Acylated Aminooligosaccharides with Inhibitory Effects against α-Amylase from Streptomyces sp. HO1518

Five new acylated aminooligosaccharides (1–5), together with one known related analogue (6), were isolated from Streptomyces sp. HO1518. Their structure was identified by extensive spectroscopic analysis, including 1D and 2D NMR data and high resolution electrospray ionization mass spectrometry (HRESIMS), and by comparison with those reported in the literature. All of the new compounds showed more promising porcine pancreatic α-amylase (PPA) inhibitory activities than the clinical drug acarbose, indicating them as potential pharmaceutical drug leads toward type II diabetes.


Introduction
α-Amylase, belonging to glycoside hydrolase family 13 (GH 13), is widely found in organisms, including plants, animals, bacteria, and fungi [1]. It is an endoamylas, which catalyzes the hydrolysis of α-(1 → 4)-D-glucosidic bind of starch, amylase, amylopectin, glycogen, and various maltodextrins into smaller oligomers [2]. The inhibitors of α-amylase are of pharmacological importance, as α-amylase is considered as an attractive target for treating elevated post-prandial blood glucose levels resulting in type II insulin-independent diabetes, obesity, and other related secondary symptoms [3]. The well-known acarbose, originated from Actinoplanes species, is recognized as one of the most clinically important α-amylase inhibitor [4]. It is a member of the aminooligosaccharides family exclusively produced by microorganisms, particularly soil bacteria of the order of Actinomycetes. Structurally, aminooligosaccharides contain a single or repeated pseudo-trisaccharide core(s) with D-glucose units attached to the reducing and non-reducing end through α-(1 → 4) glycosidic bond [4,5]. Pseudo-trisaccharide core is formed by an acarviosine moiety and a D-glucopyranose group through an α-(1 → 4) quinovosidic bond; acarviosine is composed of an unsaturated C 7 N aminohexitol (valienamine) unit and a 4,6-dideoxy-D-glucopyranose (4-deoxy-D-quinovopyranose) unit; and the amino-hexitol bond is defined as pseudo-glycosidic bond. Therefore, aminooligosaccharides as acarviostatins followed by a Roman numeral and two numbers, i.e., acarviostatin I01 (acarbose). Acarvios originates from the acarviosine core; the Roman numeral represents the number of the pseudo-trisaccharide cores; the middle digit indicates the number of glucose residues at the noreducing end and the last digit infers the number of glucose residues at the reducing end. Generally, an extensive MS study is helpful to discriminate the structure of aminooligosaccharides, due to their characteristic positive-ion ESIMS/MS fragmentation pattern [6][7][8][9][10][11][12][13][14][15][16]. The formation of bi and yj fragment ions is typical of glycosidic bond dissociation of protonated oligosaccharides. Every glycosidic bond could be dissociated, including the pseudo-glycosidic bond within the acarviosin moiety. Moreover, the cleavage of the quinovosidic bond in the pseudo-trisaccharide core happened more readily than that of an ordinary glycosidic bond in the molecule. Until now, guided by the MS study, hundreds of aminooligosaccharides have been quickly identified, including trestatins [6,7], isovalertatins [8,9], butytatins [10], acarviostatins [11][12][13][14][15], SF638-1 [16], as well as artificial acarbose analogues (i.e., G6-Aca, G12-Aca) [2]. Many have shown more potent porcine pancreatic α-amylase (PPA) activities than acarbose, among which acarviostatins III03 [11] was the most effective PPA inhibitor known to data. It was reported that the acarviosine unit was postulated to be essential for its biological activity [17]. In addition, the number of the pseudo-trisaccharide cores along with the Dglucose residues also affected the PPA inhibitory activity [18].
As part of our ongoing search for novel biologically active natural products from marine actinomycetes [19][20][21][22], we chemically investigated Streptomyces sp. HO1518 from sediments collected off Yellow Sea, close to Rizhao, Shandong province, China. Fractionation of the resins-absorbed extract of the liquid fermentation broth of the strain led to the isolation of five new metabolites 1-5 ( Figure 1), namely acylated aminooligosaccharides, (4), and D6-O-β-hydroxybutyryl-acarviostatin I03 (5), as well as one known related analogue, acarviostatin II03 (6). This paper describes the isolation, structural elucidation, and PPA inhibitory activity of these metabolites.

