GB2380998A

GB2380998A - D-amino acid peptide for security tagging

Info

Publication number: GB2380998A
Application number: GB0119053A
Authority: GB
Inventors: Marc Kvansakul; Niren Thanky; Nayen Thanky
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-08-03
Filing date: 2001-08-03
Publication date: 2003-04-23
Anticipated expiration: 2021-08-03
Also published as: GB0119053D0; GB2380998B

Abstract

The present invention relates to the labelling of objects for verifying authenticity, in particular to the use of selectively perceptible marks for labelling objects. The present invention provides a permanent tagging material comprising a synthetic polypeptide composed of a specific sequence of D amino acids with an attachment moiety. The use of and method for identifying the tagging material is also dislcosed. The polypeptide may contain tryptophan residues which fluoresce when exposed to light of 280nm wavelength. The material may also comprise fluorescent microspheres.

Description

MATERIAL The present invention relates to the labelling of objects for verifying authenticity and more particular to the use of selectively perceptible marks for labelling of objects. Authenticity implies both that the goods are genuine and that they are in the proper channels of commerce. If the goods are not genuine, then product counterfeiting has occurred. The present invention presents the ability to determine whether or not goods are genuine. If the goods have been diverted from their intended channel of commerce by, for example, entering into a country where the goods are prohibited, or for example, by contract or by law, then the goods have been subject to product diversion. Again, the present invention presents the ability to determine whether genuine goods have been improperly diverted. Finally, diverted goods also include genuine goods that have been stolen and the identification of the goods is at issue.

Many objects require verification for authentication purposes. Such objects include paintings, sculptures, sports and other collectibles, and like works of art; DVD players, televisions, and like household objects; and computers, printers and other office and business equipment. Other instances of identification in order to verify ownership include for example records, audio and video tape cassettes, computer software, perfumes, designer clothes, handbags, automobile or airplane parts, securities (e. g. stock certificates), wills, identification cards, passports, drivers licences, credit cards and like objects.

Many industries have been plagued by a piracy explosion over the past decade involving many of the aforementioned products. Often, these products have no serial number or other unique means of identification, or the number can be

removed easily following a theft. Alternatively, counterfeiting of such objects has become a thriving business and the need to identify authenticity from counterfeit objects is of great importance.

In a related scenario genuine goods are limited to being shipped and sold in selected jurisdictions, e. g. countries, for example by law or by contract. When genuine goods are diverted to countries where their presence is not authorised, product diversion has occurred. Product diversion can lead to price inequities in certain markets as well as loss of exclusivity by some manufacturers or distributors. This situation is often referred to as grey market goods. Since the goods are genuine, it is quite difficult to determine whether the goods have been improperly diverted. This is especially true for a variety of the goods such as clothing.

The economic damages incurred by counterfeiting is rising steadily and posipg an enormous problem to a huge variety of industry sectors. Furthermore, governments are equally affected due to the loss of tax income on counterfeit products and diverted goods.

Various parties have proposed tagging of objects with various chemical and biological tags. U. S. Pat. No. 4441943 uses synthetic polypeptides for labelling explosives. The present invention is significantly different from this prior art, as it uses a combination of fluorescent detection and non-natural D-amino acid peptides as tagging material. Furthermore, the method described herein includes a fast analysis procedure based on mass spectrometry, whereas US4441943 is based on the older Edman degradation method.

A whole range of patents, e. g. British Pat. No. 2209831, US5451505, US5139812, W09617954, US5942444 and W009934984 all propose tagging methods based on DNA with various information encoding schemes combined with an analysis method based on the Polymerase Chain Reaction (PCR). The present invention is significantly different as the peptides within the tagging material are far more robust than nucleic acids. DNA molecules are inherently more prone to strand breakage and environmental degradation, whereas peptides can withstand considerably adverse circumstances, as is clearly demonstrated in the classical Edman degradation procedure, where temperatures in excess of 100 C and extremely acidic conditions (6 M HCI) are required to break the peptide down.

