcombe; David Mark (Northwich, GB);
Graham; Duncan (Glasgow, GB);
Smith; William Ewen (Glasgow, GB)
|
| Assignee:
|
University of Strathclyde (Glasgow, GB)
|
| Appl. No.:
|
700732 |
| Filed:
|
May 20, 1999 |
| PCT Filed:
|
May 20, 1999
|
| PCT NO:
|
PCT/GB99/01597
|
| 371 Date:
|
March 19, 2001
|
| 102(e) Date:
|
March 19, 2001
|
| PCT PUB.NO.:
|
WO99/60157 |
| PCT PUB. Date:
|
November 25, 1999 |
Foreign Application Priority Data
| Current U.S. Class: |
435/6; 435/320.1; 435/252.8; 435/174; 435/183; 382/129; 382/133; 382/153; 382/173; 382/286; 382/291; 702/19; 702/22; 935/10; 935/24; 935/72; 536/22.1 |
| Current Intern'l Class: |
C12Q 1/68 (20060101); C12N 15/00 (20060101); C12N 15/63 (20060101); C12N 1/20 (20060101); C07H 21/04 (20060101) |
| Field of Search: |
435/6,911,912,5
536/243
935/6,77,78
436/518,164,173,525,801,805
|
References Cited [Referenced By]
U.S. Patent Documents
| 5266498 | Nov., 1993 | Tarcha.
| |
| 5306403 | Apr., 1994 | Vo-Dinh.
| |
| 5721102 | Feb., 1998 | Vo-Dinh.
| |
| 6127120 | Oct., 2000 | Graham.
| |
| Foreign Patent Documents |
| 0 667 398 | Aug., 1995 | EP.
| |
| 0 838 528 | Apr., 1998 | EP.
| |
| WO 96/4118/1 | Dec., 1996 | WO.
| |
| WO 97/0528/0 | Feb., 1997 | WO.
| |
Other References
Kneipp, K. et al., "Surface Enhanced Raman Scattering (SERS) of nucleic Acids
Adsorbed on Colloidal Silver Particles"; Journal of Molecular Structure, 145: 173-179
(1986) [Abstract].
Munro, C.H. et al., "Qualitative and Semi-quantitative Trace Analysis of Acidic
Monoazo Dyes by Surface Enhanced Resonance Raman Scattering"; Analyst, 120: 993-1003 (1995).
Helmenstine, A. et al., "Measurement of DNA Adducts Using Surface-Enhanced Raman
Spectroscopy"; Journal of Toxicology and Environmental Health, 40: 195-202 (1993).
Mirkin, C.A. et al., "A DNA-based method for rationally assembling nanoparticles
Into macroscopic materials"; Nature, 382: 607-609 (1996).
Storhoff, J.J. et al., "One Pot Colormetric Differentiation of Polynucleotides
with Single Base Imperfections Using Gold Nanoparticle Probes"; J. Am. Chem. Soc.,
120: 1959-1964 (1998) [Abstract].
Bethell, D. et al., "Nanotechnology and nucleotides"; Nature, 382: 581 (1996).
Alivisatos, A.P. et al., "Organization of 'nanocrystal molecules' using DNA";
Nature, 382: 609-611 (1996).
Munro, C.H. et al., "Characterization of the Surface of a Citrate-Reduced Colloid
Optimized for Use as a Substrate for Surface-Enhanced Resonance Raman Scattering";
Langmuir, 11: 3712-3720 (1995).
Bertoluzza, A. et al., "Raman and Infrared Spectra of Spermidine and Spermine
and their Hydrochlorides and Phosphates as a Basis for the Study of the Interactions
Between Polyamines and Nucleic Acids"; Journal of Raman Spectroscopy, 14(6): 386-394 (1983).
Cotton, T.M., "Application of Surface-Enhanced Raman Spectroscopy to Biological
Systems"; Journal of Raman Spectrocopy, 22: 729-742 (1991).
Egholm, M., "Spectrometry senses more than a small difference"; Nature Biotechnology,
15: 1346 (1997).
Graham, D. et al., "Selective Detection of Deoxyribonucleic Acid at Ultralow
Concentrations by SERRS"; Analytical Chemistry, 69(22): 4703-4707 (1997).
Rodger, C. et al., "Surface-enhanced resonance-Raman scattering: an informative
probe of surfaces"; J. Chem. Soc. Dalton Trans., 791-799 (1996).
|
Primary Examiner: Fredman; Jeffrey
Assistant Examiner: Chakrabarti; Arun
Attorney, Agent or Firm: Dann, Dorfman, Herr & Skillman, P.C.
Claims
What is claimed is:
1. A method for determining the presence or absence of a target nucleic acid
sequence in a sample nucleic acid, the method comprising:
(a) exposing the sample to a detection agent comprising at least two separate
components, including a first agent having a metal surface associated with a first
target binding species (TBS) and a second agent having a metal surface associated
with a second TBS, different from said first TBS, at least one of said metal surfaces
being associated with a SER(R)S-active species (SAS), each of said first and second
TBS being effective to bind to the target sequence, and wherein the binding of
the first and second TBS to the target sequence causes aggregation of the metal
surfaces associated with said TBS, thereby causing surface enhancement of a SAS
associated with one or both of the metal surfaces, said metal surfaces being ineffective
to cause surface enhancement in the form in which they are present in the detection
agent to which said sample is exposed, and aggregation of said metal SER(R)S surface
being dependent on the presence of said target nucleic acid in said sample; and,
(b) observing the sample/agent mixture using SER(R)S to detect any said surface
enhancement.
2. The method as claimed in claim 1 wherein each component of said detection
agent comprises monodisperse unaggregated colloidal metal particles associated
with a TBS comprising a nucleic acid or nucleic acid analog which is complementary
to all or part of the target sequence.
3. The method as claimed in claim 2 wherein the TBS comprises propargyl amino
modified nucleic acid or peptide nucleic acid.
4. The method as claimed in claim 2 wherein there are up to 20 TBS per metal
colloid particle.
5. The method as claimed in claim 1 wherein a surface seeking group (SSG) is
used to promote chemisorption of at least one of the SAS and TBS to the metal surface.
6. The method as claimed in claim 5 wherein the SSG comprises a triazole group.
7. The method as claimed in claim 5 wherein the SSG is modified with a dye which
is a SAS.