Results and Discussion
A large-scale fermentation broth of Streptomyces sp. HO1518 was collected and absorbed with Amberlite XAD-16 resins to produce a crude extract. The subsequent fractionation by repeated column chromatography over ODS-C18 and semi-preparative reversed-phase HPLC afforded the pure acylated aminooligosaccharides 1-5, together with one known related one (6 All the new compounds showed similar IR absorptions indicative of the presence of oligosaccharide (~3300 and ~1000 cm −1 ), ester carbonyl (~1720 cm −1 ), and double bond(s) (~1640 cm −1 )

Results and Discussion
A large-scale fermentation broth of Streptomyces sp. HO1518 was collected and absorbed with Amberlite XAD-16 resins to produce a crude extract. The subsequent fractionation by repeated column chromatography over ODS-C 18 and semi-preparative reversed-phase HPLC afforded the pure acylated aminooligosaccharides 1-5, together with one known related one (6 All the new compounds showed similar IR absorptions indicative of the presence of oligosaccharide (~3300 and~1000 cm −1 ), ester carbonyl (~1720 cm −1 ), and double bond(s) (~1640 cm −1 ) and they also exhibited similar positive-ion HRESIMS/MS fragmentation pattern, which further divided them into two groups. As for 1 and 2 ( Figure 2, Figures S19 and S41), their common fragment ion peaks at m/z 304 (b2), 466 (b3), 624 (b4), and 769 (b5) were immediately discriminated, the same as those of co-occurring acarviostatin II03 (6), while the fragment ion peaks at m/z y5-y8 and b6-b8 in 1 and 2, along with their pseudo-molecular ion peaks, were 42 and 86 mass units more than those of 6 ( Figure 2 and Figure S105), respectively. According to 3-5 ( Figure 3, Figures S60, S79 and S98), they possessed the sole mutual fragment ion at m/z 304 (b2), with their primary MS ion peaks and all the other secondary MS/MS ion peaks at m/z y4, y5 and b3-b5 in 3-5 being 42, 56, and 86 mass units more than those of the model acarviostatin I03 (7) (Figure 3 and Figure S107), previously obtained from the soil Streptomyces coelicoflavus ZG0656 [11], respectively. Moreover, their NMR data was also reminiscent of those of acarviostatin II03 (6) and acarviostatin I03 (7). In fact, 1 and 2 like co-occurring 6 exhibited the same acarviostatin II03-type core, with their acyl units linked to the C-D6 ester differing, while 3-5 showed the same acarviostatin I03-type core as in model 7, differ from each other also in acyl units linked to the C-D6 ester. Further alkaline hydrolysis of 1 and 2 afforded precursor 6, while treatment of 3-5 with methanolic ammonium hydroxide provided precursor 7.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 14 and they also exhibited similar positive-ion HRESIMS/MS fragmentation pattern, which further divided them into two groups. As for 1 and 2 (Figures 2, S19 and S41), their common fragment ion peaks at m/z 304 (b2), 466 (b3), 624 (b4), and 769 (b5) were immediately discriminated, the same as those of co-occurring acarviostatin II03 (6), while the fragment ion peaks at m/z y5-y8 and b6-b8 in 1 and 2, along with their pseudo-molecular ion peaks, were 42 and 86 mass units more than those of 6 ( Figures 2 and S105), respectively. According to 3-5 (Figures 3, S60, S79 and S98), they possessed the sole mutual fragment ion at m/z 304 (b2), with their primary MS ion peaks and all the other secondary MS/MS ion peaks at m/z y4, y5 and b3-b5 in 3-5 being 42, 56, and 86 mass units more than those of the model acarviostatin I03 (7) (Figures 3 and S107), previously obtained from the soil Streptomyces coelicoflavus ZG0656 [11], respectively. Moreover, their NMR data was also reminiscent of those of acarviostatin II03 (6) and acarviostatin I03 (7). In fact, 1 and 2 like co-occurring 6 exhibited the same acarviostatin II03-type core, with their acyl units linked to the C-D6 ester differing, while 3-5 showed the same acarviostatin I03-type core as in model 7, differ from each other also in acyl units linked to the C-D6 ester. Further alkaline hydrolysis of 1 and 2 afforded precursor 6, while treatment of 3-5 with methanolic ammonium hydroxide provided precursor 7.      D6-O-acetyl-acarviostatin II03 (1) was isolated as a white amorphous powder, and its molecular formula, C58H96N2O41 was established by its HRESIMS of pseudo-molecular ion peak [M + H] + (m/z 1477.5564, cald 1477.5561), implying 12 site of unsaturation. The 13 C NMR (Table 1) and DEPT spectra revealed the following carbon types: Three methyl, seven sp 3 methylene, two sp 2 methine, forty-three sp 3 methine, and three sp 2 quaternary carbons. Three of the twelve degrees of unsaturation were accounted for by the two tri-substituted double bonds (δ 139. 