With respect to detection using optical systems, the method proposed in W009934984 operates in the infrared region of the spectrum, whereas the present invention utilises shorter wavelengths of 280-800 nm.

The present invention provides a permanent tagging material comprising a synthetic polypeptide composed of a specific sequence of D-amino acids and an attachment moiety. The tagging material is applied onto an object to allow subsequent or retrospective unambiguous identification. In this context,"permanent"means that the label is incapable of being removed from the object in the ordinary course of intended handling and usage of the object for a time adequate for identification and/or verification of the object to occur and/or is placed on the object at a location that is seldomly, if ever, accessed by the user in the ordinary course of using the object.

The tagging material is preferably a liquid that can be sprayed, stamped, stencilled or painted onto the object that is to be labelled. Alternatively the tagging material can be

used as an ink in a pen to allow easy application of the material onto the object to be labelled, without contamination of any surrounding objects.

In one embodiment of the invention the polypeptide includes one, two or three tryptophan residues. These residues can be consecutive or non-consecutive, and positioned anywhere within the polypeptide. Tryptophan exhibits considerable fluorescence when exposed to light with a wavelength of 280nm, allowing the presence of the tagging material to be detected visually.

In a second preferred embodiment the tagging material further comprises a fluorescent microsphere. This microsphere preferably emits at a wavelength of 280-SOOnm.

The attachment moiety is preferably colourless, so that it is invisible to the human eye once an object has been labelled with the tagging material. Suitable attachment moieties currently available that can be used, such as the Blak- RayTM fluorescent inks (Ultra-Violet Products Ltd, Cambridge, UK. ) The only criterion for the attachment moiety is good adhesive properties for the object that is to be tagged.

The invention also provides a method of identifying the permanent tagging material of the present invention on a tagged object comprising the followirg steps: (a) Detecting the presence of the tagging material by means of fluorescence using a hand held detector; (b) Analysing the polypeptide sequence of the tagging material using mass spectrometry;

(c) Comparing the polypeptide sequence of the tagging material to a database of the polypeptide sequences utilised to identify the origin of the tagging material.

The method comprises application of a synthetic polypeptide together with a fluorescent microsphere in a suitable colourless/invisible attachment moiety as tagging material and subsequent detection of the tagging material and precise composition of the polypeptide. The tagging material is readily detected with handheld detectors using the fluorescence of the tagging material.

The polypeptide is synthesized to have a specific undisclosed sequence of amino acids which can be readily identified from minute amounts using presently available analytical methods and apparatus. The polypeptide chain can consists of any number of residues, preferably 15 to 30.

In another aspect the present invention provides compositions comprising the tagging material of the invention. Suitable compositions include paints, aerosol sprays, inks, or gels.

In a further aspect the present invention provides the use of the tagging material of the invention for labelling an object to allow subsequent identification. The process of identification will allow the owner or origin of the labelled object to be traced.

The invention will now be described in more detail with reference to the following figures:

Figure 1 shows a Jablonski diagram, illustrating by a simple electronic state diagram, the process responsible for fluorescence of fluorophores.

Figure 2 shows the excitation of fluorophores at three different wavelengths.

The present invention describes a highly accurate and secure tagging method provided by a combination of a polypeptide and a fluorescent microsphere. The fluorescent microsphere emits at a wavelength in the region of 280-800 nm and affords the detection of the tagging material using suitable handheld devices.

Polypeptides are compounds of two or more amino acids which contain one or more peptide groups. The amino acids that may be used to form the polypeptide belong to the class of D-amino acids, as listed in Table 1.

