COMPOUND AND DIAGNOSTIC SYSTEM COMPRISING SAID COMPOUND FOR THE GUSTATORY DETECTION OF INFLAMMATIONS IN THE ORAL CAVITY

Abstract

The present invention relates to compounds comprising denatonium linked to the C-terminus of a protease-sensitive peptide; or a salt thereof. The compounds are useful in the diagnosis of inflammatory conditions of the oral cavity.

Claims

1. A compound having the structure Den-R.sup.1—NH—R.sup.2, or a salt thereof, wherein Den is denatonium, R.sup.1 is an optionally substituted C.sub.1-3 alkylene group R.sup.2 is a protease-sensitive peptide characterized by a C-terminal amino acid that forms an amide bond with the —NH— group of said compound.

2. The compound or salt of claim 1, having the formula [I]: ##STR00031## wherein R.sup.1 and R.sup.2 are as defined in claim 1.

3. The compound or salt of claim 1, wherein the compound has the formula [Ia]: ##STR00032## wherein R.sup.1 and R.sup.2 are as defined in claim 1.

4. The compound or salt of claim 1, wherein R.sup.1 is —CH.sub.2—.

5. The compound or salt of claim 1, wherein the protease-sensitive peptide comprises at least 4 amino acids.

6. The compound or salt of claim 1, wherein the C-terminal amino acid of the protease-sensitive peptide is selected from the group consisting of alanine, arginine, glutamine, leucine, methionine and phenylalanine.

7. The compound or salt of claim 1, wherein the compound has the formula [BRS-1]: ##STR00033##

8. The compound or salt of claim 1, wherein the protease is a pathogen-specific protease.

9. The compound or salt of claim 1, wherein the protease-sensitive peptide is not QPVV, DAPV or GPQGIAGA.

10. The compound or salt of claim 1, wherein said compound is a salt comprising a counter ion selected from the group consisting of F.sup.−, Cl.sup.−, Br.sup.−, ##STR00034##

11. A bioresponsive sensor comprising the compound or salt of claim 1.

12. A method of detecting an inflammation in the oral cavity of a human patient comprising the step of applying a compound or salt according to claim 1 or a bioresponsive sensor comprising such a compound or salt to the oral cavity of said human patient.

13. A method for the preparation of the bioresponsive sensor according to claim 11, said method comprising the step of linking an amino-modified denatonium compound to the C-terminus of a protease-sensitive peptide.

14. The method of claim 13, wherein said method further comprises the performance of one or both of the following steps prior to said linking step: preparing a protease-sensitive peptide by solid phase peptide synthesis, and protecting the N-terminus of the protease-sensitive peptide with a protecting group, preferably acetic anhydride.

15. A diagnostic chewing gum or a diagnostic confectionary comprising the compound or salt of claim 1.

16. The compound or salt of claim 5, wherein the prof ease-sensitive peptide comprises 4 to 15 amino acids.

17. The compound or salt of claim 5, wherein the protease-sensitive peptide comprises 5 to 12 amino acids.

18. The compound or salt of claim 5, wherein the protease-sensitive peptide comprises 6 to 10 amino acids.

19. The compound or salt of claim 5, wherein the protease-sensitive peptide consists of 7 to 9 amino acids.

20. The method according to claim 13, wherein the amino-modified denatonium compound is a compound of formula [II] or formula [IIa-1].

Description

BRIEF DESCRIPTION OF THE FIGURES

[0080] FIG. 1 shows as graphical representation of the cleavage rate of [BRS-1] and [CC1] accompanied by the standard deviations.

[0081] FIG. 2 is a graphical representation of the cleavage rate of [IIa-1]+AA of 16 of the 20 proteinogenic amino acids.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0082] In a first aspect, the present invention relates to a compound comprising denatonium linked to the C-terminus of a protease-sensitive peptide; or a salt thereof. Preferably, the compound is a compound according to formula [I]:

##STR00011##

[0083] wherein

[0084] R.sup.1 is an optionally substituted C.sub.1-3 alkylene group, and

[0085] R.sup.2 is the protease-sensitive peptide; or a salt thereof. Preferably R.sup.1 is an unsubstituted C.sub.1-3 alkylene group. More preferably R.sup.1 is methylene, ethylene or propylene. Most preferably, R.sup.1 is methylene.