4   D6-O-acetyl-acarviostatin II03 (1) was isolated as a white amorphous powder, and its molecular formula, C 58 H 96 N 2 O 41 was established by its HRESIMS of pseudo-molecular ion peak [M + H] + (m/z 1477.5564, cald 1477.5561), implying 12 site of unsaturation. The 13 C NMR (Table 1) and DEPT spectra revealed the following carbon types: Three methyl, seven sp 3 methylene, two sp 2 methine, forty-three sp 3 methine, and three sp 2 quaternary carbons. Three of the twelve degrees of unsaturation were accounted for by the two tri-substituted double bonds (δ 139.4 and 129.2; δ 141.8 and 126.6) and one carbonyl group (δ C 177.0) observed in the 13 C NMR spectrum. Consequently, 1 should have nine rings (residues A-I). The NMR data of 1 was similar to those of co-occurring known acarviostatin II03 (6), showing the characteristic terminal unit of acarviosin in the non-reducing end, the typical reducing D-glucose terminus, as well as an inner acarviosin moiety. In fact, careful comparison of the NMR data of 1 and 6 revealed that the only difference between them resided in an additional acetyl group resonating at δ H 2.17 (3H, s) and δ C 177.0 (qC) and 23.1 (CH 3 ) in 1, in agreement with the 42 mass units difference between them, while the rest of the structure of 1 was the same as in 6. Due to the effect of the acetyl group, a series of 1 H NMR data changes associated with the inner pseudo-trisaccharide core (residues D-F) were clearly detected in 1. The usually overlapped methylene signal in the D-glycopyranose residue D was obviously downfield shift from , and 304 (b2) formed by the cleavage of quinovosidic, pseudoglycosidic, and glycosidic bonds. The complete 1 H and 13 C assignment of the 1 was inferred by combing information from the 1D-selective TOCSY, HSQC, HSQC-TOCSY, and HMBC experiments. Finally, the configuration of the glycosidic bonds of 1 was deduced to be α-(1 → 4), the same as that of 6, confirmed by the 1 H-1 H coupling constants of the anomeric protons and the NOESY experiment and proved by the chemical conversion between 1 and 6. Therefore, the structure of D6-O-acetyl-acarviostatin II03 (1) was established, as shown in Figure 1.   . The presence of the β-hydroxybutyryl group was supported by the newly appeared CH 3 CH(OH)CH 2− spin-spin coupling system in the TOCSY spectrum ( Figure 5) and by the HMBC correlation ( Figure 5) between the oxy-methine (δ H 4.27) and the ester carbonyl (δ C 176.6, qC). Accordingly, the 13 C NMR data in the inner pseudo-trisaccharide core (residues D-F) in 2 remained unaffected, although different acyl groups, β-hydroxybutyryl group in 2 vs. acetyl group in 1, were esterified at C-D6. Moreover, the similar 13 (Table 2) spectrum revealed 39 carbon resonances, which were classified by DEPT and HSQC experiments as a ester carbonyl (δ 177.0), a trisubstituted double bond (δ 142.3 and 126.1), two methyls, five sp 3 methylenes, and twenty-nine sp 3 methines. The olefin and carbonyl groups accounted for two of the eight degrees of unsaturation, therefore, 3 should be in hexacyclic ring system (residues A-F). Its 1 H and 13 C NMR data ( Table 2) closely resembled those of 1, suggesting that 3 possessed a single pseudo-trisaccharide core with three D-glucose units on the reducing end and also an acetyl ester at C-D6, in analogy to 1. In fact, 3 differs from 1 only by the absence of the repeated pseudo-trisaccharide core in the non-reducing end in 1, in agreement with 465 mass units less than that of 1, which was supported by the disappearance of the characteristic NMR signals of residues F-H in 1 resonating at δH 3.53 (H-F1)/δC 57.8 (C-F1),  Figures 3A,B and S60) at m/z 854 (y5), 832 (b5), 670 (b4), 508 (b3), and 304 (b2) allowed to locate  (Table 2) spectrum revealed 39 carbon resonances, which were classified by DEPT and HSQC experiments as a ester carbonyl (δ 177.0), a tri-substituted double bond (δ 142.3 and 126.1), two methyls, five sp 3 methylenes, and twenty-nine sp 3 methines. The olefin and carbonyl groups accounted for two of the eight degrees of unsaturation, therefore, 3 should be in hexacyclic ring system (residues A-F). Its 1 H and 13 C NMR data ( Table 2) closely resembled those of 1, suggesting that 3 possessed a single pseudo-trisaccharide core with three D-glucose units on the reducing end and also an acetyl ester at C-D6, in analogy to 1. In fact, 3 differs from 1 only by the absence of the repeated pseudo-trisaccharide core in the non-reducing end in 1, in agreement with 465 mass units less than that of 1, which was supported by the disappearance of the characteristic NMR signals of residues F-H in 1 resonating at δ  Figure S60) at m/z 854 (y5), 832 (b5), 670 (b4), 508 (b3), and 304 (b2) allowed to locate the acetyl group at OH-D6, thus completely defining the structure of 3. Finally, the small 1 H-1 H coupling constants of the anomeric protons and its ROESY spectrum indicated the glycosidic bonds to be α-(1 → 4), identical to those in 7, which was supported by chemical conversion of 3 to 7. Therefore, compound 3 is an acetyl derivative of 7, namely as D6-O-acetyl-acarviostatin I03.    Figure S79). The configuration of glycosidic bonds in 4 were deduced to be α-(1 → 4) by analysis of the coupling constants of the anomeric protons and its ROESY spectrum, the same as those in 3, thereby completing the structure of 4.