Table with D-amino acids used in tag fl]

Ammo acid Single letter code Residue mass Glycine557. 052 Alanine A 71.079 Serine S 87.078 Proline P 97. 117 Valine V 99. 133 Threonme T 101.105 Cysteine C 103.144 Isoleucine I 113.16 Leucine L 113.16 Asparagme N 114.104 Aspartic acid D 115.089 Glutamine Q 128.131 Lysine K 128.174 Glutamic acid E 129.116 Methionin M 131.198 Histidine H 137.142 Phenylalanine F 147.177 Arginine R 156.188 Tyrosine Y 163.17 Tryptophane W 186. 213 Mass is given in atomic mass units The use of D-amino acids is favourable over the naturally occurring L-form, as it is virtually resistant to any naturally occurring protease, thus considerably improving the polypeptide durability in the natural environment.

Methods and apparatus for the synthesis of polypeptides are well known to those skilled in the art. For examples of synthesizing, see [2-4]. A comprehensive review of the methods in use can be found in [5-7].

Peptide synthesis can be performed by two very different approaches-the classical in solution synthesis and on a solid phase support. Classical in solution methods are labour, time, and skill intensive largely due to the unpredictable

solubility characteristics of intermediates. Solid-phase peptide synthesis (SPPS), as developed by R. B. Merrifield, has proven to be the method of choice for producing peptides and small proteins of specific sequences.

The concept of the solid-phase approach involves covalent attachment (anchoring) of the growing peptide chain to an insoluble polymeric support (resin carrier), so that excess soluble reagents can be removed by simple filtration and washing without manipulative losses. Subsequently, the insoluble peptide-resin is extended by a series of additional cycles, which are required to proceed with exquisitely high yields and fidelities. Excess soluble reagents are used to drive reactions to completion.

Because of the speed and simplicity of the repeated steps, the major portion of the solid-phase procedure is amenable to automation. Once chain elaboration has been accomplished, it is necessary to release (cleave) the crude peptide from the support under conditions that are minimally destructive towards

sensit ve resi sensitive residues in the sequence. Finally, prudent purification and scrupulous characterisation of the synthetic peptide product must follow to ensure and verify that the desired structure is indeed the one obtained.

A more demanding part of the discipline of peptide synthesis is the necessity to block those functional groups which should not participate in peptide bond forming reactions usually called coupling. Blocking groups (or protecting groups/masking groups) are needed for the amino functionality of the amino acid which lends its (activated) carboxyl group to the coupling reaction (the "carboxyl component"), and also needed for the carboxyl group of the amino acid which will be acylated in its amino group (the"amine component"). In

order to lengthen the resulting dipeptide one of the masking groups has to be removed and this should be possible without harming the peptide bond or the "semi-permanent"protecting group which has to stay in place until a later stage when its cleavage becomes necessary. Some of the maskings are mandatory, others optional. For SPPS, three types of protection are required. The functional groups of the amino acid side-chains must be protected with groups which are stable to the repeated treatments necessary for removal of the N-amino protecting group of the growing peptide chain and for repeated amino acid couplings. Such side-chain protecting groups are usually called"permanent" protecting groups. The third type of protection may be considered to be peptideresip anchorage which protects the C-terminus of the peptide throughout the various synthetic processes required to elaborate the desired sequence, until such time as its detachment from the resin.

A desirable protecting group for amino protection should be easily removable but stable enough to survive the conditions of the coupling reaction and other manipulations. For the SPPS, the N-protecting group is almost always a urethane derivative. The most important Na-protecting groups are the Boc and Fmoc groups. The t-Butoxycarbonyl group (Boc) is one of the most popular aamino protecting groups. A Boc group can be introduced easily using tbutoxycarbonyl azide, di-tert-butyldicarbonate and several similar reagents. This group can be removed easily under mild acidic conditions such as trifluoroacetic acid (50% TFA in dichloromethane), hydrogen chloride in acetic acid/dioxane/ethylacetate etc. It is completely stable towards catalytic hydrogenation and, therefore, can be used orthogonally along with the Z group for the side-chain protection.