8. The method as claimed in claim 7 wherein the modified SSG is an azobenzotriazole.
9. The method as claimed in claim 7 wherein the modified SSG is used to associate
the TBS to the metal surface.
10. The method as claimed in claim 9 wherein the modified SSG is conjugated to
the TBS via a linker group.
11. The method as claimed in claim 1 wherein the SAS is present in an amount
of up to 100 fold excess over the TBS.
12. A method as claimed in claim 1 wherein more than one target sequence is determined
using detection agent components having distinguishable SAS.
13. A method as claimed in claim 12 wherein the target sequences share sequence
identity, and wherein a common first agent is used in conjunction with specific
distinguishable second agents which can discriminate between the remainder of the
target sequences.
14. A method for detecting the presence of, or selecting, or identifying, or
phylogenetically classifying, an organism,, the method comprising use of a method
as claimed in claim 1 wherein the target nucleic acid sequence is associated with
that organism.
15. A method for diagnosing a disease, the method comprising use of a method
as claimed in claim 1 wherein the target nucleic acid sequence is associated with
that disease.
16. A method for isolating a nucleic acid encoding a specific gene, the method
comprising use of a method as claimed in claim 1 wherein the target sequence corresponds
to a sequence associated with, or within, that gene.
17. The method as claimed in claim 6 wherein said triazole group is the benzotriazole group.
Description
TECHNICAL FIELD
The present invention relates to methods and materials for detecting or identifying
particular nucleic acid sequences in a sample using surface enhanced resonance
Raman scattering ('SERRS').
PRIOR ART
There is currently a great demand for technologies which can detect either
specific sequences, or single point mutations or polymorphisms in a targeted sequence
of nucleic acid from a particular source. Information derived from such methods
can be used in numerous aspects of genetic investigation.
Thus the identification of particular target sequences may be used in diagnosis
and detection of particular agents containing that sequence (e.g. invasive pathogens
such as a viruses) or to isolate larger sequences containing the target sequence.
The detection of nucleic acid variants is used in evolutionary and population
structure studies, forensics, and the analysis and diagnosis of genetic disease.
Variations in DNA sequence between individuals may be used to identify or isolate
genes or subsequences associated with particular traits, for instance disease traits
within organisms of interest such as humans.
Some current methodologies used for detecting (scanning or scoring) nucleic
acid variants are reviewed by Schafer & Hawkins (1998) Nature Biotechnology 16: 33-39.
These methods include single strand conformation polymorphism analysis (SSCP),
heteroduplex analysis (HA), denaturing gradient gel electrophoresis (DGGE), duplex
cleavage (e.g. using RNase, chemistry, or endonucleases). Scoring methods include
minisequencing, nuclease assays or standard Sanger sequencing.
Most of these methods rely on the specific binding of probe or primer to the
targeted sequence, followed by the detection of the binding event (e.g. by stability,
mobility, or the presence of a label). Owing to the sensitivity of the detection
methods, amplification of the sample (e.g. by PCR) is usually required before hybridisation.
This is undesirable because of the possibility that errors may occur during the
amplification process, leading to false positive or negative results.
A particularly sensitive method for identifying labelled nucleic acids is disclosed
by Graham et al (1997) Anal Chem 69: 4703-4707. This relies on the use of surface
enhanced resonance Raman scattering (SERRS), which is in turn a development of
surface enhanced Raman scattering (SERS).
Briefly, a Raman spectrum arises because light incident on an analyte is
scattered due to excitation of electrons in the analyte. "Raman" scattering occurs
when an excited electron returns to an energy level other than that from which
it came—this results in a change in wavelength of the scattered light and
gives rise to a series of spectral lines at both higher and lower frequencies than
that of the incident light. The scattered light can be detected orthogonally to
the incident beam.
Normal Raman lines are relatively weak and Raman spectroscopy is therefore
too insensitive, relative to other available detection methods, to be of use in
chemical analysis. Raman spectroscopy is also unsuccessful for fluorescent materials,
for which the broad fluorescence emission bands (also detected orthogonally to
the incident light) tend to swamp the weaker Raman emissions.
However, a modified form of Raman spectroscopy, based on SERS, has proved
to be more sensitive and hence of more general use. The analyte whose spectrum
is being recorded is closely associated with a roughened metal surface. This leads
to a large increase in detection sensitivity, the effect being more marked the
closer the analyte sits to the "active" surface (the optimum position is in the
first molecular layer around the surface, i.e., within about 2 nm of the surface).
The theory of this surface enhancement is not yet fully understood, but it is
thought that the higher valence electrons of the analyte associate with pools of
electrons (known as "plasmons") in pits on the metal surface. When incident light
excites the analyte electrons, the effect is transferred to the plasmons, which
are much larger than the electron cloud surrounding the analyte, and this acts
to enhance the output signal.
A further increase in sensitivity can be obtained by operating at the resonance
frequency of the analyte (in this case usually a dye attached to the target of
interest). Use of a coherent light source, tuned to the absorbance maximum of the
dye, gives rise to a 10
3-10
5-fold increase in sensitivity.
This is termed "resonance Raman scattering" spectroscopy. In certain embodiments
the laser excitation may be set to the maximum of the plasmon resonance. In certain
cases the plasmon resonance and dye maxima may coincide.
When the surface enhancement effect and the resonance effect are combined, to
give SERRS, the resultant increase in sensitivity and robustness is more than additive.
Moreover, the sensitivity does not seem to depend so critically on the angle of
orientation of the analyte to the surface, as is the case with SERS alone. A SERRS
signal can be more easily discriminated from contamination and background and tends
to be less variable with local conditions (e.g., ionic strength or pH when an analysis
is carried out in solution). Fluorescence is also quenched, giving cleaner Raman
spectra and allowing fluorescent dyes to be used as detectable analytes. Generally,
the signal enhancement means that a much larger range of analytes may be usefully
detected than using normal Raman spectroscopy. Furthermore, the enhancement means
that a less powerful light source is required to excite the analyte molecules.
With SERRS, detection limits down to one molecule have been achieved for compounds
which absorb light in the visible wavelength region or the electromagnetic spectrum
(see Emory & Nie (1997) "Near-Field Surface-Enhanced Raman Spectroscopy on Single
Silver Nanoparticles", Anal. Chem. 69: 2631-2635). This technique is therefore
more sensitive than fluorescence (see eg, C Rodger et al,
J. Chem. Soc. Dalton
Trans. (1996), pp791-799) and furthermore, the SERRS spectra obtained contain
molecular information which permit compound identification and discrimination.