[0086] Preferred embodiments of the compound according to formula [I] are selected from the following group consisting of [Ia] to [Ic], wherein [Ia] is particularly preferred:

##STR00012##

[0087] wherein R.sup.1 and R.sup.2 are as defined above.

[0088] In one embodiment, the compound according to formula [Ia] is selected from the group consisting of [Ia-1] to [Ia-3], wherein formula [Ia-1] is preferred:

##STR00013##

[0089] wherein R.sup.2 is the protease-sensitive peptide.

[0090] In another embodiment, the compound according to formula [Ib] is selected from the group consisting of [Ib-1] to [Ib-3]:

##STR00014##

[0091] wherein R.sup.2 is the protease-sensitive peptide.

[0092] In another embodiment, the bioresponsive sensor according to formula [Ic] is selected from the group consisting of [Ic-1] to [Ic-3]:

##STR00015##

[0093] wherein R.sup.2 is the protease-sensitive peptide.

[0094] The amino acid sequence of the protease-sensitive peptide is not particularly limited as long as it is susceptible to cleavage by an endopeptidease. Preferably the protease-sensitive peptide has a length of at least 4 amino acids, preferably it has a length of 4 to 15, more preferably 5 to 12, even more preferably 6 to 10, yet even more preferably 7 to 9, most preferably 8 amino acids.

[0095] The amino acids of the protease-sensitive peptide are independently selected from the group consisting of alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0096] In one embodiment, the amino acids are independently selected from the group consisting of alanine (A), arginine (R), aspartic acid (D), glutamine (Q), glutamic acid (E), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0097] In another embodiment, the amino acids are independently selected from the group consisting of alanine (A), glutamine (Q), glycine (G), isoleucine (I), proline (P).

[0098] To obtain maximum gustatory perception, the amino acid directly attached to the nitrogen atom of the amino-modified denatonium, i.e. the C-terminal amino acid of the protease-sensitive peptide, is preferably selected from the group consisting of alanine (A), arginine (R), aspartic acid (D), glutamine (Q), glutamic acid (E), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0099] More preferably, the C-terminal amino acid of the protease-sensitive peptide is selected from the group consisting of alanine (A), arginine (R), aspartic acid (D), glutamine (Q), glutamic acid (E), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), tryptophan (W) and tyrosine (Y).

[0100] More preferably, the C-terminal amino acid of the protease-sensitive peptide is selected from the group consisting of alanine (A), arginine (R), glutamine (Q), leucine (L), methionine (M) and phenylalanine (F).

[0101] In another preferred embodiment, the C-terminal amino acid of the protease-sensitive peptide is selected from the group consisting of alanine (A), arginine (R), glutamic acid (E), leucine (L), methionine (M) and phenylalanine (F).

[0102] In one embodiment, the protease is a pathogen-specific protease. The pathogen may be selected from the group consisting of virus, bacterium, protozoa, prion, fungus and combinations thereof. According to a preferred embodiment of the present invention, the protease or proteolytic enzyme is released or, in case of a virus or a prion, upregulated, by pathogens, preferably by bacteria, viruses, protozoa or fungi, more preferably the following class, order, genera, family of species of herpes, varicella, parvovirus, papillomavirus, polyomavirus, adenovirus, hepadnavirus, variolavirus, picornavirus, aso- and caliciavirus, human cytomegalovirus, hepatitis-A-virus, hepatitis-C-virus, hepatitis-E-virus, togavirus, flavivirus, coronavirus, retrovirus, HIV, reovirus, orthomyxovirus, bunyavirusarenavirus, human rhinovirus, dengue virus, varicella-zoster virus, paramyxovirus, rubulavoris, morbillivirus, west nile virus, yellow fever virus, pneimovirus, non classified paramyxovirus, rhabdovirus, folovirus, viroids and prions, staphylococcus, streptococcus and enterococcus, bacillus, listeria, erysipelothrix, garderella, corynebacterium, actinomyces, mycobacterium, nocardia, neisseria, acinetobacter and moraxella, enterbacteriacea including salmoneslla shigella, yersinia, E. coli and vibrio, aeromonas, plesiomonas, haemophilus, pasteurella, campyhlobacter, heliobacter, spirillum, pseudomonas, stenotropomonas, burkholderia, legionella, brucella, bordetella francisella, bacteriodaceae ioncluding trepponema, borrelia peptospira rickettsia, coxiella, orientia, ehrlichia, baronella afipia, chlamydia, mycoplasma and histoplasma, coccidioides, blasomyces, paracoccidioides, candida, aspergillus, Cryptococcus, mucor, absidia, rhizopus, phaeohyphomycetes, hyalohyphomycetes, penicillium, pneumocystis, tyrpanoma, leishmania, giradia, trichomonas, entamoeba, naegleria, toxoplasma isspora, cyclospora, sarcocystis, cryptosporidium, plasmodium, babesia, microsporida, and balantidium.