The cleavage of 9-fluorenylmethyloxycarbonyl (Fmoc) under mildly basic, nonhydrolytic conditions provides an eminently important criterion for its success as a protecting group. It is because of this property that the integrity of the peptide chain with its amide and t-butyl protected side-chain ester linkages is largely unaffected. The Fmoc group is cleavable by primary and secondary amines but is rather stable to tertiary amines. 20% piperidine in DMF, one of the most basic cyclic secondary amines, is the reagent of choice and cleaves the Fmoc group completely in 2 mins.

The number of amino acids used for the polypeptide is not critical. Typically, we envisage the use of approximately 15-30 amino acids, although any number can be used. However, the sequence of amino acids is not random. In order to allow efficient sequencing of the polypeptide, each consecutive member of the polypeptide chain has to have an atomic mass difference of at least 2 Da. This excludes a number of possible sequences, as can be seen from the masses of amino acids given in table 1. As a total of at least 18 D-amino acids can be used, a 10-mer gives a total of 1018 possible combinations.

Therefore, despite the restriction imposed by the 2 Da minimum mass difference of consecutive members in a polypeptide, an enormous range of possible chains is available which practically eliminates the possibility of duplicate codes.

The usage of a number of different amino acids for the generation of polypeptides naturally suggests the possibility of encoding information into the chain. As we intend to use at least 18 D-amino acids, a large amount of information can be encoded into a reasonably short polypeptide. We propose to

apply the Huffman Encoding Procedure [8] for the generation of a suitable code for encryption. This will ensure maximum encryption efficiency.

With reference to sequencing of the polypeptide, this is achieved by mass spectrometry. Several alternative methods exist which are labour intensive and time consuming. An example for these older methods is given in [9].

Mass spectrometers are an analytical tool used for measuring the molecular weight (MW) of a sample. For large samples such as biomolecules, molecular weights can be measured to within an accuracy of 0. 01% of the total molecular weight of the sample i. e. within a 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected, e. g. the substitution of one amino acid for another. This high accuracy of mass determination can be achieved with extremely minute quantities of sample. Accurate analysis can be achieved with quantities as low as femtograms. Nanogram quantities are now routinely analysed with very high accuracy.

A popular method for peptide analysis is called tandem mass spectrometry [10].

Fast and accurate determination of a peptide sequence is possible as peptides fragment in a reasonably well-documented manner [11-12]. The protonated molecules fragment along the peptide backbone and also show some side-chain fragmentation [13].

There are three different types of bonds that can fragment along the amino acid backbone: the NH-CH, CH-CO, and CO-NH bonds. Each bond breakage gives rise to two species, one neutral and the other one charged, and only the charged

species is monitored by the mass spectrometer. The charge can stay on either of the two fragments depending on the chemistry and relative proton affinity of the two species. Hence there are six possible fragment ions for each amino acid residue and these are labelled as in the diagram, with the a, b, and c"ions having the charge retained on the N-terminal fragment, and the x, y", and z ions having the charge retained on the C-terminal fragment. The most common cleavage sites are at the CO-NH bonds which give rise to the b and/or the y" ions. A detailed account of methods for peptide analysis that can be used is given in [14].

Once an object is chosen for tagging, the invention disclosed herein is used to allow authentication and identification at a later date. For this purpose, the object is to be permanently labelled. For some objects, it may be desirable that the label remains affixed to the object and identifiable for many years. Additionally, the precise location of labelling is not disclosed, thereby requiring a means of locating the tagging material if identification and/or verification is required.

The present invention requires detection of the tagging material on a labelled object prior to the identification and/or verification procedure by mass spectrometry. As the location of tagging material on the labelled object is not disclosed, a method of detection is required. Detection is afforded by means of fluorescence from fluorescent microspheres present in the tagging material. Polypeptide and fluorescent microspheres will be held together by a matrix provided by a colourless attachment moiety.

Fluorescence is the result of a three-stage process that occurs in certain molecules (generally polyaromatic hydrocarbons or hetero-cycles) called fluorophores or fluorescent dyes. The process responsible for the fluorescence of fluorophores is illustrated by a simple electronic-state diagram (Jablonski diagram-Figure 1).