WO 97/05280 (University of Strathclyde) discloses practical demonstrations of
the use of SE(R)RS in nucleic acid detection and sequencing. The methods disclosed
therein are based generally around the use of a labelled targetting species which
binds to a target species if present to form a complex. The complex is then associated
with a SER(R)S surface, and is detected using suitable equipment.
U.S. Pat. No. 5,721,102 (Vo Dinh et al) describes a labelled SER gene probe
which is used to hybridise to (and label) complementary sequences. Non-hybridised
material is separated from the hybridised material, and the hybridised material
is analysed.
It will be clear from the above that novel formats for detecting or identifying
particular nucleic acid sequences in a sample, particular those which have one
or more advantages over those in current use, would provide a contribution to the art.
DISCLOSURE OF THE INVENTION
The present inventors have devised a novel SERS/SERRS based method for detecting
or identifying a particular nucleic acid sequence in a sample. Not only does it
not require amplification of the sample prior to detection, but in preferred formats
the method can be carried out using simple, one pot, mixing procedures to provide
a rapid, highly-sensitive, detection of target sequences without any requirement
to separate unbound labelled targeting agent from labelled target complexes. This
is achieved by making the functionality of the SER(R)S surface dependent on the
presence of the target sample. Thus, unlike in certain existing techniques based
on labelled probes, unbound labelled target will not generate a false result if
present during detection.
The present invention may be used in either a SERS or SERRS format, and the abbreviation
SER(R)S is used hereinafter to demonstrate this. Generally speaking, owing to its
sensitivity advantages, SERRS will be preferred.
In known nucleic acid detection formats, such as those described in WO 97/05280
(University of Strathclyde), metal colloid which has been carefully aggregated
in a controlled manner is added to labelled target complex prior to detection.
In the present invention, the aggregation of colloidal SER(R)S surface is actually
dependent on the presence of target sequence, with the attendant advantages described above.
Thus in a first aspect of the present invention there is disclosed a method
for determining the presence or absence of a target nucleic acid sequence in a
sample nucleic acid, the method comprising:
(a) exposing the sample to a detection agent comprising a metal surface
associated with a SER(R)S active species (SAS) and with a target binding species (TBS),
(b) observing the sample/agent mixture to detect any surface enhancement
of the label.
The method is characterised in that it is the binding of the TBS to the target
sequence which itself causes surface enhancement of the SAS.
Equally the method differs from those in the art in that the metal surface,
in the form in which it is present in the added agent, is not itself capable of
surface enhancement. Thus any unbound detection agent present in the system following
exposure of the sample to the agent need not be removed prior to the observation
step. Thus, given that the detection agent will generally be present at great excess
over the target material, unbound agent will be present in the system during detection,
but owing to the nature of the method, will not interfere with the result. The
method is therefore a true "one pot" detection system.
The result of the observation is correlated with the presence or absence of the
target sequence, optionally by comparison with reference data.
The method is particularly susceptible to giving rapid information about whether
a known, or at least predetermined, target sequence occurs in a nucleic acid source.
The detection agent may be exposed to the sample in a number of separate steps,
or as a number of separate components, provided that ultimately all the required
components are present in the system.
In a preferred embodiment of the method, the detection agent comprises a first
agent and a second agent each having a different TBS, each TBS being capable of
binding to the target sequence, and wherein the binding of the first and second
TBS to the target sequence brings a metal surface associated with each TBS into
proximity thereby causing surface enhancement of an SAS associated with one or
both of the metal surfaces.
Generally the first and second TBS will bind adjacent each other in order
to bring their respective metal surfaces into contact or near contact.
Oligonucleotides have been used previously to assemble metal clusters
(gold colloid) into superlattices (see Bethell and Schiffrin (1996) Nature Vol
382: pg 581, plus also Mirkin et al and Alivisatos et al on pgs 607-609 and 609-611
of the same issue). However these publications were concerned generally with the
production of macroscopic materials from nanoparticles (so called 'nanotechnology'),
the nucleic acid being used to assist in the assembly process. Assembly was detected
by calorimetric differentiation. A further paper (Storhoff et al, 1998 "One pot
colorimetric differentiation of polynucleotides with single base imperfections
using gold nanoparticle probes" J Am Chem Soc 120: 1959-1964) also used calorimetric
analysis for detecting aligned gold nanoparticle probes. However no vibrational
spectroscopy was carried out on the assembled structures. No suggestion of the
technique's application in the field of raman spectroscopy was made.
The present, SER(R)S-based, invention will now be explained in more detail with
reference to some of the preferred embodiments.
The "sample nucleic acid" can be any nucleic acid, including DNA (from any source
e.g. genomic, cDNA, synthetic etc.), RNA (e.g. mRNA, tRNA, rRNA, synthetic etc.)
or derivatives of these. Generally it will be at least 16 nucleotides in length,
more preferably at least 24, 30, 40, 50, 100 or 200 nucleotides in length. The
sample can represent all or only some of the nucleic acid present in a given source.
The sample may be prepared prior to testing in order to make the sample nucleic
acid therein more available for the testing process. For instance the sample nucleic
acid may be fully or partially purified and/or fragments may be produced and separated.
As an alternative to, or in addition to, using the nucleic acid in the sample directly,
copies may be prepared and used (e.g. using PCR). The term "sample nucleic acid"
covers all of these possibilities.
Generally the sample nucleic acid will be prepared as single strand nucleic
acid prior to the sequence detection.
If desired the sample may be blotted, tethered or otherwise immobilised on a
solid
phase, optionally in the form of an array (e.g. a so called nucleic acid chip—see
e.g. Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31).
The "target" sequence itself may be any sequence of any length within the sample
which it is desired to investigate. Thus it may be any sequence found in a genome,
or subgenomic nucleic acid, chromosome, extrachromasomal vector, or gene, or motif,
or non-coding sequence, or a sequence tagged site, or expressed sequence tag. The
sequence may be derived from any source e.g. published material on a database.