D6-O-β-hydroxybutyryl-acarviostatin I03 (5) (Table 2) of 5 with those of 3 and 4 suggested that the three compounds had the same acarviostatin I03-type basic core. In fact, the only difference among them existed at C-D6, where the acetyl group in 3 or the propionyl group in 4 was replaced by the β-hydroxybutyryl group in 5. Moreover, the β-hydroxybutyryl group at C-D6 was further confirmed by comparison of its NMR data with those of 2, which showed great similarity due to the same partial structure between 2 and 5 except for the absence of the repeated pseudo-trisaccharide unit in 2. All the 1D-selective TOCSY, HSQC, HSQC-TOCSY, and HMBC experiments together with the HRESIMS/MS fragmentation pattern ( Figure 3A,D and Figure S98) unambiguously determined the structure of 5. Analogously to 2-4, the configuration of the glycoside bonds in 5 was deduced to be α-(1→4) by analysis of the 1 H-1 H coupling constants of the anomeric protons and by interpretation of its ROESY experiment. On the basis of above evidences, the structure of β-hydroxybutyryacarviostatin I03 (5) was determined as depicted.
Additionally, both 2 and 5 possess a common β-hydroxybutyryl side chain. There is a chiral carbon at the β position of the carbonyl function. To determine its absolute chemistry, the alkaline hydrolysis method followed by methyl esterification reaction [23,24] was applied to 2 and 5. Unfortunately, the efforts for obtaining its corresponding methyl 3-hydroxybutyrate failed, mainly due to the limit amount of 2 and 5 obtained. Therefore, the absolute configuration in the side chain still remains undetermined.
Aminooligosaccharides are of particular interest, due to their unique structures and promising α-amylase inhibitory activities. Although aminooligosaccharides continues to be found in recent years [14,15], acylated acarviostatins are relatively rare. Actually, to our knowledge, only eleven isovalertatins [8,9] and two butytatins M03 and M13 [10] from soil Streptomyces luteogrise, have been isolated and purified to data. All actylated acarviostatins described in the current work are C-D6 ester. The discovery of new acylated acarviostatins 1-5 from marine Streptomyces strains has added to an extremely diverse and complex array of aminooligosaccharides, which is still rapidly expanding.
As mentioned above that the inhibitors of α-amylase are of pharmacological importance against diabetes, obesity, and hyperlipidema. In our screening program to search for α-amylase inhibitors from the Yellow Sea marine actinomycetes, we evaluated compounds 1-5 for their inhibitory activity against PPA and the results revealed that all new isolates showed promising PPA inhibitory activities with IC 50 values ranging from 0.03 to 0.70 µM (Table 3), of which 1 (0.029 µM) and 2 (0.049 µM), being similar to 6, were ca. 540-and 320-fold, while 3-5 were ca. 23-to 80-fold, stronger than the positive control acarbose (15.7 µM). Interestingly, the β-hydroxybutyryl group substituted in ring D slightly increased the PPA inhibitory activity in 5 compared to 3 and 4, but it simultaneously somewhat reduced the PPA inhibitory activity in 2 compared to 1 and 6. This phenomenon suggested that the number of the pseudo-trisaccharide cores and glucose residues, as well as the bulk of the acyl groups maybe mutually affect the PPA activities. To the best of our knowledge, this is the first report of the inhibitory activities of acylated acarviostatins against PPA.