[0103] The protease may be selected from the group consisting of: KSHV-, HSV-, HAV-; HCV-, HIV-, human cytomegalovirus-, Yellow fever, CMV-, HRV14-, HRV2a-, Malaria aspartyl-, Sars protease, proteases of the S1, S2, S6, S8, S9, S33, S11, S12, S26, S18 family, streptomyces trans- and carboxypeptiidases, signal peptidase I, Omtpin and Clp, C10C11, C15, C25 cysteine proteases, Porphyromonas gingivalis cyxteine proteases, sortase, metalloproteases of the thermolysin family (m4), Metalloproteases of the M9 family inclusive of vibrio and clostridium collagenases, Serralysin and related M10 Proteases and proteases of the M12 family, bacterial metallo exopeptidases, proteases of the M19, M20, M22, M23, and M26 families, tetanus and botulinum beurotoxins as being part of a group of bacterial metalloproteases, anthrax toxin lethal factor, lysostaphin and aureolysin, and AAA proteases.

[0104] In a preferred embodiment the pathogen is a pathogen listed in Table 1 of WO 2013/132058 A1, the content of which is incorporated herein in its entirety.

[0105] The protease-sensitive peptide may be selected from SEQ ID NOs:1-141 disclosed in WO 2013/132058 A1. The amino acid sequences of SEQ ID NOs:1-141 disclosed in WO 2013/132058 A1 are incorporated herein by reference.

[0106] In a particular embodiment, the protease-sensitive peptide comprises or consists of the amino acid sequence GPQGIAGQ.

[0107] In another embodiment the protease-sensitive peptide is not GPQGIAGA, DAPV or QPVV.

[0108] In yet another embodiment, the C-terminal amino acid of the protease-sensitive peptide is not alanine or valine.

[0109] In another embodiment the compound or salt of the invention does not comprise trifluoracetate (TFA). In another embodiment the salt of the invention comprises trifluoracetate (TFA).

[0110] In one embodiment, the compound of the invention is a compound of formula [BRS-X], wherein X stands for an amino acid:

##STR00016##

[0111] In a particular preferred embodiment, the compound of the invention is a compound of formula [BRS-1]:

##STR00017##

[0112] In another aspect the present invention relates to a salt of a compound of formula [I] as defined hereinabove. The salt typically comprises a nutritionally or pharmaceutically acceptable counter ion. The counter ion is preferably selected from the group consisting of [0113] F.sup.−, Cl.sup.−, Br.sup.−,

##STR00018##

[0114] In yet another aspect the invention relates to a bioresponsive sensor comprising the compound of the present invention or the salt of the present invention. In a preferred embodiment the bioresponsive sensor does not comprise a base material or particles embedded and/or attached to said base material. In another preferred embodiment the bioresponsive sensor does not comprise particles. In another preferred embodiment the bioresponsive sensor substantially consists of the compound or salt of the invention.