The sequence may be unique within a given genome, or may have multiple occurrences
within it (the methods of the present invention may be used to determine its frequency
of occurrence). Likewise the sequence may be unique to a particular individual,
or population, or species, genus, family etc. or be present within more than one
of these groupings. The length of the target sequence may be selected on the basis
of its statistical likelihood of chance occurrence within a given size of genome.
For instance it has been suggested that a sequence of up to 16 bases in yeast,
and a few more in humans (e.g. 17-24), may be sufficient to indicate a unique sequence
in these organisms.
Particularly envisaged is the detection of nucleic acid 'variants'.
These may include single nucleotide variants (mutations or polymorphisms) or variable
number tandem repeats, or other satellite or microsatellite repeats. Thus the target
sequence in these cases may be characterised by only a single base, or numbers
of pairs of bases, within a given longer sequence.
As set out in more detail below, it may be desirable to probe several target
sequences
simultaneously using appropriate, distinctive agents.
The "exposure" of the sample to the agent can take any form which brings the
two into sufficient contact to allow binding of the agent to the target sequence
of the sample. Generally this will be mixing of solutions of these components.
The "detection agent" has a number of important attributes, although it is stressed
that it may comprise a number of discrete parts which are added simultaneously
or even sequentially.
The metal surface
The agent comprises a metal surface. As discussed above, this surface is initially
present in a form which minimises surface enhancement under the conditions selected
for the SER(R)S observation step, but becomes SER(R)S active when the TBS binds
to the target sequence.
Thus the methods of the present invention exploit the fact that the properties
of the surface to which a Raman active label is associated, have a profound effect
on the degree of surface enhancement which can be achieved. Using unaggregated
silver colloid, for example, surface enhancement is at a minimum and results in
unrecognisable, indistinct signals at wavelengths commonly used in SER(R)S detection
formats. By contrast, once aggregated, the SERRS signals obtained are strong, definable
and characteristic of a specific analyte. This effect is discussed generally in
Munro et al (1995) Langmuir 11: 3712-3720. In essence these authors showed that
monodisperse colloidal silver particles (of around 27 nm in size) have a maximum
absorption wavelength of around 400 nm, which is consistent with the excitation
of dipolar surface plasmons. Aggregated colloid (for instance consisting of two
or more silver particles in close association) shows a clear shift in absorbance
to higher wavelengths, with a much higher absorbance above 500 nm than was exhibited
by the monodisperse particles. The visible absorbance spectra for aggregated and
unaggregated particles is shown in FIG. 1. It is this region (e.g. 500 to
600 nm) which is much more useful for carrying out SER(R)S. SER(R)S surfaces are
also discussed by Rodger et al (1996) J Chem Soc Dalton Trans, pg 791-799.
The surface may be provided by a naked metal or may comprise a full or partial
coating. It may include, for instance, a metal oxide layer, or an organic coating
such as citrate, or borohydride.
Generally the metal surface will be provided by unaggregated colloidal
metal particles. For instance silver, gold or copper particles. Processes for preparing
such unaggregated colloids are now well known in the art. They involve, for instance,
the reduction of a metal salt (eg, silver nitrate) with a reducing agent such as
citrate, to form a stable microcrystalline suspension (see P C Lee & D Meisel,
J. Phys. Chem. (1982), 86, p3391).
The colloid particles are preferably monodisperse in nature. Preferably they
will be about 20-35 nm in diameter, though this will depend on the type of metal.
Preferably, the metal surface is provided by discrete silver colloid
particles, which are preferably substantially hexagonal in shape and of about 35
nm maximum diameter.
In embodiments employing metal colloid, the binding of the TBS to the target
sequence
causes individual colloidal metal particles to be brought into proximity, thereby
aggregating them, or at least mimicing the effects of aggregation, such as to cause
surface enhancement of an SAS which is associated with the "aggregated" particles
i.e. causing an enhancement of signal at the selected wavelength.
As used hereinafter, unless context demands otherwise, the terms "aggregation"
or "aggregates" describe this effect.
TBS
The TBS of the agent will generally be based on a nucleic acid, or modified nucleic
acid, or nucleic acid analog, which is complementary to all or part of the target sequence.
Under certain circumstances (when the TBS is not being synthesised to order
for instance) it may not be necessary to know its sequence. For instance nucleic
acid may be taken from a (known) source, cleaved, and the cleaved portions can
be used to prepare the detection agent of the present invention. Thus the target
(original source) is predetermined, even if the sequence is not established.
By "complementary" is capable of specific base pairing with the target sequence
whereby A is the complement of T (and U); G is the complement of C. Generally complementary
nucleic acids run anti-parallel i.e. one runs 5′ to 3′, while the
other 3′ to 5′. Where modified nucleic acid, or nucleic acid analog
is used, the base pairing is between corresponding modified or analog bases and
the complementary target sequence as appropriate.
It will be understood by those skilled in the art that, for a given target sequence
and target binding species, 100% complementarity of the full length of the sequences
may not be required to ensure hybridisation between the two (see e.g.
Molecular
Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring
Harbor Laboratory Press, or later editions of that work, for a discussion of appropriate
conditions to achieve nucleic acid hybridisation). Thus sequences which are only
substantially complementary may also hybridise under appropriate conditions, thereby
causing the aggregative effect discussed above and hence surface enhancement of
the SAS.
It is known that normally nucleic acid hybridisation conditions require the presence
of salt to prevent the repulsion of the negative phosphate backbones. However if
salt is added to the colloidal suspension then aggregation may occur producing
a false result. This may be avoided by modification of the nucleic acid and/or
colloid, or use of a nucleic acid analog.
For instance it is known that DNA forms which are neutral, or at least zwitterionic,
do not require high salt concentrations for hybridisation to occur. One possible
example of this is propargyl amino modified base as described by Cruickshank &
Stockwell (1988) Tetrahedron Letters 29: 5221-5224, and later in Graham et al (1997)
Anal Chem 69: 4703-4707. This particular modification is also believed to promote
greater specificity of base pairing (see Wagner et al (1993) Science 260: 1510-1513).