Stage 1 : Excitation A photon of energy hVEx is supplied by an external source such as an incandescent lamp or a laser and absorbed by the fluorophore, creating an excited electronic singlet state (sol'). This process distinguishes fluorescence from chemiluminescence, in which the excited state is populated by a chemical reaction.

Stage 2: Excited-State Lifetime The excited state exists for a finite time (typically 1-10 x 10-9 seconds). During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. These processes have two important consequences.

First, the energy of Sl'is partially dissipated, yielding a relaxed singlet excited state (SI) from which fluorescence emission originates. Second, not all the molecules initially excited by absorption (Stage 1) return to the ground state (So) by fluorescence emission. Other processes such as collisional quenching, fluorescence energy transfer and intersystem crossing (see below) may also depopulate Sl. The fluorescence quantum yield, which is the ratio of the number of fluorescence photons emitted (Stage 3) to the number of photons

absorbed (Stage 1), is a measure of the relative extent to which these processes occur.

Stage 3: Fluorescence Emission A photon of energy HVEM is emitted, returning the fluorophore to its ground state So. Due to energy dissipation during the excited-state lifetime, the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon hvEx The difference in energy or wavelength represented by (huer hvEM) is called the Stokes shift. The Stokes shift is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a low background, isolated from excitation photons. In contrast, absorption spectrophotometry requires measurement of transmitted light relative to high incident light levels at the same wavelength.

Fluorescence Spectra The entire fluorescence process is cyclical. Unless the fluorophore is irreversibly destroyed in the excited state (an important phenomenon known uS photobleaching), the same fluorophore can be repeatedly excited and detected.

For polyatomic molecules in solution, the discrete electronic transitions represented by hVEx and hvEM in Figure 1 are replaced by rather broad energy spectra called the fluorescence excitation spectrum and fluorescence emission spectrum, respectively. The height and widths of these spectra are parameters of particular importance for applications in which two or more different fluorophores are simultaneously detected (see below). With few exceptions, the fluorescence excitation spectrum of a single fluorophore species in dilute

solution is identical to its absorption spectrum. Under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength, due to the partial dissipation of excitation energy during the excited-state lifetime as illustrated in Figure 1. The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength (Figure 2).

A more detailed description of fluorescence and its potential applications is given in"Introduction to fluorescence techniques"by Molecular Probes Inc.

[15] and several textbooks [16, 17, 19] and other publications [18].

The presented invention proposes to use fluorescent microspheres with an emission and excitation wavelength of 280-800 nm to afford the detection of tagging material. The fluorescent microspheres proposed for detection of tagging material are covered by various patents owned by companies such as Molecular Probes Inc. A complete list of patents is found [20], covering synthesis, production and various other aspects of the spheres. Any comparable fluorescent microspheres by any suitable manufacturer can be used instead of the aforementioned types.

As detection of tagging material is of utmost importance for successful identification and/or verification, a second route for fluorescence detection is available. In its oxidised form, the amino acid Tryptophane (Trp) exhibits considerable fluorescence when excited with light at 280 nm. This fluorescence is the basis for protein concentration determination based on the Beer-Lamberts Law using UV spectrophotometers.

Polypeptides including 1-3 Trp exhibit sufficient fluorescence when excited at 280 nm to be detectable. This provides a ready alternative for detection based on fluorescent microspheres.

In summary, the present invention discloses a biomolecular tag that can be readily and selectively detected using fluorescence provided by either associated fluorescent microspheres or using intrinsic polypeptide fluorescence due to the incorporation of Trp residues. This will allow the detection of labelled objects and verification/authentication of such items. Furthermore, the precise origin of such labelled items can be determined by mass spectrometric analysis of the polypeptide by revealing the precise sequence of the polypeptide and therefore the parties associated with the particular sequence, and any encoded information within the peptide sequence.

References : 1. Appendix 6, Methods of Enzymology V 01193, p 888.