[0115] In yet another aspect the invention relates to the compound, salt or bioresponsive sensor of the present invention for use in a method of detecting an inflammation in the oral cavity of a human patient. In accordance with the invention, the detection occurs directly in the oral cavity of the patient by the use of the human tongue (sense of taste) and the nose (sense of smell) as a detector, in particular by the sense of taste. If the patient suffers from an inflammation of the oral cavity, the bioresponsive diagnostic sensor comprised e.g. in a chewing gum comes into contact with the saliva containing the matrix metalloproteinases (MMP) long enough to react. Accordingly, it is possible for the patient himself to detect an inflammation in the oral cavity, e.g. periodontal disease and/or peri-implantitis by gustatory detection of a bitter taste. Therefore, a person using the inventive chewing gum knows when detecting a bitter taste, that an inflammation in the oral cavity, e.g. periodontal disease and/or peri-implantitis is present.

[0116] In another aspect the present invention provides a diagnostic chewing gum comprising the compound, salt or bioresponsive sensor of the present invention. The diagnostic chewing gum is typically used for detecting an inflammation, e.g. periodontal disease and/or peri-implantitis.

[0117] The present invention is further directed to a method of providing the inventive compound, salt or bioresponsive sensor. It is further directed to a method of providing a chewing gum comprising the inventive compound, salt or bioresponsive sensor.

[0118] According to the method of the invention for the preparation of a bioresponsive sensor, the protease-sensitive peptide is typically synthesized by solid-phase peptide synthesis (SPPS). Since sensory measurements with an electronic tongue showed that already four amino acids (AA) effectively mask the bitter taste of denatonium, no poly(methylmethacrylate) PMMA particles are needed anymore. The N-terminus of the protease-sensitive peptide is acetylated after the solid-phase peptide synthesis (SPPS) with acetic anhydride to prevent attack of the aminopeptidase (AP), and to prevent side reactions in further synthetic steps. Cleaved from the resin, the acetylated protease-sensitive peptide is now linked to Den-CH.sub.2—NH.sub.2 via 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU). Thereby, a bioresponsive sensor with the general formula [BRS-x] is obtained, wherein X is selected from the group of the 20 proteinogenic amino acids independently from each other:

##STR00019##

[0119] In another aspect, the present invention relates to a compound of formula [II]

##STR00020##

[0120] wherein

[0121] R.sup.1 is an optionally substituted C.sub.1-3 alkylene group. The preferred embodiments of R.sup.1 in formula [II] correspond to the preferred embodiments of R.sup.1 as defined above for formula [I].

[0122] The compound according to formula [II] can be coupled to a protease-sensitive peptide to provide the compound of formula [I]. When the compound according to formula [I] comes into contact with the saliva of a person having a disease of the oral cavity, e.g. periodontitis or peri-implantitis, the compound according to formula [II] is released.

[0123] Preferred embodiments of the compound according to formula [II] are selected from the following group consisting of [IIa] to [IIc], wherein [IIa] is particularly preferred:

##STR00021##

[0124] wherein

[0125] R.sup.1 is an optionally substituted C.sub.1-3 alkylene group.

[0126] In one embodiment, the compound of formula [IIa] is selected from the group consisting of [IIa-1] to [IIa-3], wherein formula [IIa-1] is particularly preferred:

##STR00022##

[0127] In another embodiment, the bioresponsive sensor according to formula [IIb] is selected from the group consisting of [IIb-1] to [IIb-3]:

##STR00023##

[0128] In another embodiment, the bioresponsive sensor according to formula [IIc] is selected from the group consisting of [IIc-1] to [IIc-3]:

##STR00024##

[0129] In a preferred embodiment the invention relates to a compound having the formula [IIa-1]:

##STR00025##

[0130] The compound is a stable Den-CH.sub.2—NH.sub.2 compound which comprises a methylamine group with which it can be attached to the peptide chain of a protease-sensitive peptide.

EXAMPLES

Example 1: Synthesis of Denatonium-CH.SUB.2.—NHBoc

[0131] 1.2 g lidocain (5.2 mmol) and 1.0 g tert-butyl-4-(bromomethyl)benzylcarbamate (3.4 mmol) were heated to 80° C. until the formation of a yellow melt. The melt then became highly viscous, up to solid, and subsequently, after a resting period of 10 minutes at 80° C., the obtained yellow solid was treated with 40 mL of a mixture of ethyl acetate and n-hexane (1:1). To obtain the protected product, the yellow solid was stirred for 10 minutes at 80° C. in the mixture. The resulting white residue was filtered off and washed with a mixture of ethyl acetate and hexane (1:1). 1.6 g of the protected product were obtained.