In another embodiment peptide nucleic acid (PNA) is used for the probes which
assemble the colloid. PNAs do not require salt for hybridisation due to their neutral
backbone and bind with a greater specificity of base pairing and will not tolerate
mismatches. Also, the subsequent duplex formed displays much greater stability
than a DNA/DNA duplex. These properties mean a strand of PNA shorter than the corresponding
DNA probe can be used to much greater effect. The use of PNA in the context of
MALDI-TOF mass spectrometry for sequence identification is briefly reviewed by
Egholm (1997) Nature Biotechnology 15: pg 1346. In that process an amplification
step is advocated. PNA in chip technology is discussed by Marshall & Hodgson (1998)
Nature Biotechnology 16: pg 27-31.
As discussed above, generally two different (non-complementary to each other)
TBS will be used which are capable of binding adjacent, or at least closely, to
each other in the target sequence (but will not be brought together in the absence
of the target sequence).
Preferably, when the first and second TBS are bound to the target sequence
they are adjacent, or separated by 1, 2, 3, 4, 5, fewer than 10, 20 or 30 bases,
in order to effect aggregation.
It may be desirable to have several TBS per agent (e.g. spaced around a metal
colloid particle) for instance more than 1, 2, 3, 4, 5, 10, or 20 per agent. Agents
of this sort may be cross-linked in the presence of the target sequence. This can
generate larger aggregates of metal surface, with a corresponding shift in the
plasmon resonance wavelength.
Association of TBS with metal surface
The interaction between the SSG and the metal surface will typically be by chemi-sorption
of the complex onto the surface, or by chemical bonding of the complex with a coating
on the surface.
This is preferably achieved by means so called "surface seeking groups" (SSGs).
These bind extremely tightly to the metal surface, and are discussed in detail
in WO 97/05280 (University of Strathclyde).
SSGs will generally be either complexing or chelating in nature, or will comprise
bridging ligands or polymer forming groups.
Naturally the choice of the SSG will depend on the nature of the surface
(e.g. its charge and the presence or absence of an oxide or other layer) and of
any surface coatings or other species (such as citrate reducing agents) associated
with it, and also on the nature of the TBS. For most useful surfaces, the functional
group preferably comprises a Lewis base. Ideally, it is actively attracted to the
surface in use. For gold surfaces phosphorus and sulphur containing groups may
be particularly preferred, as discussed by Bethell & Schiffin cited supra.
Thus suitable groups by which the agent may be bound to the active surface include
complexing groups such as nitrogen, oxygen, sulphur and phosphorous donors; chelating
groups; bridging ligands and polymer forming ligands.
Some example SSGs are shown in FIG. 2.
The triazole group (Formula A1) is rich in nitrogen lone pairs and seems to have
a particular affinity for certain metal colloids. Thus, incorporation of this group
in the agent is particularly preferred, since it can increase the proximity of
the label to the surface, and thereby the surface enhancement effect which occurs
when the TBS binds the target sequence.
The agent preferably contains the benzotriazole group (Formula A2), particularly
when the metal surface is silver- or copper-based, which has a high degree of conjugation
(especially when deprotonated) and is thus particularly amenable to SE(R)RS detection
which relies on label resonance.
Benzotriazole derivatives (such as that shown in Formula A3) may be
readily obtained and can be coupled with existing labels (such as azo dyes) to
give appropriately modified labels.
In preferred forms, the SSG is modified to be SE(R)RS active and this is used
to conjugate the TBS to the metal surface. Examples of such groups include azobenzotriazoles,
typically formed by combining azo substrates with benzotriazole derivatives. Examples
of azobenzotriazoles include 9-(carboxyethyl)-3-hydroxy-6-oxo-6H-benzotriazole,
and substituted benzoic and naphthoic acid azo derivatives coupled to benzotriazole.
An example structure for use as an agent in the present invention is the azobenzotriazole
shown in Formula A4. The compound comprises an azo chromophore which increases
the wavelength of the absorbance maximum of the label.
In all example structures in which it appears, R
9 represents the TBS
(e.g. PNA), optionally via a linker. The linker may be used to affect the distance
between discrete metal surfaces following succesful binding, and the rigidity with
which the surfaces are held. Generally a linker of less than 5, 10 or 15 carbons
in length will be preferred. Different R
9 groups (e.g. different linkers)
can also provide the agents with molecularly specific SE(R)RS spectra.
Suitably compounds for use in the agents of the present invention labels
are encompassed by the Formulae A5 and A6 wherein:
In all cases the TBS or linker will constitute one or more of R
1-R
6
groups, preferably selected from groups R
1-R
5. The following
preferences therefore refer to those groups from R
1-R
6 not
being the TBS/linker.
Thus the remaining R
1-R
6 can represent any appropriate
groups (including hydrogen), preferably selected from those listed below the formulae.
W, X, Y and Z are defined below the formulae. A more preferred sub-set of such
compounds is those in which R
1, R
2, R
3, R
4
and R
6 are independently selected from hydrogen, C
1-C
6
alkyl, C
1-C
6 alkoxy, 6-membered aromatic rings, halogen,
—COOH, —SO
3H, —PO
4, —SH, —PO,
—NR
7 and R
8; R
5 can be as R
1 or
alternatively —NH
2 or functionalised —COOH such as —(CH
2)
n—COOH
where n is an integer from 1 to 6; and R
7 and R
8 are independently
selected from hydrogen, C
1-C
6 alkyl (linear or branched chain)
and unsaturated cyclic alkyl rings.
Most preferred forms of such labels are those in which R
1 and R
2
are both hydrogen, R
3 and R
4 are independently selected
from hydrogen and methoxy, R
5 is either —OH or -amino and R
6
is hydrogen.
The agents prepared in Examples 1 to 3 (see FIG. 6B and 6C), are
examples of falling within these formulae.
Formula A7 provides an alternative to the benzotriazole-based agents.
Functional groups on the surface seeking portion of the agents may include
charged polar groups (eg, amine, carboxyl, phosphate, thiol, hydroxyl), attracted
to the surface or surface coating (e.g., to free amine groups in a polyamine coating).
Examples of these are shown in Formula A8, wherein R
9 is as discussed
above, and R
10 is independently selected from the groups listed in the
figure, with no more than 3 of the R
10 groups in the formula being H.
Preferably the R
10 groups in the formula, other than those which are
H, are all the same, as exemplified by Formula A9 and A10.
Further alternative surface seeking groups are shown by Formulae A11, A12
and A13.
Other suitable surface seeking groups for the agent include the calixerines
and the mercapto benzotriazoles.