2. Stewart et al (1969) Solid phase peptide synthesis (W. H. Freeman and Co., San Fransisco 3. Hunkapillar et al (1980) Science, 207,523-525

4. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, p 2149-2154 5. Barany, G. and Merrifield, R. B. (1979), in The Peptides (Gross, E. & Meienhofer, J., eds. ), vol. 2, p. 1-284, Academic Press, New York.

6. Balkenkohl, F. , von dem Bussche-Huennefeld, C., Lansky, A. and Zechel, C. (1996) Combinatorial Synthesis of Small Organic Molecules.

Angewandte Chemie Int Ed Engl Vol 35, p 2288-2337

7. Lam, K. S. , Lebl, M. and Krchnak, V. (1997) The"One-Bead-One- Compound"combinatorial library method. Chem. Rev. 97, p 411-448.

8. D. A. Huffman (1952) A method for the Construction of Minimum Redundancy Codes. Proceedings of the IRE 40, p 1098-1101 9. Edman et al (1967) A protein sequenator. European J. Biochem, 1, p 80- 91 10. Klaus Biemann (1990) Sequencing of Peptides by Tandem Mass Spectrometry and High-Energy Collision-Induced Dissociation by.

Methods of Enzymology 193, p 455-479.

11. P. Roepstorrf, 1. Fohlmann (1984) Biomed. Mass Spectrom. 11, p 601- 601 12. R. S. Johnson, K. Biemann (1989) Biomed. Environ. Mass Spectrom 18, p 945-957.

13. Alison E. Ashcroft (1997) Ionization Methods in Organic Mass Spectrometry by, The Royal Society of Chemistry, UK, 14. Greer, F. M. and Morris, H. R (1997) Fast-atom bombardment and electrospray mass spectrometry of peptides, proteins, and glycoproteins by. Methods Mol Bioi 64, p 147-63 15. http ://www. probes. com 16. Brand, L. and Johnson, M. L. , Fluorescence Spectroscopy (Methods in Enzymology, Volume 278), Academic Press (1997).

17. Cantor, C. R. and Schimmel, P. R., Biophysical Chemistry Part 2, W. H.

Freeman (1980) p 433-465.

18. Oldham, P. B. , McCarroll, M. E. , McGown, L. B. and Warner, I. M. (2000) Molecular Fluorescence, Phosphorescence, and Chemiluminescence Spectrometry. Anal Chem 72,197R-209R.

19. Berlman, I. B., Handbook of Fluorescence Spectra of Aromatic Molecules, Second Edition, Academic Press (1971) 20. http : //www. probes. com/handbook/sections/0051. html

Claims

1. A permanent tagging material comprising a synthetic polypeptide composed of a specific sequence of D-amino acids and a attachment moiety.

2. A tagging material as claimed in claim 1 wherein said polypeptide includes one, two, or three tryptophan residues.

3. A tagging material as claimed in claim 1 or claim 2 further comprising a fluorescent microsphere.

4. A tagging material as claimed in any one of claims 1 to 3 wherein said fluorescent microsphere emits at a wavelength of 280-800nm

5. A tagging material as claimed in any one of claims 1 to 4 wherein said polypeptide is composed of 15 to 30 amino acids.

6. A tagging material as claimed in any one of claims 1 to 5 wherein the attachment moiety is invisible to the human eye.

7. A method of identifying the permanent tagging material as defined in any one of claims 1 to 6 comprising the following steps: (a) Detecting the presence of the tagging material by means of fluorescence using a hand held detector; (b) Analysing the polypeptide sequence of the tagging material using mass spectrometry;

8. A composition comprising the tagging material as claimed in any one of claims 1 to 6.

9. A composition as claimed in claim 9, wherein said composition is a liquid.

10. A composition as claimed in claim 8 or claim 9 wherein said composition is a paint, aerosol spray, ink or gel.

11. The use of the tagging material as claimed in any one of claims 1 to 6, or the composition as claimed in any one of claims 8 to 10, for labelling an object to allow subsequent identification of said object.