[0132] Sum formula: C.sub.27H.sub.40N.sub.3O.sub.3.sup.+Br.sup.−

[0133] Molecular mass: 534.5 g/mol

[0134] Yield: 1.6 g (91%).

Example 2: Synthesis of a Denatonium-CH.SUB.2.—NH.SUB.2 .Salt

[0135] For the subsequent deprotection, 228 mg of the product (0.4 mmol) obtained from Example 1 was dissolved in 1 mL of trifluoroacetic acid and shaken at room temperature for 1 hour. The raw product was precipitated in diethylether and filtered off. The product was purified by chromatography.

[0136] Sum formula: C.sub.22H.sub.33N.sub.3O.sup.2+2 C.sub.2F.sub.3O.sub.2.sup.−

[0137] Molecular mass: 581.6 g/mol

[0138] Yield: 175 mg (75%).

[0139] .sup.1H-NMR (DMSO, δ [ppm], J [Hz]): δ 10.14 (s, 1H), 8.30 (s, 3H), 7.62 (m, 4H), 7.20-7.10 (m, 3H), 4.84 (s. 2H), 4.18 (s, 2H), 4.12 (q. .sup.3J.sub.H,H=5.8, 2H), 3.54-3.48 (m, 4H), 2.20 (s, 6H), 1.42 (t, .sup.3J.sub.H,H=7.1, 6H).

[0140] .sup.13C-NMR (DMSO, δ [ppm], J [Hz]): δ 162.7 (s, 1C), 136.8 (s, 1C), 135.5 (s, 2C), 133.7 (s, 1C), 133.6 (s. 2C), 129.9 (s, 2C), 128.5 (s, 2C), 128.2 (s, 1C), 127.7 (s, 1C), 61.6 (s, 1C), 56.0 (s, 1C), 54.9 (s, 2C), 42.2 (s, 1C), 18.6 (s, 2C), 8.3 (s, 2C).

Example 3

[0141] The protease-sensitive peptide was synthesized using solid-phase peptide synthesis to 2-chlorotrityl chloride resin. For the proof-of-concept of the system, the amino acid sequence GPQGIAGQ was prepared. [0142] H-G-P-Q-G-I-A-G-Q-OH [0143] [PSP-1] (m/z=726.37)

[0144] This sequence was acetylated at the N-terminus according to a method known per se, so that a protected protease-sensitive peptide 2 [PSP-2] was obtained: [0145] Ac-G-P-Q-G-I-A-G-Q-OH [0146] [PSP-2] (m/z=768.38)

[0147] Using HATU/DIPEA, [PSP-2] was then linked to compound [IIa-1]. Thereby the bioresponsive sensor 1 [BRS-1] was obtained.

##STR00026##

Example 4: Cleavage Experiments

[0148] After incubation of the construct with MMP 1, 8 and 9, the following cleavage products CP1 and CP2 were obtained.

##STR00027##

[0149] In the cleavage experiments, compound [BRS-1] was incubated with an equimolar mixture of MMP 1, 8 and 9 at 37° C. in MMP buffer (200 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl.sub.2), 1 mM ZnCl.sub.2, 0.05% Brij 35, pH 7.0).

[0150] For comparison, a comparative construct [CC1], according to the prior art (Ritzer, J et al. Nat. Commun. volume 8, Article number: 264 (2017)) was incubated to evaluate the cleaving efficiency of the new system. In [CC1] denatonium is connected via a carboxylic acid group to the N-terminus of a protease-sensitive peptide, as given below:

##STR00028##

[0151] The concentration of the two compounds was 0.1 mM. The cleavage efficiency was measured using a Hitachi Elite LaChrom HPLC system (VWR, Darmstadt, Germany) with a ZORBAX Eclipse XDB-C18 column (4.6 mm internal diameter, 150 mm length (Agilent, Santa Clara, Calif.)), eluent A (0.1%). TFA in water, (v/v)) and eluent B (0.1% trifluoroacetic acid (TFA) in acetonitrile (v/v)) with a gradient of 5 to 95% eluent B over 55 min. The UV absorption was measured at I=214 nm (Table 1, FIG. 1).