SER(R)S active species
The agent comprises an SAS. This can be provided by a separate label in instances
when the TBS is non-UV absorbing. Suitable labels are discussed in detail in WO
97/05280 (University of Strathclyde), and also in the SER(R)S literature. The label
can be associated with the metal surface either as part of the TBS (optionally
as part of a SSG), or quite separately from it.
In preferred embodiments of the invention, as discussed in more detail below,
there will be more than one molecule of SAS per agent. Indeed the number is preferably
maximised such that when an aggregated metal surface is formed as a result of a
target sequence/TBS binding event, the maximum number of SAS molecules are surface
enhanced by that event.
Preferably a single molecule target sequence is capable of surface enhancing
more than 10, 20, 30, 40, 50, preferably more than 100, 150, or 200 SAS molecules
which are associated with the metal surface of the agent binding that target sequence
via the TBS.
Examples of suitable SE(R)RS-active species include fluorescein dyes, such
as 5- (and 6-) carboxy-4′,5′-dichloro-2′,7′-dimethoxy
fluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein
and 5-carboxyfluorescein; rhodamine dyes such as 5- (and 6-) carboxy rhodamine,
6-carboxytetramethyl rhodamine and 6-carboxyrhodamine X; phthalocyanines such as
methyl, nitrosyl, sulphonyl and amino phthalocyanines; azo dyes such as those listed
in C H Munro et al,
Analyst (1995), 120, p993; azomethines; cyanines and
xanthines such as the methyl, nitro, sulphano and amino derivatives; and succinylfluoresceins.
Each of these may be substituted in any conventional manner, giving rise to a large
number of useful labels.
The choice of label in any given case will depend on factors such as the resonance
frequency of the label, the other species present, label availability, choice or
laser excitation equipment etc. In particular it may be selected such as to maximally
distinguish between SAS associated with the metal surface which is not capable
of surface enhancement, and that associated with the metal surface which is capable
of surface enhancement (i.e. part of a target sequence/TBS complex.
It may be preferred that the SAS is an azo group, which can be very easily derivatised.
The skilled person will appreciate, however, that other SAS may also be readily
employed in the invention.
The dye may be associated with the metal surface using either covalent or non-covalent
interactions. Particular preferred is the use of SSGs as described above
SER(R)S detection
This can be by conventional methods, for instance as disclosed in WO 97/05280
(University of Strathclyde).
Thus in SE(R)RS the primary measurements are of the intensity of the scattered
light and the wavelengths of the emissions. Neither the angle of the incident beam
nor the position of the detector is critical. With flat surfaces an incident laser
beam is often positioned to strike the surface at an angle of 60° with detection
at either 90° or 180° to the incident beam. With colloidal suspensions
detection can be at any angle to the incident beam, 90° again often being employed.
Several devices are suitable for collecting SE(R)RS signals, including wavelength
selective mirrors, holographic optical elements for scattered light detection and
fibre-optic waveguides. The intensity of a SE(R)RS signal can be measured using
a charge coupled device (CCD), a silicon photodiode, or photomultiplier tubes arranged
either singly or in series for cascade amplification of the signal. Photon counting
electronics can be used for sensitive detection. The choice of detector will largely
depend on the sensitivity of detection required to carry out a particular assay.
For multiple, different analytes, a complex SE(R)RS spectrum across a range of
wavelengths will be obtained. Although analysis by eye may be possible, methods
for obtaining and/or analysing a SE(R)RS spectrum will preferably include the use
of some form of data processor such as a computer.
Note that the methods of the invention may involve either obtaining a full SE(R)RS
spectrum across a range of wavelengths, or selecting a peak and scanning only at
the wavelength of that peak (i.e., Raman "imaging").
Preferably the excitation beam is selected to maximally distinguish between
the SAS associated with the metal surface which is not capable of surface enhancement,
and that associated with the metal surface which is (i.e. part of a target sequence/TBS complex.
For instance if the plasmon resonance of the metal surface (following aggregation)
is 600 nm, and the SAS is also active in this region, then the excitation beam
will be selected to be in this region to maximise surface enhancement and resonance
effects. When using multiple, differing, SAS groups, excitation frequency may be
chosen to closely match the absorbance maxima of the SAS in order to provide sharp
signals thereby improving the molecular specificity of detection.
Preferred formats
Certain preferred formats are discussed below. Naturally the skilled person
will appreciate that these are not obligatory, and that other methods which also
achieve the advantages of the present invention, and are based on the disclosure
herein, may be equally be used if preferred.
Arrangements of SER(R)S dye, TBS, and metal surface.
Some of these are illustrated in FIG. 3.
Two agents comprising different TBS may be used—each TBS may be attached
to the metal surface (for instance of prepared monodisperse, unaggregated colloids)
via a linker and an SSG (e.g. based on benzotriazole) incorporating an SAS (e.g.
an azo group). The components of the agents are prepared together in situ or are
pre-mixed, and then added to the sample. Raman imaging or spectroscopy is then
carried out as desired.
Alternatively, for each of the two agents, separate TBS and SAS, each
incorporating an SSG, may be pre-mixed in the desired proportions, and the mixture
applied to metal colloid to coat it. The two agents are then added to the sample,
and the observation for surface enhancement is made.
The preferred ratio TBS:SAS may be greater or less than 1:1. However, preferably
the SAS is present in excess over the TBS, for instance greater than 10, 20, 30,
40, or 50 fold excess. The preferred ratio of TBS-SSG: SAS-SSG is about 1:100.
As discussed above, generally speaking, sensitivity will be improved by maximising
the number of molecules of SAS which are surface-enhanced by a single binding event
(i.e. two TBS molecules to one molecule of target sequence). This is achieved by
maximising the number of molecules of SAS which are present on the metal colloid
particles brought together by the binding event. However care must be taken (e.g.
for reasons of economy) to ensure that at least some TBS-SSG, preferably evenly
distributed, is present on each metal/SAS complex, in order to give it functionality.
Multiplexing
Raman signals consist of a series of discrete spectral lines of varying intensity.
The frequencies and the relative intensities of the lines are specific to the label
being detected and the Raman signal is therefore a "fingerprint" of the SAS.
If the analyzer is being used to quantitate the detection of one (e.g. as described
above) label then it will only be necessary to detect signal intensity at a chosen
spectral line frequency.