TABLE-US-00001 TABLE 1 cleavage rate (%) of [BRS-1] and [CC1] accompanied by the standard deviations. [BRS-1] [CC1] Cleaved construct after 10 min 28.0% ± 10.9% 41.6% ± 6.29%  Cleaved construct after 30 min 49.3% ± 11.7% 68.4% ± 0.587% Cleaved construct after 120 min 81.8% ± 5.09% 80.2% ± 1.78%  Cleaved construct after 120 min  98.0% ± 0.0583% 89.2% ± 0.888%

[0152] The cleavage efficiency of both systems was comparable. The slightly better cleavage rate of [CC1] is caused by the fact that it was not the final construct that was analyzed here. Due to the lack of coupling with a spatially demanding PMMA particle, the overall system is further enlarged and thus less susceptible to MMP. On the other side, [BRS-1] is the final construct used as bioresponsive sensor in the detection of inflammations in the oral cavity.

[0153] When [CP2] is obtained after cleavage of [BRS-1] it is now possible for the aminopeptidase to attack the free N-terminus of [CP2], so that further cleavage to [IIa-1] occurs. Previously, in the full construct [BRS-1] this further cleavage process is prevented by the acetyl protecting group. Therefore, no degradation occurs in [BRS-1], when no MMP is present as it is the case in healthy individuals. [CC1] is cleaved by aminopeptidase analogously. However, in the case of [CC1] an amino acid (in this case lysine) remains on the denatonium-COOH, since it is attached to the ε-position. This significantly reduces the gustatory perception compared to free denatonium.

[0154] To trigger maximum gustatory perception, the last amino acid from the modified denatonium must also be removed by the aminopeptidase. Not all amino acids are equally well suited for this purpose. To evaluate the cleavage efficiency, 20 constructs consisting of one proteinogenic amino acid coupled to compound [IIa-1] were synthesized, thereby obtaining compound [Ia-X], wherein X is an amino acid:

##STR00029##

[0155] The resulting constructs were incubated with AP for 24 h and analyzed by LC/MS for complete degradation (Table 2).

TABLE-US-00002 TABLE 2 Cleavage Experiments of different compounds with AP und their detectability after 24 h. + stands for detectability, − stands for full degradation to [IIa-1]. Detectable after 24 h [Ia-X] with X= incubation with AP Alanine − Arginine − Asparagine + Aspartic acid + Cysteine − Glutamine + Glutamic acid − Glycine + Histidine + Isoleucine + Leucine − Lysine − Methionine − Phenylalanine − Proline + Serine − Threonine + Tryptophan + Tyrosine − Valine +

[0156] Subsequently, cleavage experiments were performed for 20 min, 120 min and 24 h with an aminopeptidase concentration of 940 ng/ml of compound Ia-X. Only 16 out of 20 constructs were used, as lysine, asparagine, cysteine, and histidine did not produce significant synthesis yields, which also would interfere with subsequent peptide coupling.

Example 5: Synthesis Example

[0157] The invention is further exemplified by the following example of the synthesis of [BRS-1]:

##STR00030##

[0158] [PSP-2] (71.3 mg, 0.0928 mmol) was dissolved with HATU (34.0 mg, 0.0894 mmol) in anhydrous dimethylformamide (DMF) (2 mL). Subsequently, [IIa-1] (63.0 mg, 0.178 mmol) was added and after complete dissolution DIPEA (31.9 μL) was added. The mixture was stirred for 18 h protected from light at room temperature. Then ice-cold diethyl ether (20 mL) was added. After centrifugation and decantation of the supernatant, the residue was dried overnight.

[0159] The residue was purified using a FPLC system (Äkta purifier, GE Healthcare) using reversed phase chromatography (RPC) on a C18 column (Phenomenex®) (eluent A: 0.1% TFA in water, eluent B: 0.1% TFA in acetonitrile). After subsequent lyophilization, [BRS-1] was obtained as a colorless (white) powder. The overall yield of high purity [BRS-1] was 5.32 mg (5.19%).