However more than one detection agent may be used simultaneously to probe
for more than one target sequence by using detection agents having distinctive,
distinguishable, SAS.
If the analyzer is being used to quantitate the detection of several labels,
each
of which has a unique spectral line, then it will only be necessary to detect signal
intensity at several chosen spectral line frequencies. Otherwise, if a SE(R)RS
analyzer is being used selectively to detect one or more 'bound' agents out of
a mixture, it will be necessary to detect the entire "fingerprint" spectrum for
identification purposes.
In cases where the target sequences share some sequence identity (e.g. distinguishing
target sequences containing single nucleotide polymorphisms), a common first agent
may be used, provided that the second agent can in each case discriminate between
the remainder of the target sequence, and can itself be distinguished from the
other second agents by use of a distinctive SAS.
Alternatively, for detecting several quite distinct sequences, several
pairs of agents may be used, provided only that they are not self-complementary.
Further aspects of the invention
As discussed in the Introduction, the methods may have numerous applications
in
genomics, whereby they can be used analogously to existing methods which employ
a step of in which nucleic acid sequence is analysed (see e.g. "Principles of Genome
Analysis" by S B Primrose, Pub. Blackwell Science, Oxford, UK, 1995).
Some specific applications are as follows. Generally speaking all of these can
be carried out using the single target sequence, or multiplexing approach. In the
latter case, the combination of various results may be used to make a determination:
(i) Detection of the presence of an organism (e.g. virus, provirus, virion, prokaryote
(such as bacterium), eucaryote (such as protozoan)) in a sample wherein the presence
of the target sequence is associated with the presence of the organism, for instance
because the sequence is unique to that organism.
Even in cases where the sequence probed may not actually be unique to the organism,
its presence (in conjunction with other diagnostic information e.g. immunological,
behavioural etc.) may be used to increase the certainty of a determination of its
presence of absence. The detection may be confirmed where still further certainty
is required by full sequencing.
The sample in this case can be anything suspected of containing the organism
e.g. a sample taken from a different organism, a foodstuff, an environmental sample
(e.g. soil, water etc.) The organism may be pathogenic, or may simply be associated
with some other quality of interest.
(ii) Diagnosis of a disease associated with a pathogenic organism, by carrying
out a determination as described above. The sample may be in vitro or in vivo.
The test may be carried out in conjunction with other diagnostic techniques, or
an assessment of symptoms etc.
(iii) Diagnosis of a disease associated with a DNA variation, by detecting
the presence of the DNA variant comprising use of a method as discussed above wherein
the target sequence corresponds to the sequence in which the variation occurs.
The test may be carried out in conjunction with other diagnostic techniques, or
an assessment of symptoms etc.
(iv) A method of selecting an organism having a particular phenotypic trait
whereby the target sequence corresponds to a sequence associated with that trait.
(v) A method of isolating a nucleic acid encoding a specific gene whereby the
target sequence corresponds to a sequence associated with, or within, that gene.
(vi) A method of phylogenetic classification, wherein the target sequence is
associated with a particular individual, population, species, genus etc.
(vii) A method of identifying an individual wherein the target sequence is
associated with that individual. Generally speaking this may entail scoring a number
of discrete polymorphisms (see e.g. WO 96/01687 of Tully et al for sequences used
in forensic typing and matching).
(viii) A method of expression profiling a cell or tissue. In this case the
sample nucleic acid is mRNA, or is derived from it (e.g. cDNA).
In a further aspect of the invention there is disclosed a method of producing
a detection agent comprising: combining unaggregated metal particles with a SER(R)S
active species (SAS) and a target binding species (TBS), whereby said SAS and TBS
combine with said metal particles via a surface seeking group.
In a further aspect of the invention there is disclosed a detection agent comprising:
an unaggregated metal particle, being associated with a SER(R)S active species
(SAS) and with a target binding species (TBS).
The various components of the agent may be any of those discussed above. In particular
the SAS and the TBS are preferably bound to metal particle via an SSG, optionally
in the form of a single molecule. Preferably the TBS and SSG are discrete molecules.
Preferably the TBS is PNA or propargyl amino modified DNA. Preferably the SAS is
an azo group and the SSG is benzotriazole.
In one embodiment the agent comprises a first agent and a second agent each having
a different TBS.
In a further aspect there is disclosed a composition comprising two or more detection
agents as described above, each having a distinctive SAS.
The agents or compositions of the present invention will generally be provided
as solutions.
In a further aspect there is disclosed a kit comprising the agents or compositions
of the present invention, plus one or more additional materials for practising
the methods of the present invention e.g. target nucleic acid for control experiments.
In a further aspect there is disclosed a system comprising an agent or composition
described above plus a nucleic acid sample, which is preferably a sample of DNA
or RNA, most preferably extracted from a cell taken from or constituting an organism.
Such a system may particularly comprise:
- (i) a reaction vessel,
- (ii) an agent as described above,
- (iii) a nucleic acid.
preferably in a homogenous format.
In a further aspect there is disclosed an apparatus comprising a SERRS analyser
plus an agent, composition or system as described above, and methods of use of
such an apparatus, comprising (for instance) the steps of preparing and monitoring
(e.g. at between 500 and 600 nm) a homogenous system in order to detect a SER(R)S signal.
The invention will now be further explained with reference to the following non-limiting
Figures and Examples. Other embodiments falling within the scope of the present
invention will occur to those skilled in the art in the light of these.
FIGURES
FIG. 1: this shows the visible absorption spectra for a citrate colloid
aggregated with (a) nitric acid, and (b) poly(L-lysine) and ascorbic acid. The
dotted line represents the spectrum of unaggregated, monodisperse, citrate silver
colloid prior to aggregation.
FIG. 2: this shows Formulae A1-A13 representing various SSGs which may
be employed in the present invention.
FIG. 3: this shows various different formates for performing the present invention.
In (a)(i) an agent, which itself comprises two types of silver colloid particle
is used. Each particle carries a different PNA probe (the TBS) which are designated
X and Y respectively. These are associated with the metal surface via different
dyes (the SAS) designated A and B, which interact with the metal surface via linkers
and surface seeking groups (not shown). When combined with appropriate genomic
material having a target sequence X′-Y′ (containing portions complementary
to X and Y) the silver colloid particles are aggregated, this aggregation can be
monitored via one or both of dyes A and B (one of which could be omitted if not required).
In (b) an alternative agent is shown. In this case the dye (A) and PNA probe
(X)
are separately associated with the metal surface, each via a linker and surface
seeking group (not shown).
In (c) a method for distinguishing and scoring polymorphisms is shown. In this
case the possible target sequences are X′-Y′ and X′-Y2′.
These can be distinguished by using an additional colloid particle type, having
an appropriate PNA probe (designated Y2) and a dye which is distinguishable from
B (designated C). By observing which of B and C is surface enhanced, the target
sequence can be elucidated. It may be desirable to label common sequence colloid
particle (X) with dye A as a control, since this should be detectable for either polymorphism.
In (d) a method for probing discrete sites is shown. This is similar to (c) but
there is no common sequence between the target sequences, therefore four different
types of colloid particle are used. The detection of dye D indicates the presence
of target sequence V′-W′.
FIG. 4: this shows a pathway for producing an amino derivative of benzotriazole
dye [N
1-[4-(5-Azobenzotriazoyl)phenyl]-ethane-1,2-diamine—Compound(1)],
and a carboxylic acid derivative of this [N
1-[4-(5-Azobenzotriazoyl)phenyl
amino-ethyl]-succinamic acid—Compound(2)]. The carboxylic acid derivative
may be attached to DNA (Scheme 1) or used to produce an active ester (Scheme 2)
for subsequent attachment.
FIG. 5: this shows a SERRS spectrum of a benzotriazole dye labelled 26mer
DNA oligonucleotide.
FIG. 6: this shows a SERRS spectrum of benzotriazole dye labelled PNA
oligonucleotides in presence and absence of aggregating agent.
FIG. 7: this shows a SERRS spectrum of benzotriazole dye labelled PNA
oligonucleotides in presence and absence of complementary target sequences.
EXAMPLES
Example 1
Overview of Synthesis of Labelled PNA Probes and Subsequent Use
Basic synthesis involves the addition of a carboxylic acid or an active ester
form of a benzotriazole dye to the amino terminus of a PNA probe, optionally while
the PNA is on a solid support used for synthesising it. The probe consists of eight
or more bases complementary to part of a target sequence found in genomic DNA.
Thus the bases will still be protected, but the primary amine is free for reaction.
a) Synthesis of Benzotriazole Carboxylic Acid or Active Ester
An amino alkylated aromatic amine, such as N-(1-naphthyl)ethylenediamine dihydrochloride,
is coupled to aminobenzotriazole via a diazonium coupling to produce a monoazo
dye. The free amine then reacts with succinic anhydride to provide a carboxylic
acid (see FIG. 4).
In more detail, to provide N
1-[4-(5-Azobenzotriazoyl)phenyl]-ethane-1,2-diamine
(=Compound 1), 5-Aminobenzotriazole (0.854 g, 1.1 eq, 6.37 mmol) was dissolved
in HCl (5ml, 50% v/v) and diazotised by dropwise addition of sodium nitrite (0.484
g, 1.2 eq, in 5 ml H
2O) at 0° C. An excess of sodium nitrite was
detected using starch iodide paper. A dark blue colour indicated excess nitrous
acid which inferred the formation of the diazonium salt. Separately N-(1-Naphthyl)
ethylenediamine dihydrochloride (1.500 g, 5.79 mmol) was dissolved in sodium acetate
buffer (1.0M, 60 ml, pH 6.0) and acetone (80 ml). Diazotised aminobenzotriazole
(1.1 eq) was added to this solution dropwise at 0° C. with stirring over 1
hour after which the solution was neutralised by addition of sodium hydroxide (2M).
The solid produced was isolated by filtration and washed with sat. KCl (3×50
ml) prior to purification by trituration using methanol and diethyl ether to produce
the title compound as an orange solid in 66% yield Rf (EtOAc/CH
3OH/NH
3
5/1/1) 0.14; d
H (270 MHz, CD
3OH) 3.00 (2H, t, CH
2)
3.48 (2H, t, CH
2) 6.69 (1H, dd, arH) 7.51 (1H, t, arH) 7.63 (1H, t,
arH) 7.87 (1H, d, arH) 7.94 (2H, m, arHs) 8.13 (1H, d, arH) 8.34 (1H, s, arH) 9.02
(1H, d, arH); d
c(270 MHz, CD
3OD) 41.40 (CH
2) 47.01
(CH
2) 104.26 (CH) 114.93 (CH) 115.13 (CH) 116.67 (CH) 117.47 (CH) 122.20
(CH) 124.26 (C) 124.73 (CH) 126.03 (CH) 127.91 (CH) 134.62 (C) 139.91 (C) 146.12
(C) 146.76 (C) 148.98 (C) 151 28 (C); FAB ms m/z 332.1621 [C
18H
18N
7
(M+1) <0.1 ppm].
In order to provide N
1-[4-(5-Azobenzotriazoyl)phenyl amino-ethyl]-succinamic
acid (=Compound 2), Compound (1) (1.000 g, 3.02 mmol) was dissolved in DMF (100
ml) and left to stir. Succinic anhydride (0.363 g, 1.2 eq, 3.63 mmol) dissolved
in acetone (10 ml) was then added dropwise over one hour. After 18 hours the solvent
was removed in vacuo and the product isolated by column chromatography (pre-absorbed
onto Na
2SO
4) eluting with ethyl acetate, methanol and ammonia
(5/1/1) to produce an orange solid which was further purified by trituration from
diethyl ether to give 0.856 g (66% yield) of the pure product Rf (ethyl acetate/methanol/ammonia
5/1/1) 0.11; d
H (400 MHz, DMSO-d6) 2.40 (2H, dd, CH
2) 2.44
(2H, dd, CH
2) 3.42 (4H, m, 2×CH
2) 6.66 (1H, brs, NH)
6.76 (1H, d, arH) 7.32 (1H, m, arH) 7.54 (1H, t, arH) 7.67 (1H, t, arH) 7.90 (1H,
d arH) 7.99 (1H, dd, arH) 8.25 (1H, d, arH) 8.29 (1H, s, arH) 8.98 (1H, d, arH).
If an active ester is desired then the succinyl form can be produced from the
carboxylic acid by activating with hydroxy succinimide as shown in FIG. 4—scheme 2).
<
